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2012 35 María Henar Alonso Ezcurra Study of the insertion sequence IS6110 in Mycobacterium tuberculosis Beijing strains and its promoter activity Departamento Director/es Bioquímica y Biología Molecular y Celular Otal Gil, Isabel Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es María Henar Alonso Ezcurra STUDY OF THE INSERTION SEQUENCE IS6110 IN MYCOBACTERIUM TUBERCULOSIS BEIJING STRAINS AND ITS PROMOTER ACTIVITY Director/es Bioquímica y Biología Molecular y Celular Otal Gil, Isabel Tesis Doctoral Autor 2012 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA Departamento Director/es Director/es Tesis Doctoral Autor Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es UNIVERSIDAD DE ZARAGOZA  FACULTAD DE MEDICINA DEPARTAMENTO DE MICROBIOLOGÍA, MEDICINA PREVENTIVA Y SALUD PÚBLICA “Study of the Insertion Sequence IS 6110 in Mycobacterium tuberculosis Beijing strains and its promoter activity” Memoria presentada por María Henar Alonso Ezcurra Licenciada en Bioquímica Para optar al grado de Doctor por la Universidad de Zaragoza Directora: Isabel Otal Gil FACULTAD DE MEDICINA Departamento de Microbiología, Medicina Preventiva y Salud Pública D. Isabel Otal Gil, Profesora Titular del Departamento de Microbiología, Medicina Preventiva y Salud Pública, como directora de la Tesis Doctoral de María Henar Alonso Ezcurra titulada: “Study of the Insertion Sequence IS 6110 in Mycobacterium tuberculosis Beijing strains and its promoter activity” EXPONE: Que esta Tesis Doctoral corresponde con el proyecto de tesis presentado y aprobado en su momento, no habiéndose producido ninguna variación. Que esta Tesis Doctoral reúne los requisitos necesarios para optar al título de Doctor. Por lo anterior, emito el presente INFORME FAVORABLE. Zaragoza, Junio 2012 Fdo. Isabel Otal Gil Esta tesis doctoral ha sido elaborada en el Departamento de Microbiología, Medicina Preventiva y Salud Pública, adscrita al programa de Doctorado del Departamento de Bioquímica y Biología Molecular y Celular, siendo María Henar Alonso Ezcurra beneficiaria de un contrato como investigador junior del Consorcio público Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERes), de una beca predoctoral del Proyecto de la Unión Europea del VII Programa Marco (HEALTHF3-2008-200973) y de una beca predoctoral concedida por el Departamento de Ciencia, Tecnología y Universidad de la Diputación General de Aragón (referencia: B034/09). Parte de este trabajo es fruto de una estancia de tres meses en el Instituto Pasteur de Corea bajo la supervisión de la Doctora Priscile Brodin, dentro de un Proyecto Europeo para el estudio de la virulencia de las cepas Beijing. Este trabajo se ha realizado dentro de los siguientes proyectos: - Proyecto de la Unión Europea del VII Programa Marco: “Mycobacterium tuberculosis W-Beijing genetic diversity and differential virulence and host immune responses” (Referencia: HEALTH-F3-2008-200973) desde 2008 al 2011. - Proyecto del Ministerio de Ciencia y Tecnología “Estudios de los mecanismos de virulencia regulados por phoP en Mycobacterium tuberculosis. Aplicación para la construcción de una nueva generación de vacunas” (Referencia: BIO 2008-01561) desde 2009 al 2011. 7  Index of Tables Chapter I: Characterization of M. tuberculosis Beijing strain GC1237: Study of the copies of IS6110 and other genetic markers Table 1. Percentage of Beijing isolates in Canary Island from 1991 to 2008 58 Table 2. Insertion sites of IS6110 in M. tuberculosis GC1237, 210 and W 70 Table 3. Direct Repeats of IS6110 insertion sequence in M. tuberculosis GC1237 72 Chapter III: Localization of copies of IS6110 in M. tuberculosis HCS, Beijing and non-Beijing Table 4. Strains used in this study 129 Table 5. Locations of IS6110 of the eight representative Beijing strains 136 Table 6. New locations of IS6110 of the eight representative Beijing strains where IS6110 could act as a mobile promoter 138 Table 7. Points of insertion, DR, orientation and distance to neighboring genes of IS6110 located copies of the 61 studied strains 145 Mª Henar Alonso Ezcurra 8  Abreviations list A Adenine A1 (Insertion) IS6110 between dnaA-dnaN of Beijing strains ABB AnnexinV binding buffer Abs Absorbance ADC albumin-dextrose-catalase Ap Ampicillin AZYTRO Azytromycin BC Before Christ BCG vaccine or BCG “Bacille de Calmette et Guérin” vaccine bp Base Pairs BS Beijing strains BSL 3 Bio Safety Level 3 C Cytosine ºC Degree celsius CAS family Central Middle Eastern Asian family cDNA Complementary DNA CFU Colony Forming Units CM Chloramphenicol CYC Cycloserin CTAB hexadecyltrimethylammonium bromide DAT diacyltrehaloses DMEM Dulbecco’s modified Eagle medium DNA Deoxyribonucleic acid dNTPs deoxyribonucleotides DOTS Directly Observed Therapy Short-Course DR Direct Repeats DR Region Direct Repeat Region EAI family East-African-Indian family EDTA Ethylenediaminetetraacetic acid EMB Ethambutol FACS Fluorescence activated cell sorting FBS fetal bovine serum FMI Fluorescence median intensity G Guanine GFP Green Fluorescent Protein 9  Gm Gentamicin GTC buffer guanidinium thiocyanate buffer h hour HCS High copy strains, referred to high copy number of IS6110 H family Haarlem family Hyg Hygromycin INH Isoniazid IPT Isoniazid Preventive Treatment IR Inverted Repeats IS Insertion Sequence Km Kanamycin LAM family Latin-American-Mediterranean family LAM lipoarabinomannan LCS Low copy strains, referred to low copy number of IS6110 LM Lipopolysaccharides lipomannan LMPCR Ligation Mediated PCR LSP Large sequences of polymorphism m Mycolic acids mAG mycolyl arabinogalactan MANU family Indian TB family MCS Multiple Cloning Site MDR Multi Drug Resistant MH-S Murine immortalized alveolar macrophages MIC Minimum Inhibitory Concentration min minute MOI multiplicity of infection mRNA messenger RNA MSH Mycothiol MTB Mycobacterium tuberculosis MTBC Mycobacterium tuberculosis complex ml mililitres μg micrograms μl microlitres μm micrometre NBS Non-Beijing strains NaAc Sodium acetate NaCl Sodium chloride ng nanogram Mª Henar Alonso Ezcurra 10  nm nanometres NO Nitric oxide nsSNP nonsynonymous Single Nucleotide Polymorphism NTF region genetic marker specific to the Beijing genotype OADC oleic acid-albumin-dextrose-catalase OD Optical Density OP6110 Promoter Region of IS6110 ORF Open Reading frame OriC Replication origin PAT polyacyltrehaloses PAS Para-aminosalicylic Acid PAZ Pyrazinamide PBS phosphate buffered saline PCR Polymerase Chain Reaction PDIM Phthiocerol dimycocerosates PE Proline-glutamic acid PFA Paraformaldehide PPE Proline-Pproline glutamic acid PG Peptidoglycan PGG Principal genetic groups PGL Phenolic Glycolipid PIM Phosphatidylinositol mannosides plc region Phospholipase C region pmol picomol qRT-PCR Quantitative-Reverse Transcription Polymerase Chain Reaction RD Region of Difference RIF Rifampicin RFLP Restriction Fragment Length Polymorphism RNA Ribonucleic acid rRNA ribosomal RNA ROI Reactive Oxygen Intermediates rmp Revolutions per minute RQ Relative Quantification RT-PCR Reverse transcription Polymerase Chain Reaction RvD Regions absent in M. tuberculosis H37Rv s second SCGs SNP Cluster Groups s.d. Standard deviation 11  SM Streptomycin SNP Single Nucleotide Polymorphism sSNP silentSingle Nucleotide Polymorphism SL Sulfolipids ST spoligotypes T family Modern TB strains family tRNA transfer RNA TB Tuberculosis TDR Totally Drug-Resistant tsp Transcriptional start point U Unit UEPs Unique-event poliporphisms X family IS-low-banding family XDR eXtensively Drug Resistant WHO World Health Organization WT Wild Type    Resumen 13 Resumen Mycobacterium tuberculosis es el agente causal de la tuberculosis (TB). Esta enfermedad ha acompañado a la raza humana a lo largo de su historia y hoy en día sigue siendo una de las enfermedades infecciosas que causa mayor número de muertes en el mundo. Aunque se han realizado grandes avances en la lucha contra esta enfermedad, sigue siendo un problema de salud mundial. En la actualidad, una de las familias de M. tuberculosis que más atención atrae en todo el mundo es la familia Beijing, debido a que muestran importantes características patogénicas. Diferentes estudios han indicado que un tercio de los casos de tuberculosis a nivel mundial son causados por cepas de M. tuberculosis pertenecientes a la familia Beijing, siendo ésta una de las familias de M. tuberculosis con mayores tasas de morbilidad y mortalidad. Su capacidad de infección, así como su capacidad para propagarse rápidamente, probablemente se deban a ventajas genéticas y a mecanismos de virulencia aún no identificados. Entre los mecanismos propuestos causantes de las diferencias de virulencia entre los fenotipos de las cepas de M. tuberculosis se encuentran inserciones de la secuencia IS6110, deleciones duplicaciones y reorganizaciones genéticas. Se ha demostrado que esta secuencia puede aumentar la expresión de genes adyacentes actuando como un promotor móvil localizado en su extremo 3’. El hecho de que las cepas de la familia Beijing tengan un mayor número de copias de IS6110 que otras familias, podría estar relacionado con las características especiales de virulencia y de transmisión de esta familia. La cepa de M. tuberculosis GC1237, perteneciente a la familia Beijing, ha demostrado una elevada capacidad de diseminación y transmisión dentro de una comunidad. Con el fin de comprender mejor algunos de los mecanismos de virulencia de las cepas Beijing y relacionarlos con la secuencia de inserción IS6110, se caracterizó el aislado clínico de M. tuberculosis GC1237. Se localizaron todas las copias de este elemento en el genoma de la cepa GC1237 y se realizó un análisis más detallado de una copia de IS6110 localizada a 31 nucleótidos del inicio del gen esencial Rv2179c y próxima al gen esencial aroG. Mediante el uso de varias técnicas se confirmó que esta copia de IS6110 está actuando como un promotor móvil de Rv2179c, tanto en cultivo líquido como en el interior del macrófago. A partir de estos resultados podemos sugerir que la sobre-expresión de este gen podría ser ventajosa para la cepa Beijing GC1237, al menos en ciertos entornos, como en el interior de las células infectadas. Además, esta localización resultó ser única de esta cepa lo que permitió el diseño de un test rápido para su detección. Mª Henar Alonso Ezcurra 14 Por otro lado, en anteriores investigaciones se estudió la actividad promotora de IS6110 en la copia de IS6110 localizada en la región promotora del gen phoP en M. bovis MBZ. Debido a la condición MDR esta cepa, este estudio se llevó a cabo mediante el uso de dos plásmidos recombinantes en el interior M. smegmatis mc2155, en cual se observó un incremento en la transcripción del gen phoP en la construcción que contenía la IS6110. Con el objetivo de profundizar en el estudio del efecto de esta copia de IS6110 sobre el gen phoP, se realizaron nuevas construcciones de plásmidos que contenían esta región y se introdujeron en M. tuberculosis. A continuación, se analizó la actividad promotora de IS6110 por varios métodos y en condiciones diferentes, tanto en cultivo líquido como en el interior de macrófagos. Los resultados obtenidos confirmaron que esta copia de IS6110 está actuando como secuencia promotora del gen phoP. Este hecho cobra gran importancia ya que el gen phoP codifica para un factor de transcripción que regula ~2% del genoma de M. tuberculosis y está implicado en virulencia. Por último, dado que las cepas de M. tuberculosis Beijing contienen un mayor número de copias de IS6110 lo que podría aportar alguna ventaja a este genotipo, se realizó una búsqueda de puntos de inserción de IS6110 en 61 aislados clínicos, entre ellos 17 cepas Beijing y 44 cepas no-Beijing, los cuales presentaban un alto número de copias de este elemento. La ubicación de las copias de esta secuencia permitió la detección de los puntos de mayor frecuencia de inserción de IS6110, o “hot-spots”, en las cepas Beijing. Estas localizaciones podrían ser la clave de algunas ventajas del genotipo Beijing y su estudio debe de ser tenido en cuenta en una futura investigación.   Summary 15 Summary Mycobacterium tuberculosis is the aetiological agent of tuberculosis (TB). This disease has walked together with human beings along our history, and nowadays is still one of the infectious diseases that causes more deaths in the world. Although in the last century great advances have been done in our fight against TB, this disease is still a worldwide health problem. One of the families of M. tuberculosis currently attracting considerable worldwide attention is the Beijing family as they display important pathogenic features. Different studies have indicated that one-third of global TB cases are caused by this family assigning this lineage to one of the most successful mycobacterial families in terms of morbidity and mortality. The capacity of infection and the ability of M. tuberculosis Beijing strains to spread rapidly probably result from genetic advantages and unidentified mechanisms of virulence not yet thoroughly investigated. Among the mechanisms proposed to be responsible for differences in the virulence phenotypes of M. tuberculosis strains we find IS6110 insertions, genetic reorganizations, genetic duplications and deletions, which have strong influence on fitness. It has been demonstrated that IS6110 may increase the expression of neighboring genes through an outward-directed promoter in its 3’ end. The fact that the Beijing lineage contains a larger number of IS6110 copies than other lineages could be related with the special characteristics of this family in terms of virulence and capacity for rapid dissemination. The M. tuberculosis GC1237 strain, which belongs to Beijing family, has demonstrated an enhanced capacity to spread and transmit within a community. In order to better understand some mechanisms of virulence of Beijing strains and related them with the insertion sequence IS6110, we have characterized the clinical isolate M. tuberculosis GC1237 strain locating all the copies of this element. We focused in the characterization of one copy located 31 bp upstream of the essential gene Rv2179c and close to the essential gene aroG and, by several methods, we confirmed that this copy of IS6110 is acting as a mobile promoter both in broth and inside macrophages. From our results we can hypothesize that the over-expression of this gene could be advantageous for GC1237 at least in certain environments such as infecting macrophages. In addition, this location was unique of this strain allowing us the design of a rapid test for its detection. On the other hand, in previous investigations, the promoter activity of IS6110 was studied in the copy of IS6110 located in the promoter region of phoP gene of M. bovis MBZ. Due to the MDR condition of this strain, this study was carried out by using two recombinant plasmids inside M. smegmatis mc2155 obtaining an increment in the Mª Henar Alonso Ezcurra 16 transcription of phoP gene in the construction containing IS6110. To depth in the study of the promoter activity of this IS6110 in phoP gene, new plasmid constructions containing this region were made and introduced in M. tuberculosis. After that, the promoter activity was analyzed by several methods and in different conditions both in broth and inside macrophages. Our results confirmed that this copy of IS6110 is acting as a promoter of phoP gene. This fact could be important as this gene codifies for a transcription factor that regulates ~2% of M. tuberculosis genome and is involved in virulence. Finally, as M. tuberculosis Beijing contain a larger number of IS6110 copies and these could provide some advantages to this genotype, a scrutiny of points of insertion of IS6110 was carried out in 61 clinical isolates with high number of copies of this element, 17 Beijing and 44 non-Beijing strains. The location of the copies of this sequence allowed us to detect new hot-spots for the insertion of IS6110 specific of Beijing strains. These locations could be the key of some advantages of the Beijing genotype and its study should be taken into account in further research. General Introduction 23 Koch announced the discovery of a vaccine against tuberculosis consisting of glycerol extracts of pure cultures of tubercle bacilli. The vaccine candidate was assayed in a clinical trial with disastrous results but in 1908, Charles Mantoux found that it was an effective intradermic test for diagnosing tuberculosis. Regardless of this failure, Koch’s observations settled the basis for microbiology known as the Koch’s postulates. These comprise identification of the pathogen in affected tissue, growth of single clones of the pathogen in vitro, and establishment of a similar disease in the experimental animals by means of the pure culture (64). With Edward Jenner’s successful invention, showing that infection with cowpox would give immunity against smallpox in humans, many doctors placed their hopes on the use of Mycobacterium bovis (the agent that causes bovine TB) for the development of a vaccine against human TB. In 1908 Albert Calmette and Camille Guérin started the development of a vaccine against tuberculosis. They isolated from a dead cow M. bovis and for the next 13 years, these scientists grew every fortnight a new batch of bacteria in a solution of beef bile and potato. Eventually, the bacteria lost their ability to cause disease, but were still capable of stimulating the immune system to protect mice, pigs, guinea pigs and monkeys from tuberculosis. The vaccine was called “Bacille de Calmette et Guérin” or “BCG” and it was first administered to an infant in 1921 (Figure 1). A total of 8 million babies and nearly 14 million people were given the BCG vaccine in the International Tuberculosis Campaign, which ran through 1951. The project initially began in Europe in the aftermath of World War II. However, the program extended beyond Europe when UNICEF contributed two million dollars to expand the program to other continents. Since then more than a billion people have been vaccinated with BCG (9). In 1943, Selman A. Waksman, who had been working for decades to find an effective antibiotic against Mycobacterium tuberculosis, was finally successful (Figure 1). Streptomycin purified from Streptomyces griseus was first administered to a human on November 20th, 1944. The results were extremely impressive. The disease immediately stopped its progression, the bacteria disappeared from the patient's sputum, and he recovered fully. A rapid succession of anti-TB drugs appeared in the following years: para-aminosalicylic acid (PAS, 1949), isoniazid (INH, 1952), pyrazinamide (PAZ, 1954), ethambutol (EMB, 1962) and rifampicin (RIF, 1963). This advance was very important because with the streptomycin (SM) treatment, resistant mutants appeared within a few months, endangering the success of antibiotic therapy. However, it was soon proven that using a combination of drugs would solve this problem (59). Mª Hena r 24 The un a The ho p with its and the In res p declara t half a m occur w with tu b is calle d it is kn o (72, 12 7 Figure 1 against t Tuberc u only su inciden t negativ e HIV-as s r Alonso Ezcu a voidable p es that th e dramatic r e deadly for m p onse to t h t ion of a gl m illion new c w orldwide. E b erculosis t h d TDR, for T o wn. The fir s 7 ). 1 : Timeline i t his disease. u losis is to d rpassed b y t cases of T e people a n s ociated T B rra return of T e disease c e surgence, m of MDR a h is resurg e obal healt h c ases of m E ven more h at has be c T otally Dru g s t cases of i n tubercul o Adapted fr o d ay the se c y HIV/AIDS T B, 1.1 mil l n d an addi t B (134). T B c ould be co the rise p r a ssociated w e nce, the W h emergenc ultidrug-re s alarming i s c ome resist a g -Resistant these TDR o sis resear c o m (64). c ond majo r . In 2010, t l ion (range , t ional 0.35 mpletely el i r evalence o w ith HIV/AI D W orld Hea y in 1993 ( s istant tube r s the existe a nt to all th e . In other w were repo r c h. It shows r cause of t here were , 0.9–1.2 m million (ra n i minated w e o f multidru g D S (48). lth Organi z ( 135) (Figu r r culosis (M D nce of at l e e drugs us e w ords, they a r ted in Iran the most rel death from 8.8 million m illion) deat n ge, 0.32– 0 e re dashed g -resistant s z ation (W H r e 1). Ever y D R-TB) ar e e ast 12 pa t e d against t a re untreat a in 2009 an d evant highli g any infec t (range, 8. 5 hs from T B 0 .39 million ) in the 19 8 s trains (M D H O) issued y year, ne a e estimated t ients infect t he disease a ble as far d Italy in 20 g hts in the fi g t ious disea s 5 –9.2 milli o B among H I ) deaths fr o 8 0s R) a a rly to ed . It as 07 g ht s e, o n) I Vo m General Introduction 25 II.The disease: Pathogenesis and intracellular lifestyle of M. tuberculosis Pathogenesis of M. tuberculosis Tuberculosis is a contagious disease (113). Infection with M. tuberculosis occurs through inhalation of aerosols containing the bacteria which are spread by persons with active pulmonary TB. After inhalation, bacteria are settled in the alveoli and disseminated by the lymphatic circulation. Further dissemination to other parts of the lung and occasionally to other organs is achieved by haematogenous circulation. The most common form of the disease is pulmonary tuberculosis, although TB-meningitis, miliary (disseminated) tuberculosis, lymphadenitis, osteomyelitis and Pott’s disease (affected bones) also occur (113). Primary infection leads to active disease in about 10% of infected individuals, in 80% of the cases in the period of two years (125). In the remaining 90%, the immune system controls the infection, and the individual is noninfectious and asymptomatic. In this clinical state the TB bacilli can lie dormant for years (latent TB) (102). However, when the immune system is weakened, the latent infection can reactivate (113, 125). In an HIV-infected person the risk of reactivation of latent TB is higher than 10% per year, compared to a lifetime risk of 10-20% for HIVnegative individuals (23, 111, 125). Life inside the macrophages Once the bacteria are phagocytosed by alveolar macrophages, mediated by specific macrophage receptors (36, 38, 62, 110), a localized proinflammatory response that leads to recruitment of mononuclear cells from neighboring blood vessels is induced. These cells will build the granuloma (necessary to contain infection) which consists of a group of infected macrophages, surrounded by foamy giant cells (multinuclear macrophages loaded with lipids) and macrophages with a layer of lymphocytes delineating its periphery (101-103) (Figure 2). Activated macrophages promote phagosomal maturation by a process that finally involves fusion with lysosomes and formation of the phagolysosome. These vesicles provide a hostile environment for the bacilli including acid pH, reactive oxygen intermediates (ROI), lysosomal enzymes and toxic peptides. M. tuberculosis has evolved to survive within macrophages by arresting the normal phagosomal maturation at an early stage, thereby restricting its acidification and limiting fusion with lysosomes (90, 98). Modulation of phagosome maturation seems to be mainly mediated by both mycobacterial cell-wall lipids and other bacterial effectors (e.g., SapM or PknG) (46, 132, 133). Additionally, the screening of shotgun libraries has enabled the isolation of several M. tuberculosis mutants unable to induce phagosomal arrest (96, 118). Apart Mª Henar Alonso Ezcurra 26 from surviving within the macrophage, pathogenic mycobacteria have also been suggested to escape into the cytosol in an ESX-1-dependent manner (128). In addition, M. tuberculosis reduces phagosome acidification by the exclusion of the protonATPase (119) and decreases ROI and nitric oxide (NO) synthesis and cytokine production by modulating Ca2+ fluxes in infected macrophages (76). Moreover, M. tuberculosis has evolved multiple strategies to detoxify ROI and RNI (37). Recently, authophagy (29, 57) and apoptosis (68) have been postulated as effective antimycobacterial mechanisms. Figure 2: Progression of TB granuloma. In the early stage, the granuloma has a core of infected macrophages enclosed by foamy macrophages and other mononuclear phagocytes, surrounded by lymphocytes. As the granuloma matures, it develops a fibrous capsule that encases the macrophage core and excludes the majority of lymphocytes from the center of the structure. Adapted from (102). Measures against tuberculosis: ¾ Antibiotic therapy Tuberculosis treatment is difficult and requires long courses (around 6 to 12 months) of multiple antibiotics. It consist of an initial intensive phase of treatment (2 months) with RIF, INH, PAZ and either EMB or SM designed to kill actively growing and semidormant bacilli, followed by a continuation phase (4 months) with RIF and INH to eliminate residual bacilli and reduce the number of relapses. To complete the treatment it is critical to reduce the development of acquired drug resistance and therefore trained individual personnel supervises that the patient takes each dose of medication. This strategy is known as directly observed treatment short-course (DOTS) and was promoted as the official policy of the WHO in 1991 (136). General Introduction 27 Isoniazid Preventive Treatment (IPT) for six months is recommended for high risk individuals, like HIV infected individuals, or children who are in close contact with an infectious person with the aim of decreasing the risk of primary infection with M. tuberculosis (63). Another major challenge is the ever-increasing resistance to anti-TB drugs. Multidrugresistant TB refers to TB caused by M. tuberculosis isolates that are resistant to the most effective drugs, INH and RIF. MDR strains take longer to treat (up to two years) with second-line drugs, which are more than 100 times more expensive, less effective and also much more toxic to the patients (49). The virtually untreatable extensively drug-resistant TB (XDR-TB) is defined as MDR-TB which is also resistant any of the fluoroquinolones and second-line anti-TB injectable drugs (Amikacin, Kanamycin (Km) or Capreomycin). ¾ Vaccination BCG is one of the most widely used vaccine today (> 80 % of neonates and infants in some countries). Although it does not prevent primary infection or reactivation of latent pulmonary TB, the principal source of bacillary spread in the community, BCG protects against disseminated forms of TB, especially against meningitis in children. For that reason, BCG vaccination is still recommended by the WHO and applied widely (115, 123). After its first use in humans in 1921, propagation of BCG in non-standardized conditions in different laboratories all around the world gave rise to different variant strains that differ in genotype and phenotypic characteristics (9). Strain differences could be one cause of the variable protective efficacy afforded by BCG (44), but other factors such as population genetics or exposure to environmental mycobacteria could be implicated (28). Due to this inconsistency in protective efficacy, and to the inability of BCG to prevent pulmonary forms of tuberculosis and consequently, to prevent transmission of the disease, great efforts are being made by governments, research institutions and private foundations for constructing and testing new promising vaccine candidates. Mª Henar Alonso Ezcurra 28 III.Biology of the bacillus and mycobacterial genetics General characteristics of mycobacteria Mycobacterium genus comprises a number of Gram-positive, acid-fast, rod-shaped aerobic bacteria and it is the only member of the family Mycobacteriaceae within the order Actinomycetales (117). Like other closely related Actinomycetales, such as Nocardia and Corynebacterium, mycobacteria have unusually high genomic DNA GC content and are capable of producing mycolic acids (m) as major components of their cell wall. Phenotypically M. tuberculosis is catalase and nitrate reductase positive, nonmotile and non-sporulating of 2-5μm in length and 0.2-0.5μm in width. To date, more than 100 species of mycobacteria have been described (122). Most of these species are environmental saprophytes. Phylogenetic analyses and phenotypic characteristics, such as growth rate and pigmentation, can be used to classify these species. The classical distinction between rapid and slow growth is based on the ability of strains to develop clearly visible colonies in less or more than 7 days, respectively. Rapid growers (< 7 days) are free, environmental, saprophytic species. Interestingly, the slow growing mycobacteria comprise most of the pathogenic species, including M. tuberculosis which has a generation time of ~24 hours. This contributes to the chronic nature of the infection, requires long-term treatment to fully eradicate the pathogen and imposes a barrier for microbiologists since it takes up to four weeks for a single M. tuberculosis cell to become a colony on solid media. The various etiologic agents of TB are clustered in the M. tuberculosis complex (MTBC), which currently includes eight species: The human-adapted strains M. tuberculosis, M. africanum and M. canettii (being M. canettii the most divergent within the MTBC) and MTBC also includes the animal-adapted strains M. bovis (primarily a pathogen of cattle), M. caprae (a pathogen of goats), M. microti (a pathogen of rodents) and M. pinnipedii (a pathogen of seals and sea lions) and, recently M. mungi was isolated from mongoose (2, 89). Mycobacteria grouped in MTBC are characterized by 99.9% similarity at the nucleotide level and identical 16S rRNA but that differ in terms of phenotypes and host preference (13). The envelope of mycobacteria The unique cell wall of M. tuberculosis forms a waxy envelope and has been established as an important factor leading to bacterial virulence and survival. This envelope differs considerably from cell wall of both Gram-positive and Gram-negative bacteria. Even though they are structurally more closely related to Gram-positive bacteria, mycobacteria do not fit into the Gram-positive category as the molecules General Introduction 29 attached to the cell wall are distinctively lipids rather than proteins or polysaccharides. Frequently, they do not retain the crystal violet and appear as “ghosts” after Gram staining (7). Therefore, acid-fast stains, such as the Ziehl-Neelsen stain or the fluorescent auramine-rhodamine stain are recommended for the detection of mycobacteria. A characteristic feature of their cell envelope is the high proportion of lipid-rich molecules. Schematically, the cell envelope from the inside to the outside, comprises a typical bacterial plasma membrane, a cell wall and an external capsule, which contains proteins, polysaccharides and lipids (Figure 3) (26). The outer layer presents various non-covalently attached lipids and glycolipids esterified with multimethyl-branched long-chain fatty acids (61, 86). These complex lipids are restricted, with few exceptions, to pathogenic mycobacteria and are suggested to play important roles in pathogenicity. In M. tuberculosis, these lipids include the phthiocerol dimycocerosates (PDIM) (5, 18, 24, 92, 100) and the closely related phenolic glycolipids (PGL) (93, 97, 112), the trehalose ester families that include sulfolipids (SL) (22, 31, 54), diacyltrehaloses (DAT) and polyacyltrehaloses (PAT) (69, 99), and the family of mannosyl-β-1phosphomycoketides (80). This highly hydrophobic envelope decreases the permeability and susceptibility to degradation and makes the bacterium naturally resistant to most of the antibiotics and chemical agents and confers unique staining properties. Figure 3: Arrangement of structural components in the cell envelope structure of M.tuberculosis. The cell wall consists of a peptidoglycan (PG) covalently attached to the mycolyl arabinogalactan (mAG), which is in turn esterified by mycolic acids (m). The lipopolysacharides lipomannan (LM) and lipoarabinomannan (LAM) are anchored to the plasmatic membrane through phosphatidylinositol mannosides (PIM) and they extend towards the exterior of the cell wall (86). Mª Hena r 30 Mycob a The co m M. tub e 2002 a Project ) strains, tuberc u genetic as in s e or http: / The ge n regions high G + family o content deviati o hydrop h low G + encode Figure genomi c Some o metabo the cell acid m e PPE fa 168 ge n r Alonso Ezcu a cterial ge n m plete gen o e rculosis, H nd it is in ) . Complet e CDC1551, u losis geno technique s e veral onli n / /www.micr o n ome com p show a s k + C content o f PE or P P are those o n to low h obic amin o + C content 3,989 prot e 4: Functio n c content of M o utstandin g lism which envelope. e tabolism, c mily of pro t n es. The n rra n omics o me seque 37Rv, was continuou e genome F11, H37 R mes is in s (70). Thi s n e databas o besonline. p rises 4,41 1 k ew in this (> 80%) a P E proteins. coding fo r G+C cont e o acids, es s codons. T e ins and 71 n al classifi c M . tuberculo s g features i is indicati v There are g c ompared t o t eins, whic h ames com e nce of the m obtained i n s revision sequence s R a, H37Rv progress p s informatio es (http://t u org/). 1 ,532 bp, w G+C conte a ppear to b In turn, th e r transme m e nt is beli e s ential in a T he last re - stable RN A c ation of M s is H37Rv. A i nclude the v e of the hi g enes enc o o only 50 i n h represen t e from Pr o m ost studi e n 1998 (11 3 (as of Au g s are now and KZN1 4 p roviding a n is availa b u berculist.e w ith a G+C nt. A cons p b e unique i e few gene s m brane prot e ved to b e ny transm e - annotation A s (Figure 4 M . tubercul o A dapted fro m great ab u ghly active o ding for 2 5 n the geno m t s approxi m o -Glu (PE) a e d and the b 3 ) and revi g ust 2010; available f 4 35, and s e a valuable b le in sequ e pfl.ch/, htt p content of p icuous gr o i n mycoba c s with parti c eins or po l e a conse q e mbrane d o identified 4 ) (70). o sis H37R v m (70). u ndance of metabolis m 5 0 distinct e m e of Esch e m ately 4% o a nd Pro-P r b est under s sed and r e NCBI En t f or five M. e quencing o tool for re e ncing we b p ://www.nc b 65.9% (21 o up of gen e c teria and b c ularly low l yketide sy n q uence of o main, that 4090 gen e v genes. T genes in v m mainly i n e nzymes in v e richia coli ( o f the gen o r o-Glu (PP E s tood strain e -annotated t rez Geno m tuberculo s o f 71 other search usi b sites, as w b i.nlm.nih.g o ). Only a f e e s with a v e b elong to t (< 50%) G + n thases. T h the requi r are coded e s thought imeline of t v olved in li p n relation w v olved in f a ( 20). The P o me, includ E ) sequen c of in m e s is M. ng w ell o v/ e w e ry he + C h is r ed by to t he p id w ith a tty P Ees c es General Introduction 31 found in the two conserved N-terminal regions in each of these protein families that are approximately 110 and 180 amino acids long, respectively. Although the function of the members of the PE and PPE protein families has not been established, they are suggested to be involved in antigenic variation and disease pathogenesis (29). Tubercle bacilli also contain complex regulatory machinery comprising 11 two component systems, 11 serine/threonine protein kinases, 13 σ factors, and over 100 repressors and activators, suggesting a tight regulation of transcription in response to different stimuli. Interestingly, the analysis of the DNA metabolic system of M. tuberculosis indicates a very efficient DNA repair system, in other words, replication machinery of exceptionally high fidelity. The genome of M. tuberculosis lacks the MutSbased mismatch repair system. However, this absence is overcome by the presence of nearly 45 genes related to DNA repair mechanisms (87), including three copies of the mutT gene. This gene encodes the enzyme in charge of removing oxidized guanines whose incorporation during replication causes base-pair mismatching (20, 87). Repetitive DNA is very important in mycobacterial genomes. Elements like Tandem Repeats, Interspersed Repeats, and mobile genetic elements are common. In H37Rv, 56 loci with similarity to IS elements have been found that can be classified into major IS families (IS3, IS5, IS605 or IS21). Mobile genetic elements will be widely analyzed in the following section as they could have an important role in genome plasticity and the virulence of the bacilli. Mobile genetic elements in mycobacteria Insertion sequence (IS) is a short DNA mobile genetic element coding for proteins involved in the transposition activity, which allows it to spread within the genome. ISs are widely distributed in prokaryotes and can be grouped into different families based on structure characteristics and transposase similarities (75). More than 46 ISs from different species have been located and identified in Mycobacterium, mostly on the basis of sequence similarities (15). In the genome of the members of MTBC it has been possible to find dispersed IS elements that could be included in several of the following families attending to their characteristics: IS3, IS5, IS21, IS30, IS110, IS256; IS1535, ISL3 and other IS-like elements (53). The IS3 family consist of an extensive set of insertion elements in bacteria. The features that characterize this family are their length, between 1200 and 1600 bp, and their inverted repeats (IRs), between 20 and 40 bp long, as well as the presence of two overlapping open reading frames (ORFs: orfA and orfB) (75, 81). After the insertion, a duplication of 3 or 4 bp occurs at the insertion point (85). Recombination is also Mª Henar Alonso Ezcurra 32 another mechanism participating in the changes of the location of ISs along the genomes. All those mechanisms lead to IS-mediated gene rearrangements, inversions, deletions and duplications in the bacterial genomes. Examples of sequences of this family in MTBC are IS1540, IS1604, IS1556/990 and IS6110. The IS1540, IS1604 and IS1556/990, have missed the IRs or contain mutations in orfB making them supposedly inactive and non-functional (34, 82). However, the most representative member of this family, IS6110, has the two overlapping ORFs intact and a functional transposase. The insertion sequence IS6110 IS6110 was initially named IS986. Subsequently, a related element from M. bovis BCG was sequenced by Hermans et al. (58) and is referred to as IS987. The three sequences IS6110, IS986 and IS987 were practically identical and to avoid confusion, it was decided to use the same name IS6110 (81, 120). IS6110 is a genomic insertion element 1361 bp long, it shows 28 bp imperfect IRs, and due to the mechanism of transposition it generates duplications of 3 or 4 bp (known as Direct Repeat, DR) next to the insertion site (Figure 5). It may be present up to 25 copies per genome in M. tuberculosis (15) and only a few number of strains have no copies of this element (73). Recently, it has been suggested variations into the sequence of IS6110 copies from different strains of M. tuberculosis, which could have implications in its usefulness as target of PCR detection (109). Figure 5: Complete sequence of IS6110. The direct repeats (DR) (red) and the invert repeats (IR) (green) flanking the transposase gene are underlined. General Introduction 39 Spoligotype of M. tuberculosis Based on the genotype technique spoligotyping, we can group the global and local geographical structures of MTBC populations in families (16). Generally, a strain family can be described as a group of isolates that share specific biomarkers or properties indicative of a recent ancestor. MTBC strains contain a distinct chromosomal region consisting of multiple 36-bp direct repeats (DRs) interspersed by unique spacer DNA sequences (35 to 41 bp). Spoligotyping is based in the detection of 43 interspersed spacer sequences. Membranes spoted with 43 synthetic oligonucleotides are hybridized with labeled PCR-amplified DR region resulting in a pattern that can be detected by chemiluminiscence. The results are highly reproducible, and the binary (present/absent) data generated can be easily interpreted and computcrized and are amenable to intralaboratory comparisons (79). SpolDB4, the international spoligotyping database, contains 1939 different spoligotypes (ST), representing a total of 39,295 M. tuberculosis strains from 122 countries, and these are organized into 22 lineages/sublineages (16). Among these families we can enhance the Beijing family, the CentralAsian (CAS) family with two principal sublineages (CAS1 and CAS2), Zero, whose name comes from the absence of IS6110 insertion element (“Zero copy”), MANU (subdivided into MANU1 to MANU3), East-African-Indian (EAI) family, Haarlem family (H) with 4 sub lineages (H1-H4), Latin-American-Mediterranean (LAM), T family (modern TB strains) which is stratified into 5 sub-clades (T1-T5) and X (X1-X3 sub-lineages) which is well characterized as IS6110 low-banding family. ¾ Variation in spoligotype among the IX genetic cluster groups and the SCGs. Analysis of the relationship between the SNP-based phylogenetic trees (17, 55, 79) showed that some spoligotype clades were phylogenetically accurate with the SNPderived phylogenetic framework trees while others were less so. For example, the spoligotype-defined Beijing clade was exclusively present in SCG-2 and exclusive to cluster II of the IX cluster groups described by Gutaker (Figure 10). Mª Hena r 40 Figure Spoligot y clusters. groups spoligot y Althou g transmi s shown h r Alonso Ezcu 10: Distrib u y pe pattern s A dapted fr o (SCGs). Is o y pe clade as g h there a r s sion capa c h igh trans m rra u tion of th s superimp o o m (79). (B) o lates are i signment. A d r e few stu d c it y , it is no m issibilit y a n e spoligot y o sed on the Distribution ndicated b y d apted from d ies showi n w clear tha t n d virulenc e y pe clades SNP-deriv e of the spoli y a dot, w h (43). n g a clear t the M. tu b e capacity m on the S N d phylogen t gotype patt e ich is colo r associatio b erculosis B m ore than a n N P-based p t etic tree b a e rns among r coded ac c n between B eijing famil y n y other lin e hylogeny. ( a sed on the the ten clu s c ording to t lineage a y globally h e age. ( A) IX s ter t he nd h as General Introduction 41 The M. tuberculosis Beijing family The M. tuberculosis Beijing genotype family, also named East Asian Lineage (47), constitutes a homogeneous group of strains sharing a closely related IS6110 RFLP patterns containing a high number of bands, identical spoligotyiping (deletion of spacers 1 to 34 in the Direct Repeat region) (130), an insertion of IS6110 between dnaA-dnaN genes (called insertion A1) (67) and one or two IS6110 copies in a DNA region called NTF locus (66, 67). The insertion of IS6110 in these regions subdivide Beijing genotype in ancestral or atypical and modern or typical being the modern sublineage which contains at least one copy of IS6110 upstream of the NTF region (88). Moreover, the deletion of the regions of difference (RD) 105 and RD207, which are LSP markers, are present in all Beijing strains (124) (detailed in chapter 1). A few outliers that do not meet all of the above-suggested criteria for identification have also been described, as strains with “Beijing-like” spoligo patterns (25) (lacking one or more of the last nine spacers) and strains that harbor the Beijing genotype and the A1 insertion but which differ significantly in their IS6110 banding patterns from the regular Beijing strains (67). Strains of Beijing genotype were first described in China and neighbouring countries in 1995. The name comes from the finding that more than 80% of strains from the Beijing area were of this type (130). However, currently they are widespread in many regions of the world and frequently cause epidemic outbreaks (10, 19, 42, 50). Different studies have indicated that one-third of global TB cases is caused by Beijing family strains assigning this lineage to one of the most successful mycobacterial families in terms of morbidity and mortality (10). In some areas, such as Vietnam, Cuba, and Estonia, Beijing strains were found to be strongly associated with drug resistance (4, 12, 30, 65). This is consistent with the prolific spread of the (multidrug)-resistant strain W and its variants in the 1990s in Northern America (11). This strain and its variants exhibit highly similar IS6110 RFLP patterns and an identical spoligo pattern. It is now known that the W family and the Beijing family represent the same genotype which was concurrently identified in North America (12) and Asia (130). Both groups of strains are referred as Beijing family (Beijing/W or W-Beijing). A second group of strains with distantly related IS6110 RFLP profiles have been determined to belong to the Beijing lineage by other molecular techniques and are referred to as ancestral strains (88). They share a common predecessor with Beijing family strains, as illustrated by the extensive shared molecular characteristics, including chromosomal insertions and deletions, and single nucleotide polymorphisms (35). Mª Henar Alonso Ezcurra 42 The widely distributed association of drug resistance and the Beijing genotype suggest that these strains may have a particular propensity for acquiring drug resistance (51). It was reported that Beijing strains carry mutations in putative mutators genes, and this may explain a higher adaptability of these bacteria to stress conditions such as exposure to antituberculosis drugs and the hostile intracellular environment (35). A number of selective advantages have been associated with its success including a lower efficacy of BCG for Beijing strains (74, 130), higher virulence (74, 78), ability to induce a differential immune response (33, 77), higher transmissibility (10) and enhanced capacity to grow in human macrophages and monocytes (71, 137) and an enhanced capacity to acquire drug resistance (1, 11, 12). However, unidentified virulence factors and the modulation of specific host responses are not yet thoroughly investigated. General Introduction 43 References 1. Agerton, T. B., S. E. Valway, R. J. Blinkhorn, K. L. Shilkret, R. Reves, W. W. Schluter, B. Gore, C. J. Pozsik, B. B. Plikaytis, C. Woodley, and I. M. Onorato. 1999. Spread of strain W, a highly drug-resistant strain of Mycobacterium tuberculosis, across the United States. Clin Infect Dis 29:85-92; discussion 93-5. 2. Alexander, K. A., P. N. Laver, A. L. Michel, M. Williams, P. D. van Helden, R. M. Warren, and N. C. Gey van Pittius. Novel Mycobacterium tuberculosis complex pathogen, M. mungi. Emerg Infect Dis 16:1296-9. 3. Alonso, H., J. I. Aguilo, S. Samper, J. A. Caminero, M. I. Campos-Herrero, B. Gicquel, R. Brosch, C. Martin, and I. Otal. Deciphering the role of IS6110 in a highly transmissible Mycobacterium tuberculosis Beijing strain, GC1237. Tuberculosis (Edinb). 4. Anh, D. D., M. W. Borgdorff, L. N. Van, N. T. Lan, T. van Gorkom, K. Kremer, and D. van Soolingen. 2000. Mycobacterium tuberculosis Beijing genotype emerging in Vietnam. Emerg Infect Dis 6:302-5. 5. Astarie-Dequeker, C., L. Le Guyader, W. Malaga, F. K. Seaphanh, C. Chalut, A. Lopez, and C. Guilhot. 2009. Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids. PLoS Pathog 5:e1000289. 6. Baker, L., T. Brown, M. C. Maiden, and F. Drobniewski. 2004. Silent nucleotide polymorphisms and a phylogeny for Mycobacterium tuberculosis. Emerg Infect Dis 10:1568-77. 7. Barrera, L. 2007. The basics of clinical bacteriology. In J. C. Palomino, S. CardosoLeao, and V. Ritacco (ed.), Tuberculosis. From basic science to patient care. 8. Beggs, M. L., K. D. Eisenach, and M. D. Cave. 2000. Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis. J Clin Microbiol 38:2923-8. 9. Behr, M. A. 2002. BCG--different strains, different vaccines? Lancet Infect Dis 2:86-92. 10. Bifani, P. J., B. Mathema, N. E. Kurepina, and B. N. Kreiswirth. 2002. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol 10:45-52. 11. Bifani, P. J., B. Mathema, Z. Liu, S. L. Moghazeh, B. Shopsin, B. Tempalski, J. Driscol, R. Frothingham, J. M. Musser, P. Alcabes, and B. N. Kreiswirth. 1999. Identification of a W variant outbreak of Mycobacterium tuberculosis via populationbased molecular epidemiology. JAMA 282:2321-7. 12. Bifani, P. J., B. B. Plikaytis, V. Kapur, K. Stockbauer, X. Pan, M. L. Lutfey, S. L. Moghazeh, W. Eisner, T. M. Daniel, M. H. Kaplan, J. T. Crawford, J. M. Musser, and B. N. Kreiswirth. 1996. Origin and interstate spread of a New York City multidrugresistant Mycobacterium tuberculosis clone family. Jama 275:452-7. 13. Brosch, R., S. V. Gordon, M. Marmiesse, P. Brodin, C. Buchrieser, K. Eiglmeier, T. Garnier, C. Gutierrez, G. Hewinson, K. Kremer, L. M. Parsons, A. S. Pym, S. Samper, D. van Soolingen, and S. T. Cole. 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A 99:3684-9. 14. Brosch, R., W. J. Philipp, E. Stavropoulos, M. J. Colston, S. T. Cole, and S. V. Gordon. 1999. Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain. Infect Immun 67:5768-74. 15. Brosch, R. G., S.V.; Eiglmeier, K.; Garnier, T.; Tekaia, F.; Yeramian, E. & Cole, S.T. . 2000. Genomics, biology, and evolution of the Mycobacterium tuberculosis complex. Molecular Genetics of Mycobacteria, ISBN 1-55581-191-4:19-36. 16. Brudey, K., J. R. Driscoll, L. Rigouts, W. M. Prodinger, A. Gori, S. A. Al-Hajoj, C. Allix, L. Aristimuno, J. Arora, V. Baumanis, L. Binder, P. Cafrune, A. Cataldi, S. Cheong, R. Diel, C. Ellermeier, J. T. Evans, M. Fauville-Dufaux, S. Ferdinand, D. Garcia de Viedma, C. Garzelli, L. Gazzola, H. M. Gomes, M. C. Guttierez, P. M. Hawkey, P. D. van Helden, G. V. Kadival, B. N. Kreiswirth, K. Kremer, M. Kubin, S. Mª Henar Alonso Ezcurra 44 P. Kulkarni, B. Liens, T. Lillebaek, M. L. Ho, C. Martin, I. Mokrousov, O. Narvskaia, Y. F. Ngeow, L. Naumann, S. Niemann, I. Parwati, Z. Rahim, V. RasolofoRazanamparany, T. Rasolonavalona, M. L. Rossetti, S. Rusch-Gerdes, A. Sajduda, S. Samper, I. G. Shemyakin, U. B. Singh, A. Somoskovi, R. A. Skuce, D. van Soolingen, E. M. Streicher, P. N. Suffys, E. Tortoli, T. Tracevska, V. Vincent, T. C. Victor, R. M. Warren, S. F. Yap, K. Zaman, F. Portaels, N. Rastogi, and C. Sola. 2006. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6:23. 17. Brudey, K., I. Filliol, S. Ferdinand, V. Guernier, P. Duval, B. Maubert, C. Sola, and N. Rastogi. 2006. Long-term population-based genotyping study of Mycobacterium tuberculosis complex isolates in the French departments of the Americas. J Clin Microbiol 44:183-91. 18. Camacho, L. R., D. Ensergueix, E. Perez, B. Gicquel, and C. Guilhot. 1999. Identification of a virulence gene cluster of Mycobacterium tuberculosis by signaturetagged transposon mutagenesis. Mol Microbiol 34:257-67. 19. Caminero, J. A., M. J. Pena, M. I. Campos-Herrero, J. C. Rodriguez, I. Garcia, P. Cabrera, C. Lafoz, S. Samper, H. Takiff, O. Afonso, J. M. Pavon, M. J. Torres, D. van Soolingen, D. A. Enarson, and C. Martin. 2001. Epidemiological evidence of the spread of a Mycobacterium tuberculosis strain of the Beijing genotype on Gran Canaria Island. Am J Respir Crit Care Med 164:1165-70. 20. Cole, S. T. 1999. Learning from the genome sequence of Mycobacterium tuberculosis H37Rv. FEBS Lett 452:7-10. 21. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, 3rd, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, A. Krogh, J. McLean, S. Moule, L. Murphy, K. Oliver, J. Osborne, M. A. Quail, M. A. Rajandream, J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares, S. Squares, J. E. Sulston, K. Taylor, S. Whitehead, and B. G. Barrell. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-44. 22. Converse, S. E., J. D. Mougous, M. D. Leavell, J. A. Leary, C. R. Bertozzi, and J. S. Cox. 2003. MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence. Proc Natl Acad Sci U S A 100:6121-6. 23. Corbett, E. L., C. J. Watt, N. Walker, D. Maher, B. G. Williams, M. C. Raviglione, and C. Dye. 2003. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 163:1009-21. 24. Cox, J. S., B. Chen, M. McNeil, and W. R. Jacobs, Jr. 1999. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402:79-83. 25. Chan, M. Y., M. Borgdorff, C. W. Yip, P. E. de Haas, W. S. Wong, K. M. Kam, and D. Van Soolingen. 2001. Seventy percent of the Mycobacterium tuberculosis isolates in Hong Kong represent the Beijing genotype. Epidemiol Infect 127:169-71. 26. Daffe, M., and P. Draper. 1998. The envelope layers of mycobacteria with reference to their pathogenicity. Adv Microb Physiol 39:131-203. 27. Daniel, T. M. 2006. The history of tuberculosis. Respir Med 100:1862-70. 28. Demangel, C., T. Garnier, I. Rosenkrands, and S. T. Cole. 2005. Differential effects of prior exposure to environmental mycobacteria on vaccination with Mycobacterium bovis BCG or a recombinant BCG strain expressing RD1 antigens. Infect Immun 73:2190-6. 29. Deretic, V., S. Singh, S. Master, J. Harris, E. Roberts, G. Kyei, A. Davis, S. de Haro, J. Naylor, H. H. Lee, and I. Vergne. 2006. Mycobacterium tuberculosis inhibition of phagolysosome biogenesis and autophagy as a host defence mechanism. Cell Microbiol 8:719-27. 30. Diaz, R., K. Kremer, P. E. de Haas, R. I. Gomez, A. Marrero, J. A. Valdivia, J. D. van Embden, and D. van Soolingen. 1998. Molecular epidemiology of tuberculosis in General Introduction 45 Cuba outside of Havana, July 1994-June 1995: utility of spoligotyping versus IS6110 restriction fragment length polymorphism. Int J Tuberc Lung Dis 2:743-50. 31. Domenech, P., M. B. Reed, C. S. Dowd, C. Manca, G. Kaplan, and C. E. Barry, 3rd. 2004. The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis. J Biol Chem 279:21257-65. 32. Donoghue, H. D., M. Spigelman, C. L. Greenblatt, G. Lev-Maor, G. K. Bar-Gal, C. Matheson, K. Vernon, A. G. Nerlich, and A. R. Zink. 2004. Tuberculosis: from prehistory to Robert Koch, as revealed by ancient DNA. Lancet Infect Dis 4:584-92. 33. Dormans, J., M. Burger, D. Aguilar, R. Hernandez-Pando, K. Kremer, P. Roholl, S. M. Arend, and D. van Soolingen. 2004. Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model. Clin Exp Immunol 137:460-8. 34. Dziadek, J., A. Sajduda, J. W. Dale, and J. J. McFadden. 1998. IS990, a new species-specific insertion-sequence-related element of the Mycobacterium tuberculosis complex. Microbiology 144 ( Pt 12):3407-12. 35. Ebrahimi-Rad, M., P. Bifani, C. Martin, K. Kremer, S. Samper, J. Rauzier, B. Kreiswirth, J. Blazquez, M. Jouan, D. van Soolingen, and B. Gicquel. 2003. Mutations in putative mutator genes of Mycobacterium tuberculosis strains of the WBeijing family. Emerg Infect Dis 9:838-45. 36. Ehlers, M. R., and M. Daffe. 1998. Interactions between Mycobacterium tuberculosis and host cells: are mycobacterial sugars the key? Trends Microbiol 6:328-35. 37. Ehrt, S., and D. Schnappinger. 2009. Mycobacterial survival strategies in the phagosome: defence against host stresses. Cell Microbiol 11:1170-8. 38. Ernst, J. D. 1998. Macrophage receptors for Mycobacterium tuberculosis. Infect Immun 66:1277-81. 39. Fang, Z., C. Doig, D. T. Kenna, N. Smittipat, P. Palittapongarnpim, B. Watt, and K. J. Forbes. 1999. IS6110-mediated deletions of wild-type chromosomes of Mycobacterium tuberculosis. J Bacteriol 181:1014-20. 40. Fang, Z., and K. J. Forbes. 1997. A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome. J Clin Microbiol 35:479-81. 41. Fang, Z., D. T. Kenna, C. Doig, D. N. Smittipat, P. Palittapongarnpim, B. Watt, and K. J. Forbes. 2001. Molecular evidence for independent occurrence of IS6110 insertions at the same sites of the genome of Mycobacterium tuberculosis in different clinical isolates. J Bacteriol 183:5279-84. 42. Filliol, I., J. R. Driscoll, D. van Soolingen, B. N. Kreiswirth, K. Kremer, G. Valetudie, D. A. Dang, R. Barlow, D. Banerjee, P. J. Bifani, K. Brudey, A. Cataldi, R. C. Cooksey, D. V. Cousins, J. W. Dale, O. A. Dellagostin, F. Drobniewski, G. Engelmann, S. Ferdinand, D. Gascoyne-Binzi, M. Gordon, M. C. Gutierrez, W. H. Haas, H. Heersma, E. Kassa-Kelembho, M. L. Ho, A. Makristathis, C. Mammina, G. Martin, P. Mostrom, I. Mokrousov, V. Narbonne, O. Narvskaya, A. Nastasi, S. N. Niobe-Eyangoh, J. W. Pape, V. Rasolofo-Razanamparany, M. Ridell, M. L. Rossetti, F. Stauffer, P. N. Suffys, H. Takiff, J. Texier-Maugein, V. Vincent, J. H. de Waard, C. Sola, and N. Rastogi. 2003. Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study. J Clin Microbiol 41:1963-70. 43. Filliol, I., A. S. Motiwala, M. Cavatore, W. Qi, M. H. Hazbon, M. Bobadilla del Valle, J. Fyfe, L. Garcia-Garcia, N. Rastogi, C. Sola, T. Zozio, M. I. Guerrero, C. I. Leon, J. Crabtree, S. Angiuoli, K. D. Eisenach, R. Durmaz, M. L. Joloba, A. Rendon, J. Sifuentes-Osornio, A. Ponce de Leon, M. D. Cave, R. Fleischmann, T. S. Whittam, and D. Alland. 2006. Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol 188:759-72. Mª Henar Alonso Ezcurra 46 44. Fine, P. E. 1995. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 346:1339-45. 45. Fomukong, N., M. Beggs, H. el Hajj, G. Templeton, K. Eisenach, and M. D. Cave. 1997. Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110. Tuber Lung Dis 78:109-16. 46. Fratti, R. A., J. Chua, I. Vergne, and V. Deretic. 2003. Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest. Proc Natl Acad Sci U S A 100:5437-42. 47. Gagneux, S., K. DeRiemer, T. Van, M. Kato-Maeda, B. C. de Jong, S. Narayanan, M. Nicol, S. Niemann, K. Kremer, M. C. Gutierrez, M. Hilty, P. C. Hopewell, and P. M. Small. 2006. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 103:2869-73. 48. Gandhi, N. R., A. Moll, A. W. Sturm, R. Pawinski, T. Govender, U. Lalloo, K. Zeller, J. Andrews, and G. Friedland. 2006. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368:1575-80. 49. Geerligs, W. A., R. Van Altena, W. C. M. De Lange, D. Van Soolingen, and T. S. Van Der Werf. 2000. Multidrug-resistant tuberculosis: long-term treatment outcome in the Netherlands. Int J Tuberc Lung Dis 4:758-64. 50. Glynn, J. R., A. C. Crampin, H. Traore, M. D. Yates, F. D. Mwaungulu, B. M. Ngwira, S. D. Chaguluka, D. T. Mwafulirwa, S. Floyd, C. Murphy, F. A. Drobniewski, and P. E. Fine. 2005. Mycobacterium tuberculosis Beijing genotype, northern Malawi. Emerg Infect Dis 11:150-3. 51. Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen. 2002. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 8:843-9. 52. Gordon, S. V., R. Brosch, A. Billault, T. Garnier, K. Eiglmeier, and S. T. Cole. 1999. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 32:643-55. 53. Gordon, S. V., B. Heym, J. Parkhill, B. Barrell, and S. T. Cole. 1999. New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv. Microbiology 145 ( Pt 4):881-92. 54. Guiard, J., A. Collmann, L. F. Garcia-Alles, L. Mourey, T. Brando, L. Mori, M. Gilleron, J. Prandi, G. De Libero, and G. Puzo. 2009. Fatty acyl structures of mycobacterium tuberculosis sulfoglycolipid govern T cell response. J Immunol 182:7030-7. 55. Gutacker, M. M., B. Mathema, H. Soini, E. Shashkina, B. N. Kreiswirth, E. A. Graviss, and J. M. Musser. 2006. Single-nucleotide polymorphism-based population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites. J Infect Dis 193:121-8. 56. Gutacker, M. M., J. C. Smoot, C. A. Migliaccio, S. M. Ricklefs, S. Hua, D. V. Cousins, E. A. Graviss, E. Shashkina, B. N. Kreiswirth, and J. M. Musser. 2002. Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains. Genetics 162:1533-43. 57. Gutierrez, M. G., S. S. Master, S. B. Singh, G. A. Taylor, M. I. Colombo, and V. Deretic. 2004. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119:753-66. 58. Hermans, P. W., D. van Soolingen, E. M. Bik, P. E. de Haas, J. W. Dale, and J. D. van Embden. 1991. Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect Immun 59:2695-705. 59. Herzog, H. 1998. History of tuberculosis. Respiration 65:5-15. 60. Ioerger, T. R., Y. Feng, K. Ganesula, X. Chen, K. M. Dobos, S. Fortune, W. R. Jacobs, Jr., V. Mizrahi, T. Parish, E. Rubin, C. Sassetti, and J. C. Sacchettini. General Introduction 47 Variation among genome sequences of H37Rv strains of Mycobacterium tuberculosis from multiple laboratories. J Bacteriol 192:3645-53. 61. Jackson, M., G. Stadthagen, and B. Gicquel. 2007. Long-chain multiple methylbranched fatty acid-containing lipids of Mycobacterium tuberculosis: biosynthesis, transport, regulation and biological activities. Tuberculosis (Edinb) 87:78-86. 62. Kang, B. K., and L. S. Schlesinger. 1998. Characterization of mannose receptordependent phagocytosis mediated by Mycobacterium tuberculosis lipoarabinomannan. Infect Immun 66:2769-77. 63. Kaufmann, S., and P. v. Helden (ed.). 2008. Handbook of Tuberculosis. 64. Kaufmann, S. H. 2005. Robert Koch, the Nobel Prize, and the ongoing threat of tuberculosis. N Engl J Med 353:2423-6. 65. Kruuner, A., S. E. Hoffner, H. Sillastu, M. Danilovits, K. Levina, S. B. Svenson, S. Ghebremichael, T. Koivula, and G. Kallenius. 2001. Spread of drug-resistant pulmonary tuberculosis in Estonia. J Clin Microbiol 39:3339-45. 66. Kurepina, N., E. Likhoshvay, E. Shashkina, B. Mathema, K. Kremer, D. van Soolingen, P. Bifani, and B. N. Kreiswirth. 2005. Targeted hybridization of IS6110 fingerprints identifies the W-Beijing Mycobacterium tuberculosis strains among clinical isolates. J Clin Microbiol 43:2148-54. 67. Kurepina, N. E., S. Sreevatsan, B. B. Plikaytis, P. J. Bifani, N. D. Connell, R. J. Donnelly, D. van Sooligen, J. M. Musser, and B. N. Kreiswirth. 1998. Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaAdnaN region. Tuber Lung Dis 79:31-42. 68. Lee, J., M. Hartman, and H. Kornfeld. 2009. Macrophage apoptosis in tuberculosis. Yonsei Med J 50:1-11. 69. Lee, K. S., V. S. Dubey, P. E. Kolattukudy, C. H. Song, A. R. Shin, S. B. Jung, C. S. Yang, S. Y. Kim, E. K. Jo, J. K. Park, and H. J. Kim. 2007. Diacyltrehalose of Mycobacterium tuberculosis inhibits lipopolysaccharideand mycobacteria-induced proinflammatory cytokine production in human monocytic cells. FEMS Microbiol Lett 267:121-8. 70. Lew, J. M., A. Kapopoulou, L. M. Jones, and S. T. Cole. TubercuList--10 years after. Tuberculosis (Edinb) 91:1-7. 71. Li, Q., C. C. Whalen, J. M. Albert, R. Larkin, L. Zukowski, M. D. Cave, and R. F. Silver. 2002. Differences in rate and variability of intracellular growth of a panel of Mycobacterium tuberculosis clinical isolates within a human monocyte model. Infect Immun 70:6489-93. 72. Loewenberg, S. India reports cases of totally drug-resistant tuberculosis. Lancet 379:205. 73. Lok, K. H., W. H. Benjamin, Jr., M. E. Kimerling, V. Pruitt, M. Lathan, J. Razeq, N. Hooper, W. Cronin, and N. E. Dunlap. 2002. Molecular differentiation of Mycobacterium tuberculosis strains without IS6110 insertions. Emerg Infect Dis 8:13103. 74. Lopez, B., D. Aguilar, H. Orozco, M. Burger, C. Espitia, V. Ritacco, L. Barrera, K. Kremer, R. Hernandez-Pando, K. Huygen, and D. van Soolingen. 2003. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 133:30-7. 75. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol Mol Biol Rev 62:725-74. 76. Malik, Z. A., C. R. Thompson, S. Hashimi, B. Porter, S. S. Iyer, and D. J. Kusner. 2003. Cutting edge: Mycobacterium tuberculosis blocks Ca2+ signaling and phagosome maturation in human macrophages via specific inhibition of sphingosine kinase. J Immunol 170:2811-5. 77. Manca, C., M. B. Reed, S. Freeman, B. Mathema, B. Kreiswirth, C. E. Barry, 3rd, and G. Kaplan. 2004. Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis. Infect Immun 72:5511-4. Mª Henar Alonso Ezcurra 48 78. Manca, C., L. Tsenova, A. Bergtold, S. Freeman, M. Tovey, J. M. Musser, C. E. Barry, 3rd, V. H. Freedman, and G. Kaplan. 2001. Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta. Proc Natl Acad Sci U S A 98:57527. 79. Mathema, B., N. E. Kurepina, P. J. Bifani, and B. N. Kreiswirth. 2006. Molecular epidemiology of tuberculosis: current insights. Clin Microbiol Rev 19:658-85. 80. Matsunaga, I., A. Bhatt, D. C. Young, T. Y. Cheng, S. J. Eyles, G. S. Besra, V. Briken, S. A. Porcelli, C. E. Costello, W. R. Jacobs, Jr., and D. B. Moody. 2004. Mycobacterium tuberculosis pks12 produces a novel polyketide presented by CD1c to T cells. J Exp Med 200:1559-69. 81. McAdam, R. A., P. W. Hermans, D. van Soolingen, Z. F. Zainuddin, D. Catty, J. D. van Embden, and J. W. Dale. 1990. Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family. Mol Microbiol 4:1607-13. 82. McAdam, R. A. Q., S. & Guilhot, C. 2000. Mycobacterial Tansposons and their applications. Molecular Genetics of Mycobacteria American Society for Microbiology Press, ISBN 1-55581-191-4, Washington DC, USA.:69−84. 83. McEvoy, C. R., A. A. Falmer, N. C. Gey van Pittius, T. C. Victor, P. D. van Helden, and R. M. Warren. 2007. The role of IS6110 in the evolution of Mycobacterium tuberculosis. Tuberculosis (Edinb) 87:393-404. 84. McEvoy, C. R., R. M. Warren, P. D. van Helden, and N. C. Gey van Pittius. 2009. Multiple, independent, identical IS6110 insertions in Mycobacterium tuberculosis PPE genes. Tuberculosis (Edinb) 89:439-42. 85. Mendiola, M. V., C. Martin, I. Otal, and B. Gicquel. 1992. Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain. Res Microbiol 143:767-72. 86. Minnikin, D. E., L. Kremer, L. G. Dover, and G. S. Besra. 2002. The methyl-branched fortifications of Mycobacterium tuberculosis. Chem Biol 9:545-53. 87. Mizrahi, V., and S. J. Andersen. 1998. DNA repair in Mycobacterium tuberculosis. What have we learnt from the genome sequence? Mol Microbiol 29:1331-9. 88. Mokrousov, I., O. Narvskaya, T. Otten, A. Vyazovaya, E. Limeschenko, L. Steklova, and B. Vyshnevskyi. 2002. Phylogenetic reconstruction within Mycobacterium tuberculosis Beijing genotype in northwestern Russia. Res Microbiol 153:629-37. 89. Mostowy, S., J. Inwald, S. Gordon, C. Martin, R. Warren, K. Kremer, D. Cousins, and M. A. Behr. 2005. Revisiting the evolution of Mycobacterium bovis. J Bacteriol 187:6386-95. 90. Mueller, P., and J. Pieters. 2006. Modulation of macrophage antimicrobial mechanisms by pathogenic mycobacteria. Immunobiology 211:549-56. 91. Nerlich, A. G., C. J. Haas, A. Zink, U. Szeimies, and H. G. Hagedorn. 1997. Molecular evidence for tuberculosis in an ancient Egyptian mummy. Lancet 350:1404. 92. Onwueme, K. C., C. J. Vos, J. Zurita, J. A. Ferreras, and L. E. Quadri. 2005. The dimycocerosate ester polyketide virulence factors of mycobacteria. Prog Lipid Res 44:259-302. 93. Ordway, D., M. Henao-Tamayo, M. Harton, G. Palanisamy, J. Troudt, C. Shanley, R. J. Basaraba, and I. M. Orme. 2007. The hypervirulent Mycobacterium tuberculosis strain HN878 induces a potent TH1 response followed by rapid down-regulation. J Immunol 179:522-31. 94. Otal, I., A. B. Gomez, K. Kremer, P. de Haas, M. J. Garcia, C. Martin, and D. van Soolingen. 2008. Mapping of IS6110 insertion sites in Mycobacterium bovis isolates in relation to adaptation from the animal to human host. Vet Microbiol 129:333-41. 95. Otal, I., C. Martin, V. Vincent-Levy-Frebault, D. Thierry, and B. Gicquel. 1991. Restriction fragment length polymorphism analysis using IS6110 as an epidemiological marker in tuberculosis. J Clin Microbiol 29:1252-4. Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 55 Introduction Since the first description in 1995, M. tuberculosis Beijing strains became a main health problem worldwide. Strains of this genotype are widespread in many regions of the world and frequently cause epidemic outbreaks (5, 10, 20). The distribution of Beijing strains differs, being low in Western Europe, although a slight increase in the number of Beijing strains has been detected over time (5). Recent studies have illustrated the high frequency of this genotype in different regions of China being around 80%. Moreover, according to spoligotyping database (SpolDB4), strains from this genotype were found to be present in the largest number of countries globally (13% of global isolates) (22). This family is generally considered to be associated with drug-resistance (2, 6, 7), although this association has not been found in all geographic settings (1, 20). The predominance of this lineage probably results from genetic advantages, including unidentified virulence factors and the modulation of specific host responses not yet thoroughly investigated. For example, a study using human macrophages found that Beijing strains grew significantly faster than non-Beijing strains (49). However, when it was analyzed whether this characteristic was common to Beijing strains, it was found that not all Beijing strains grew rapidly (46). In addition to this, there are some studies that relate hypervirulence of Beijing strains with production of PGL (12, 39), which is a putative virulence factor that attenuates the host’s innate immune response and ability to control infection (39). The pks15/1 locus, described to be polymorphic among members of the MTBC (14), is involved in the biosynthesis of the PGL and it is characteristic of Beijing strains to have an intact pks15/1 region for which they could produce a functional Pks15/1 synthase (14, 43, 45). However, when monocytes were infected with a non-Beijing strain transformed with the functional pks15/1 gene, growth was similar to the wild-type non-Beijing strain, suggesting that PGL did not influence growth of M. tuberculosis (22). Another example of virulence factor could be that Beijing strains have a basal level of transcription for the dormancy regulon genes (dosR, Rv3130c, hspX, fdxA and narX) that is up to 50-fold higher than that of nonBeijing genotype strains (40). More recently, it was found that Beijing strains possess two copies of dosR which was suggested to be partly responsible for the constitutive dosR over-expression phenotype (15). Beijing strains are currently attracting considerable worldwide attention because they display important pathogenic features (30, 49). Mª Henar Alonso Ezcurra 56 Subfamilies Beijing Members of this family were subdivided into modern/typical and ancient/atypical sublineages based on the analysis of the NTF locus (34). In addition to the characteristic deletion of the DR locus, Tsolaki et al. described LSPs (RD105, RD142, RD150, and RD181) which further divided this family into four monophyletic subgroups (47). They sought LSPs that were unique-event polymorphism (UEPs) within Beijing genotype. A total of 21 distinct LSPs were identified that were present in H37Rv but absent from the Beijing strains. Based on the frequency of these 21 LSPs, three classes were identified: four common LSPs (RD105, RD149, RD152 and RD207) were found deleted in all Beijing strains. RD207 (also detected by spoligotyping), RD149 and RD152 are associated with mobile genetic elements and may not be UEPs. Three LSPs (RD181, RD142 and RD150) were found variably deleted and these are not associated with either repetitive DNA or mobile genetic elements therefore likely to represent UEPs and the remaining 14 LSPs were unique to a single strain. Based on this, Beijing lineage is principally defined by deletion RD105, which is a useful marker for the identification of this family. RD181, RD142 and RD150 further divided this family into four monophyletic subgroups. RD142 and RD150 are only observed in strains with RD181 being this the more ancestral event (47). After that, the RD207 was included within the monophyletic subgroups of the East-Asian lineage (19) (Figure 11). Figure 11: Subfamilies Beijing. (A) Schematic phylogenetic tree of the M. tuberculosis Beijing family. The name of the LSPs or regions of difference are shown in rectangles. The evolutionary process of the lineage (ancient to modern) is also indicated. (B) Genes affected for the deletion of the RD. The genes affected for the deletion of RD149 and RD152 (common in all Beijing strains) are included (*). Adapted from (19, 31, 47). Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 57 Some authors have proposed an evolutionary pathway of Beijing lineage based on RD deletions and on the IS6110 insertions in the NTF region indicating that ancient strains have neither deletion RD181 nor insertion of IS in NTF region (16). However, recent studies have shown that not all ancient/atypical strains are RD181 intact which indicates that the RD181 deletion occurred before the insertion of an IS element in the NTF locus (24, 31) (Figure 11). Moreover, it has been demonstrated that the 3’ common truncation in the gene Rv2820c (RD207, figure 11B) of Beijing strains could enhance mycobacterial virulence ex vivo and in vivo. This enhancement was not observed in non-Beijing strains with intact gene (29). IS6110 in Beijing family Three features which characterize this family are related to IS6110 insertion sequence: The presence of one copy of this element in the oriC region, the deletion of the rightsite DR spacers (the so-called deletion RD207) and similar RFLP multiband pattern profile (22). Members of this genotype usually are high copy number of IS6110 (between 15-25 copies per genome) suggesting the relevance of this element in the variability of their genomes. IS6110 insertions, genetic reorganizations and deletions (gene deletion can occur by homologous recombination between 2 flanking copies of IS6110) are some of the mechanisms proposed to be responsible for differences in the virulence phenotypes of M. tuberculosis. Supporting this possibility, sublineages of this family were identified to carry an important genome duplication that involves up to 8% of the genome (corresponding to more than 300 genes). Copies of IS6110 were identified flanking that duplication, thus suggesting the occurrence of homologous recombination event mediated by this IS (15). It has been demonstrated that IS6110 can upregulate downstream genes through an outward-directed promoter in its 3’ end (41, 44). The fact that the Beijing lineage contains a larger number of IS6110 copies than other lineages (21) could be related to the special characteristics of this family in terms of virulence and capacity for rapid dissemination. The clinical isolate M. tuberculosis GC1237 M. tuberculosis GC1237, which belongs to the Beijing family, has been responsible for different epidemic outbreaks in the Gran Canary Island. The first case recognized on the island with this strain was a refugee from Liberia (Africa) diagnosed with smearpositive pulmonary tuberculosis in 1993. Its explosive spread in this community over the next few years was reported in 2001 (11). Nowadays, this strain continues being predominant in the area due to its rapid and successful dissemination within the community (Table 1). Mª Henar Alonso Ezcurra 58 Table 1. Percentage of Beijing isolates in Canary Island from 1991 to 2008. Adapted from (11, 33). Period Method Total isolates Beijing percent (%) 1991-1992 RFLP 85 0 1993 RFLP, spoligotyping 179 5.5 1994 RFLP, spoligotyping 148 8.1 1995 RFLP, spoligotyping 110 16.4 1996 RFLP, spoligotyping 129 27.1 1999 Spoligotyping 40 22.5 2002 Spoligotyping 154 28.57 2003 Spoligotyping 49 28.57 2004 Spoligotyping 120 29.16 2007-2008 Spoligotyping 214 25.23 It was reported that one isolate with the same RFLP was detected in Madrid. Although in that study the strain did not show increased intracellular replication (THP1-cells) and it was not distinguishable from other isolates (3), the increased capacity of infection and the high success rate of this strain to spread rapidly could be a consequence of genetic advantages and unidentified mechanisms of virulence not yet thoroughly investigated. Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 59 Objectives • To classify the clinical isolate M. tuberculosis GC1237 within the Beijing family with the analysis of several Region of Differences (RD). • To localize all the copies of IS6110 insertion sequence in the genome of M. tuberculosis GC1237. • To study the direct repeats flanking each copy of IS6110 of GC1237. • To analyze the orientation and distance to neighbouring genes of each copy of IS6110 present in M. tuberculosis GC1237. Mª Henar Alonso Ezcurra 60 Material and Methods Bacterial strains, culture media, and growth conditions BACs library of M. tuberculosis GC1237 (Figure 14A) was used in this study. This library, which represents around 75% of the chromosome of GC1237, was constructed in a previous work using pBeloBAC11 plasmid. E. coli DH10B cultures were used in order to isolate the BACs. Strains were grown at 37ºC in Lurina-Bertani (LB) broth supplemented with chloramphenicol (CM) (12.5μg/ml). The clinical isolate M. tuberculosis GC1237, M. bovis BCG and the reference M. tuberculosis H37Rv strains were used in this work. Mycobacterial strains were grown at 37ºC in Middlebrook 7H9 broth supplemented with albumin-dextrose-catalase (ADC) and 0.05% Tween 80 or in Middlebrook 7H10 medium Bacto agar supplemented with oleic acid-albumin-dextrose-catalase (OADC) (Difco Laboratories, Detroit, Mich.) and 0.05% Tween 80 (23). Liquid cultures were grown to logarithmic phase for mycobacterial DNA extraction. M. tuberculosis manipulation was carried out in a biosafety level 3 (BSL3) laboratory (facilities notification A/ES/04/I-05; activity A/ES/06/03). Mycobacterial DNA extraction Genomic DNA of mycobacterial strains was isolated using the CTAB method (48). Briefly, mycobacteria were resuspended in 400μl TE (100mM Tris/HCl, 10mM EDTA, pH 8.0) and heated for 20min at 80ºC. Samples were slightly cooled at room temperature before adding 0.5mg lysozyme and were then incubated for at least 1h at 37ºC. Subsequently, 0.05mg proteinase K dissolved in 75μl 10% SDS were added and the suspension warmed for 10min at 65ºC. Hereafter, 100μl 5M NaCl and 100μl CTAB/NaCl (10% CTAB in 0.7M NaCl) pre-warmed at 65ºC were added and samples incubated for further 10min at 65ºC. Genomic DNA was extracted by adding 750μl of chloroform:isoamylalcohol 24:1 (v/v). Samples were mixed by vortexing for 10s before centrifugation (13,000rpm for 5min). The upper (aqueous) phase was transferred to a fresh tube containing 450μl isopropanol and samples incubated overnight at -20ºC. Precipitated nucleic acids were collected by centrifugation (13,000rpm for 10min at 4ºC). The pellets were dissolved in 50μl double-distilled water and extracted DNA was quantified by Abs260 readings using a ND-1000 spectrophotometer (NanoDrop Technologies). Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 61 BAC DNA extraction BAC DNA extraction was done as previously described by Birnboim et al. (8) with minor modifications. Briefly, 100ml of BAC-transformed E. coli was prepared in LB medium containing 12.5μg/ml CM, and the cultures were grown overnight at 37ºC with vigorous and continuous agitation. Then, the bacterial cells were collected by centrifugation. The bacterial pellet was softly resuspended in a solution of 5ml of 50mM glucose 10mM EDTA, 25mM Tris pH 8, 200mg lysozime was added to the tube and a solution of 4ml of ice-cold NaAc pH 4.8 was added to the mixture. The tube was placed on ice and the precipitated debris was removed by centrifugation. Then, 14ml of chloroform/isoamilic alcohol (24/1) were added to the supernatant and after centrifugation the aqueous phase was transferred to a new microfuge tube. DNA of the BACs was precipitated adding isopropanol and finally obtained by centrifugation. Identification of genomic deletions and analysis of pks15/1 region in M. tuberculosis GC1237 strain The study of the genomic deletions of the regions of difference 105, RD142, RD150, RD181 and RD207 in M. tuberculosis GC1237 strain, which identify and phylogenetically sub-classify the Beijing lineage, was performed by PCR. Other regions of difference (RD108, RD110a, RD127, RD129, RD139BW, RD149, RD152, RD165, RD166 and RD182a) were also analysed. The primers used in these amplifications were as described elsewhere (47) and are included in Table 1 of Annex I. The PCR was carried out in a total volume of 50μl, containing 0.5μg of DNA, 5μl of 10x PCR buffer, 200μM dNTPs, 12.5pmol of each primer and 1U of Taq Gold polymerase (Roche). Before the amplification, the template was initially denatured by incubation at 94ºC for 9min then the amplification was performed in 35 cycles of 94ºC for 30s, corresponding annealing temperature for 30s, and 72ºC for 2 to 3min depending on the amplified product. After the last cycle, the samples were incubated at 72ºC for 10min. The RD deletions were confirmed by DNA sequencing using H37Rv as reference genome. The pks15/1 polymorphism in this clinical isolate was determined by PCR, as above described, with the primers pks1I and pks1J (Table 2 of Annex I) and sequencing and analyzed as previously described (14). Mª Henar Alonso Ezcurra 62 Location of the copies of IS6110 insertion sequence in M. tuberculosis GC1237 • BACs with IS6110 The study of the presence of IS6110 insertion sequence in GC1237 BACs library was carried out by PCR with the specific primers of this sequence, Gab 1 and Gab 2 (42) (Table 5 of Annex I) and was performed as explained before in this chapter changing the final volume to 25μl. After that, PCR products were analyzed by 0.8% agarose gel electrophoresis and visualized by ethidium bromide staining. • Ligation mediated PCR (LMPCR) LMPCR was used to locate the copies of IS6110 as previously described by Prod’hom et al. (38). This technique amplifies both ends of each copy of IS6110. Briefly, BACs containing IS6110 and genomic DNA of M. tuberculosis GC1237 were digested with SalI or with SmaI and ligated to a linker containing a SalI restriction site or a SmaI restriction site, respectively. The resulting template was then digested by SalI or with SmaI. PCR was performed using ISA1 and ISA3 (Table 2 Annex I), specific primers for IS6110 directed outwards (32) and the common linker primer Salgd (38) (Table 2 Annex I) (Figure 12). PCR products were purified using GFX PCR DNA gel band purification kit (Amersham Pharmacia Biotech) and the restriction enzyme ExoSAP-IT® (Affymetrix). Figure 12: Ligation Mediated PCR (LMPCR). (1) Total DNA or BACs containing IS6110 are restriction digested, (2) linkers are ligated, (3) unspecified ligations are removed and (4) transposon junctions are PCR amplified. The amplified products were sequenced using CNIO service with the corresponding oligonucleotides and when a match was found for a flanking region in the databases, additional primers were designed to verify the point of insertion. PCR amplification was carried out as previously described in this chapter with genomic DNA of GC1237. PCR Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 63 products included the complete sequence of IS6110 and approximately 100-200 bp of both flanking sequences. Each amplified PCR product was sequenced and analysed for homology with Tuberculist (http://genolist.pasteur.fr/TubercuList), Bovilist (http://genolist.pasteur.fr/BoviList) and NCBI (http://www.ncbi.nlm.nih.gov/) database Blast analysis. • PCR with specific primers In addition, we designed primers to amplify the different regions that might include IS6110 based in the published genome locations of IS6110 in 210 and W Beijing strains (4). As controls H37Rv and BCG genomes were used. The primers used to localize IS6110 are collected in Table 5 of Annex I. Determination of Direct Repeats (DR) of each copy of IS6110 in GC1237 genome The DR generated by the mechanism of the transposition of IS6110 was determined with the sequence analysis of the flanking regions of each copy of IS6110 in GC1237 genome. Design of a multiplex PCR to detect the clinical isolate M. tuberculosis GC1237 To detect the clinical isolate M. tuberculosis GC1237 faster than by RFLP and spolygotyping, a multiplex PCR based on the detection of two of its IS-locations was designed in this study. The first target (fragment of 550bp) is the IS6110 located between dnaA:dnaN genes which is common to all Beijing strains (26) and it is amplified with the primers dnaII and dnaAIII (Table 3 of Annex I). This last oligonucleotide is more specific to Beijing genotype as it anneals in the 3’ end of the IS6110 and the following nucleotides of that region. The second target, amplified with primers Rv2179cfw and Rv2179crv (Table 3 of Annex I), is specific of GC1237 (see results of this chapter). In this case, the GC1237-PCR product includes the entire sequence of IS6110 (1626 bp) and the PCR fragment of the rest of the strains correspond to 261 bp. The multiplex PCR was carried out as describe above with minor modifications. Briefly, PCR was performed in a final volume of 25μl, containing 200μM dNTPs, 50mM KCl, 1.5mM MgCl2, 2.5U of puReTaq DNA polymerase (puReTaq Ready-to-go, GE Healthcare), reaction buffer, 25μM of each primer and 0.5μg of DNA. Temperature cycling conditions included 94°C for 10 min, followed by 35 cycles of 94°C for 30s, 70°C for 30s, and 72°C for 1.5min, and 10min for a final extension at 72°C. PCR Mª Henar Alonso Ezcurra 64 products were analyzed by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 71 The open reading frames represent 91% of M. tuberculosis genome (13) but in GC1237 the insertion of IS6110 into coding regions occurred in 42% of the cases then, apparently the transposition is relatively more frequent in intergenic regions. Our results agreed with other studies that found that 58% of discrete IS6110 insertion sites occurred within coding regions in M. tuberculosis (43) and in M. bovis strains (35). In this context, because of the insertion of IS6110 in possible promoter regions the probability of IS6110 influencing the expression of the neighbouring genes is increased. It is necessary to take into account that the locations of IS6110 observed in the different genomes are the result of the transposition and selection of viable infecting mycobacteria because when insertion occurs in a gene necessary for virulence, we cannot observe it. Analysis of Direct Repeats (DR) of each copy of IS6110 in GC1237 genome The flanking regions of each of the nineteen copies of IS6110 in GC1237 genome were analyzed and the presence of DR of 3-4 nucleotides at the extremities of sixteen IS6110 sequences were detected (Table 3). These DR indicate that the presence of IS6110 was due to transposition events. The other three copies analysed were not flanked by DRs. Genomic regions loss was observed in two of these locations which correspond to Rv1754c-Rv1765c (RD152) and RD207 respectively, and this is probably a consequence of recombination between two adjacent copies of IS6110 (47). The last of the three IS6110 elements without DR, which was localized between Rv0794c:Rv0797, was in the opposite orientation with respect to the IS6110 in the reference genome H37Rv. The IS6110 in H37Rv contains DR and the lack of DR in the studied IS6110 could be explained by reorganization of this region in GC1237 genome. The two copies localized in PPE34 are flanked by the direct repeat sequence TTA. These DRs were located in the 5’ end of the first IS6110 and the 3’ end of the second one, probably due to simultaneous transposition of the two copies. The DR sequences differ among each other indicating the lack of transposition specificity. There are very few studies where authors report DR flanking IS6110 (32, 35). In these studies the number of copies is low (<6) and all of them were flanked by DR, indicating that these IS6110 are consequence of transposition. In our study, because of the high number of copies of IS6110 we observed some copies flanked by DR and other copies without DR suggesting that the probability of rearrangement process between copies rises when the number of those increases producing more variability among strains. This finding is in agreement with different studies indicating that strains with a high number of IS6110 copies (>14) have lost genomic regions more often than strains with only few copies (9), as is in the case of GC1237. Mª Henar Alonso Ezcurra 72 Table 3. Direct repeats of IS6110 insertion sequence in M. tuberculosis GC1237. Genes of IS6110 in GC1237 genome DRsa Rv0001 (dnaA):Rv0002 (dnaN) ATT Rv0794c:Rv0797 - Rv0840c (pip) ACG Rv1371 GAGG Rv1469 (ctpD) CGT Rv1754c-Rv1765c (RD152) - Rv1917c (PPE34) TTA Rv1917c (PPE34) TTA Rv2016 AGG MT2080 (Mb2047c):MT2081 (Mb2048c) GAA Rv2077A:Rv2078 AGG Rv2180c AGC Rv2286c ATC Rv2353 (PPE39):Rv2356c (PPE40) CCG Rv2813-Rv2820c (RD207) - Rv3018A (PE27A):Rv3019c (esxR) GCC Rv3324A:Rv3327 (IS1547) GGC Rv3383c (idsB) ATC Rv3427c (IS1532):Rv3428c (IS1532) CCCG aThe absence of DR is indicated by minus. Study of the orientation and the distance of IS6110 to the neighbouring genes Different studies have indicated that IS6110 could up-regulate the expression of downstream genes. Previous studies have shown that when IS6110 is inserted in the same orientation as, and close enough to, a downstream gene, IS6110 could potentially function as a promoter (41). We analysed the orientation of each copy of the IS6110 in GC1237 strain and the distance to the close gene in order to test the promoter function of IS6110. We obtained 8 genes with an IS6110 inserted upstream in the right orientation but in 4 locations the distance is reasonable to act as a mobile promoter (Table 2). One of them is located 297 bp upstream of the Rv1468c gene, another 138 bp upstream of PE27A gene, the third one 41 bp upstream Rv3427c gene and the last one is located 31 bp upstream of the essential gene Rv2179c. The analysis of the natural insertion site of IS6110 upstream the gene ctpD in the M. tuberculosis Beijing strain 210 was carried out both in broth and in monocytes and compared with that in H37Rv (41). In broth, transcriptional levels of this gene were Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 73 similar in the two strains. However, the expression of ctpD was three to fivefold higher levels inside monocytes when IS was upstream (41). As GC1237 presents a copy of IS6110 in this same point and in the same orientation, the expression of ctpD in this strain was analysed (by qRT-PCR) and compared with its expression in H37Rv. Similar results to those reported were obtained both in broth and inside the cells corroborating the promoter activity of IS6110 in this location. The insertion of IS6110 in Rv2180c gene stands out as it is located 31 bp upstream of the essential gene Rv2179c and close to the essential gene AroG which is involved in the common aromatic amino acid biosynthesis in M. tuberculosis (36). This location was also investigated in other M. tuberculosis strains, including Beijing and non-Beijing strains, and surprisingly, this point of insertion is unique to GC1237 strain. For that, the promoter activity in this location will be studied widely in Chapter 2. Detection of the clinical isolate M. tuberculosis GC1237 by multiplex PCR With the aim of facilitating the detection of this isolate faster than with traditional techniques, a multiplex PCR based on the detection of two locations of IS6110 in GC1237 genome was developed in this study (Figure 15). The design of this assay was possible as we located all the copies of IS6110 in this strain. The first step of the design was to distinguish between Beijing and non-Beijing genotypes. For that, the IS6110 located between dnaA:dnaN (Target 1 in Figure 15A) and the genomic deletion of the region of difference RD105 were chosen. These two targets were selected for its specificity of Beijing genotype. Given that the presence of a copy of IS6110 between dnaA:dnaN is not exclusive of Beijing strains (28), one of the primers used for the amplification of this region was designed annealing in the 3’ end of the IS6110 and the following nucleotides (Target 1 of Figure 15A) becoming totally specific of Beijing genotype. The second step was to distinguish the GC1237 genotype among Beijing genotypes. To obtain this, screening of IS6110 locations in Beijing strains and non-Beijing strains was carried out as mentioned above and finally, the IS6110 located in Rv2180c was selected as specific of this strain (Target 2 of Figure 15A). The multiplex PCR was performed with these three targets and it was possible to differentiate GC1237 genotype from the rest of the strains used as control strains. However, this PCR was performed with pure DNA and when it was performed with DNA obtained from boiled samples (it is important to remember that the main objective was to obtain a rapid test for the identification of GC1237 genotype), the PCR results failed with high frequency. After this data, the detection of RD105 region was removed from the multiplex PCR and on this occasion, as Figure 15B shows, it was a success. Although Figure 15 only shows the PCR results obtained with M. tuberculosis (Beijing Mª Henar Alonso Ezcurra 74 and non-Beijing), this multiplex was also tested with different mycobacteria genomes as M. vaccae, M. bovis and M. smegmatis obtaining in all cases the same profile as H37Rv strain. Figure 15: Detection of GC1237 genotype by multiplex PCR. (A) Schematic representation of the two targets selected for the multiplex PCR. Target 1 (primers dnaII and dnaIII) permits to differentiate between Beijing and non-Beijing genotypes. Target 2 (primers Rv2179c fw and Rv2179c rv) was selected to identify GC1237 genotype among Beijing strains. (B) PCR analysis of the multiplex PCR. Two fragments were obtained for isolates with GC1237 genotype (1626 bp and 550 bp). Two amplicons of 550 bp and the 261 bp were obtained as expected for other Beijing isolates and only one fragment of 261 bp in H37Rv used as non-Beijing strain. Due to the high incidence of this strain in the Gran Canary Island, its rapid detection would be useful for better control of TB cases. Considering that the RFLP and spoligotyping methods are difficult to implement in the clinical setting, the described multiplex PCR could bring the possibility of in situ identification of GC1237 genotype. The incidence of this strain in this Island has been screened by spoligotyping until now. This technique is good distinguishing the Beijing lineage from the rest; however this method does not provided the information about clonality within this genotype. In addition, the better use of this method is for the study of 40 isolates at a time. The RFLP based on IS6110 give the clonality information, but this technique requires higher quantity of DNA of high quality. In contrast to spoligotyping and RFLP, this multiplex PCR can be used to identify one or more positive isolates. It is quick and inexpensive, it has a simple interpretation and it can be completed in one day. Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 75 Conclusions • Based on NTF classification, M. tuberculosis GC1237 is an ancestral strain. • The LSPs RD105, RD149, RD152, RD181 and RD207 are deleted in GC1237 strain. • The representative BAC library of M. tuberculosis GC1237 was a useful tool for locating IS6110 in a high-copy strain. • M. tuberculosis GC1237 has 19 copies of the insertion sequence IS6110. • The complementation of different techniques guaranteed the success in the search of IS6110 in GC1237 genome. • Sixteen copies of IS6110 have DR indicating that its presence was due to a transposition event. In two of the other 3 copies recombination between two adjacent copies of IS6110 was observed and the presence of the last one is implicated in genomic reorganization. • According to the locations, four of the nineteen copies could act as a mobile promoter: one of them is located 297 bp upstream of the Rv1468c gene, another 138 bp upstream of PE27A gene, the third one 41 bp upstream Rv3427c gene and the last one is located 31 bp upstream of the essential gene Rv2179c. • The copy of IS6110 locates 31 bp upstream of the essential gene Rv2179c and is specific of GC1237 strain. • The study of all the copies of IS6110 in M. tuberculosis GC1237 allowed us to design an assay based on its specific location of IS6110. • The multiplex PCR facilitates the identification of GC1237 quickly and easily. Mª Henar Alonso Ezcurra 76 References 1. 2006. European Concerted Action on new generation genetic markers and techniques for the epidemiology and control of Tuberculosis. Beijing/W genotype Mycobacterium tuberculosis and drug resistance. Emerg Infect Dis 12:736-743. 2. Agerton, T. B., S. E. Valway, R. J. Blinkhorn, K. L. Shilkret, R. Reves, W. W. Schluter, B. Gore, C. J. Pozsik, B. B. Plikaytis, C. Woodley, and I. M. Onorato. 1999. Spread of strain W, a highly drug-resistant strain of Mycobacterium tuberculosis, across the United States. Clin Infect Dis 29:85-92; discussion 93-5. 3. Alonso, M., N. Alonso Rodriguez, C. Garzelli, M. Martinez Lirola, M. Herranz, S. Samper, M. J. Ruiz Serrano, E. Bouza, and D. Garcia de Viedma. Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area. BMC Microbiol 10:151. 4. Beggs, M. L., K. D. Eisenach, and M. D. Cave. 2000. Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis. J Clin Microbiol 38:2923-8. 5. Bifani, P. J., B. Mathema, N. E. Kurepina, and B. N. Kreiswirth. 2002. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol 10:45-52. 6. Bifani, P. J., B. Mathema, Z. Liu, S. L. Moghazeh, B. Shopsin, B. Tempalski, J. Driscol, R. Frothingham, J. M. Musser, P. Alcabes, and B. N. Kreiswirth. 1999. Identification of a W variant outbreak of Mycobacterium tuberculosis via populationbased molecular epidemiology. Jama 282:2321-7. 7. Bifani, P. J., B. B. Plikaytis, V. Kapur, K. Stockbauer, X. Pan, M. L. Lutfey, S. L. Moghazeh, W. Eisner, T. M. Daniel, M. H. Kaplan, J. T. Crawford, J. M. Musser, and B. N. Kreiswirth. 1996. Origin and interstate spread of a New York City multidrugresistant Mycobacterium tuberculosis clone family. JAMA 275:452-7. 8. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513-23. 9. Brosch, R., W. J. Philipp, E. Stavropoulos, M. J. Colston, S. T. Cole, and S. V. Gordon. 1999. Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain. Infect Immun 67:5768-74. 10. Brudey, K., J. R. Driscoll, L. Rigouts, W. M. Prodinger, A. Gori, S. A. Al-Hajoj, C. Allix, L. Aristimuno, J. Arora, V. Baumanis, L. Binder, P. Cafrune, A. Cataldi, S. Cheong, R. Diel, C. Ellermeier, J. T. Evans, M. Fauville-Dufaux, S. Ferdinand, D. Garcia de Viedma, C. Garzelli, L. Gazzola, H. M. Gomes, M. C. Guttierez, P. M. Hawkey, P. D. van Helden, G. V. Kadival, B. N. Kreiswirth, K. Kremer, M. Kubin, S. P. Kulkarni, B. Liens, T. Lillebaek, M. L. Ho, C. Martin, I. Mokrousov, O. Narvskaia, Y. F. Ngeow, L. Naumann, S. Niemann, I. Parwati, Z. Rahim, V. RasolofoRazanamparany, T. Rasolonavalona, M. L. Rossetti, S. Rusch-Gerdes, A. Sajduda, S. Samper, I. G. Shemyakin, U. B. Singh, A. Somoskovi, R. A. Skuce, D. van Soolingen, E. M. Streicher, P. N. Suffys, E. Tortoli, T. Tracevska, V. Vincent, T. C. Victor, R. M. Warren, S. F. Yap, K. Zaman, F. Portaels, N. Rastogi, and C. Sola. 2006. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6:23. 11. Caminero, J. A., M. J. Pena, M. I. Campos-Herrero, J. C. Rodriguez, I. Garcia, P. Cabrera, C. Lafoz, S. Samper, H. Takiff, O. Afonso, J. M. Pavon, M. J. Torres, D. van Soolingen, D. A. Enarson, and C. Martin. 2001. Epidemiological evidence of the spread of a Mycobacterium tuberculosis strain of the Beijing genotype on Gran Canaria Island. Am J Respir Crit Care Med 164:1165-70. 12. Caws, M., G. Thwaites, S. Dunstan, T. R. Hawn, N. T. Lan, N. T. Thuong, K. Stepniewska, M. N. Huyen, N. D. Bang, T. H. Loc, S. Gagneux, D. van Soolingen, K. Kremer, M. van der Sande, P. Small, P. T. Anh, N. T. Chinh, H. T. Quy, N. T. Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 77 Duyen, D. Q. Tho, N. T. Hieu, E. Torok, T. T. Hien, N. H. Dung, N. T. Nhu, P. M. Duy, N. van Vinh Chau, and J. Farrar. 2008. The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis. PLoS Pathog 4:e1000034. 13. Cole, S. T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, 3rd, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, A. Krogh, J. McLean, S. Moule, L. Murphy, K. Oliver, J. Osborne, M. A. Quail, M. A. Rajandream, J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares, S. Squares, J. E. Sulston, K. Taylor, S. Whitehead, and B. G. Barrell. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537-44. 14. Constant, P., E. Perez, W. Malaga, M. A. Laneelle, O. Saurel, M. Daffe, and C. Guilhot. 2002. Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene. J Biol Chem 277:38148-58. 15. Domenech, P., G. S. Kolly, L. Leon-Solis, A. Fallow, and M. B. Reed. Massive gene duplication event among clinical isolates of the Mycobacterium tuberculosis W/Beijing family. J Bacteriol 192:4562-70. 16. Dou, H. Y., F. C. Tseng, C. W. Lin, J. R. Chang, J. R. Sun, W. S. Tsai, S. Y. Lee, I. J. Su, and J. J. Lu. 2008. Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei. BMC Infect Dis 8:170. 17. Fang, Z., C. Doig, D. T. Kenna, N. Smittipat, P. Palittapongarnpim, B. Watt, and K. J. Forbes. 1999. IS6110-mediated deletions of wild-type chromosomes of Mycobacterium tuberculosis. J Bacteriol 181:1014-20. 18. Fang, Z., and K. J. Forbes. 1997. A Mycobacterium tuberculosis IS6110 preferential locus (ipl) for insertion into the genome. J Clin Microbiol 35:479-81. 19. Gagneux, S., K. DeRiemer, T. Van, M. Kato-Maeda, B. C. de Jong, S. Narayanan, M. Nicol, S. Niemann, K. Kremer, M. C. Gutierrez, M. Hilty, P. C. Hopewell, and P. M. Small. 2006. Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 103:2869-73. 20. Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen. 2002. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 8:843-9. 21. Gutacker, M. M., B. Mathema, H. Soini, E. Shashkina, B. N. Kreiswirth, E. A. Graviss, and J. M. Musser. 2006. Single-nucleotide polymorphism-based population genetic analysis of Mycobacterium tuberculosis strains from 4 geographic sites. J Infect Dis 193:121-8. 22. Hanekom, M., N. C. Gey van Pittius, C. McEvoy, T. C. Victor, P. D. Van Helden, and R. M. Warren. Mycobacterium tuberculosis Beijing genotype: a template for success. Tuberculosis (Edinb) 91:510-23. 23. Jacobs, W. R., Jr., G. V. Kalpana, J. D. Cirillo, L. Pascopella, S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol 204:537-55. 24. Kang, H. Y., T. Wada, T. Iwamoto, S. Maeda, Y. Murase, S. Kato, H. J. Kim, and Y. K. Park. Phylogeographical particularity of the Mycobacterium tuberculosis Beijing family in South Korea based on international comparison among surrounding countries. J Med Microbiol. 25. Kanji, A., Z. Hasan, M. Tanveer, R. Laiq, and R. Hasan. Occurrence of RD149 and RD152 deletions in Mycobacterium tuberculosis strains from Pakistan. J Infect Dev Ctries 5:106-13. 26. Kremer, K., J. R. Glynn, T. Lillebaek, S. Niemann, N. E. Kurepina, B. N. Kreiswirth, P. J. Bifani, and D. van Soolingen. 2004. Definition of the Beijing/W lineage of Mª Henar Alonso Ezcurra 78 Mycobacterium tuberculosis on the basis of genetic markers. J Clin Microbiol 42:40409. 27. Kurepina, N., E. Likhoshvay, E. Shashkina, B. Mathema, K. Kremer, D. van Soolingen, P. Bifani, and B. N. Kreiswirth. 2005. Targeted hybridization of IS6110 fingerprints identifies the W-Beijing Mycobacterium tuberculosis strains among clinical isolates. J Clin Microbiol 43:2148-54. 28. Kurepina, N. E., S. Sreevatsan, B. B. Plikaytis, P. J. Bifani, N. D. Connell, R. J. Donnelly, D. van Sooligen, J. M. Musser, and B. N. Kreiswirth. 1998. Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaAdnaN region. Tuber Lung Dis 79:31-42. 29. Lam, J. T., K. Y. Yuen, P. L. Ho, X. H. Weng, W. H. Zhang, S. Chen, and W. C. Yam. Truncated Rv2820c enhances mycobacterial virulence ex vivo and in vivo. Microb Pathog 50:331-5. 30. Lopez, B., D. Aguilar, H. Orozco, M. Burger, C. Espitia, V. Ritacco, L. Barrera, K. Kremer, R. Hernandez-Pando, K. Huygen, and D. van Soolingen. 2003. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 133:30-7. 31. Maeda, S., T. Wada, T. Iwamoto, Y. Murase, S. Mitarai, I. Sugawara, and S. Kato. Beijing family Mycobacterium tuberculosis isolated from throughout Japan: phylogeny and genetic features. Int J Tuberc Lung Dis 14:1201-4. 32. Mendiola, M. V., C. Martin, I. Otal, and B. Gicquel. 1992. Analysis of the regions responsible for IS6110 RFLP in a single Mycobacterium tuberculosis strain. Res Microbiol 143:767-72. 33. Millan-Lou, M. I., H. Alonso, P. Gavin, M. Hernandez-Febles, M. I. Campos-Herrero, R. Copado, F. Canas, K. Kremer, J. A. Caminero, C. Martin, and S. Samper. Rapid test for identification of a highly transmissible Mycobacterium tuberculosis Beijing strain of sub-Saharan origin. J Clin Microbiol 50:516-8. 34. Mokrousov, I., H. M. Ly, T. Otten, N. N. Lan, B. Vyshnevskyi, S. Hoffner, and O. Narvskaya. 2005. Origin and primary dispersal of the Mycobacterium tuberculosis Beijing genotype: clues from human phylogeography. Genome Res 15:1357-64. 35. Otal, I., A. B. Gomez, K. Kremer, P. de Haas, M. J. Garcia, C. Martin, and D. van Soolingen. 2008. Mapping of IS6110 insertion sites in Mycobacterium bovis isolates in relation to adaptation from the animal to human host. Vet Microbiol 129:333-41. 36. Parish, T., and N. G. Stoker. 2002. The common aromatic amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis. Microbiology 148:3069-77. 37. Plikaytis, B. B., J. L. Marden, J. T. Crawford, C. L. Woodley, W. R. Butler, and T. M. Shinnick. 1994. Multiplex PCR assay specific for the multidrug-resistant strain W of Mycobacterium tuberculosis. J Clin Microbiol 32:1542-6. 38. Prod'hom, G., B. Lagier, V. Pelicic, A. J. Hance, B. Gicquel, and C. Guilhot. 1998. A reliable amplification technique for the characterization of genomic DNA sequences flanking insertion sequences. FEMS Microbiol Lett 158:75-81. 39. Reed, M. B., P. Domenech, C. Manca, H. Su, A. K. Barczak, B. N. Kreiswirth, G. Kaplan, and C. E. Barry, 3rd. 2004. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 431:84-7. 40. Reed, M. B., S. Gagneux, K. Deriemer, P. M. Small, and C. E. Barry, 3rd. 2007. The W-Beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and has the DosR dormancy regulon constitutively upregulated. J Bacteriol 189:2583-9. 41. Safi, H., P. F. Barnes, D. L. Lakey, H. Shams, B. Samten, R. Vankayalapati, and S. T. Howard. 2004. IS6110 functions as a mobile, monocyte-activated promoter in Mycobacterium tuberculosis. Mol Microbiol 52:999-1012. 42. Samper, S., I. Otal, M. C. Rubio, M. A. Vitoria, R. Gomez-Lus, and C. Martin. 1993. [Application of RFLP to typing strains of Mycobacterium tuberculosis]. Enferm Infecc Microbiol Clin 11:547-51. Chapter I: Characterization of M. tuberculosis Beijing strain GC1237 79 43. Sampson, S., R. Warren, M. Richardson, G. van der Spuy, and P. van Helden. 2001. IS6110 insertions in Mycobacterium tuberculosis: predominantly into coding regions. J Clin Microbiol 39:3423-4. 44. Soto, C. Y., M. C. Menendez, E. Perez, S. Samper, A. B. Gomez, M. J. Garcia, and C. Martin. 2004. IS6110 mediates increased transcription of the phoP virulence gene in a multidrug-resistant clinical isolate responsible for tuberculosis outbreaks. J Clin Microbiol 42:212-9. 45. Staden, R. 1996. The Staden sequence analysis package. Mol Biotechnol 5:233-41. 46. Theus, S., K. Eisenach, N. Fomukong, R. F. Silver, and M. D. Cave. 2007. Beijing family Mycobacterium tuberculosis strains differ in their intracellular growth in THP-1 macrophages. Int J Tuberc Lung Dis 11:1087-93. 47. Tsolaki, A. G., S. Gagneux, A. S. Pym, Y. O. Goguet de la Salmoniere, B. N. Kreiswirth, D. Van Soolingen, and P. M. Small. 2005. Genomic deletions classify the Beijing/W strains as a distinct genetic lineage of Mycobacterium tuberculosis. J Clin Microbiol 43:3185-91. 48. van Soolingen, D., P. E. de Haas, P. W. Hermans, and J. D. van Embden. 1994. DNA fingerprinting of Mycobacterium tuberculosis. Methods Enzymol 235:196-205. 49. Zhang, M., J. Gong, Z. Yang, B. Samten, M. D. Cave, and P. F. Barnes. 1999. Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J Infect Dis 179:1213-7. Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 87 Objectives • To evaluate the promoter activity of three natural insertions of IS6110 (before dnaN gene which is characteristic of all Beijing strains, before the gene Rv2179c in M. tuberculosis GC1237 Beijing strain and before phoP gene in M. bovis B strain) by using several GFP-constructions containing promoter regions. • To study the promoter activity inside three different mycobacteria, the reference strain H37Rv, the clinical isolate GC1237 and M. bovis BCG, to demonstrate that this activity does not depend on the genetic background in MTBC. • To verify the promoter activity of these three locations in the corresponding wild-type strain. • To analyze these promoter activity inside macrophages with the GFP-constructions and with the wild-type strains. Mª Henar Alonso Ezcurra 88 Material and methods Bacterial strains, culture media, and growth conditions The clinical isolate M. tuberculosis GC1237, the reference M. tuberculosis H37Rv, M. bovis BCG and the M. tuberculosis H37Rv ΔphoP strains were used. Mycobacterial strains were grown in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 or in Middlebrook 7H10 mediumBacto agar supplemented with OADC (Difco Laboratories, Detroit, Mich.) and 0.05% Tween 80 (9). Liquid cultures were grown to logarithmic and stationary phases to be used for macrophage infection in vitro and for mycobacterial RNA extraction. E. coli XL1 was used for cloning experiments. Strains were grown in LB broth or LB agar plates. When necessary, media were supplemented with kanamycin (Km) (20μg/ml) or hygromicing (Hyg) (50μg/ml). The plasmid pFPV27-int (Figure 18A), derived from pFPV27 (1), was used for the construction of the strains expressing GFP. Electrotransformation of E. coli and of mycobacteria To prepare E. coli competent cells, bacteria were grown to an OD600nm of 0.4 to 0.6. Then the growth was stopped for 30min on ice, and bacteria were washed twice in chilled-cold water, and once in chilled-cold 10% glycerol. Aliquots of 50μl were directly used or frozen at -80ºC. Aliquots of 50μl were electroporated with plasmid DNA in 0.2cm gap cuvettes (Bio-Rad) with a single pulse (2.5kV, 25μF, 200Ω) in a GenePulser XcellTM (Bio-Rad). Cells were resuspended in LB to a final volume of 1ml and incubated for 1h at 37ºC if required before plating. M. tuberculosis competent cells were prepared as described by Wards et al. (20). Bacteria weregrown until an OD600nm of 0.6 to 0.8.After two washes in 0.05% Tween80 and one wash in 10% glycerol-0.05% Tween-80, cells were resuspended in 2ml of 10% glycerol-0.05% Tween-80. Aliquots of 400μl were electroporated with plasmid DNA in 0.2cm gap cuvettes (Bio-Rad) with a single pulse (2.5kV, 25μF, 1000Ω) in a GenePulser XcellTM (Bio-Rad). Cells were recovered with 1ml of 7H9-ADC-0.05% Tween-80 and incubated for 24h at 37ºC, to express the antibiotic resistance, before plating several dilutions on plates containing the relevant antibiotic. Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 89 Construction of GFP-strains • From the copy of IS6110 located upstream dnaN in GC1237 strain: To construct pFPVdnaN-int plasmid, primers dnaN-EcoRI and dnaN-kpnI (Table 6 Annex I) were used to amplify a 913 bp fragment from H37Rv strain. To construct pFPVISdnaN-int plasmid, ISA4-Eco and dnaN-kpnI (Table 6 Annex I) were used to amplified 713 bp segment from GC1237 strain. The PCR products were inserted in pFPV27-int, which had been cut with EcoRI and KpnI enzymes (Figure 18B). The plasmids were sequenced and electroporated into H37Rv strain obtaining HFPVdnaN and HFPVISdnaN strains. • From the IS6110 located upstream phoP gene in MBZ strain: Plasmids pFPVSO5-int and pFPVSO7-int (Figure 18C) were constructed in a previous work (not published). Briefly, to construct pFPVSO5-int plasmid, primers BCG2B-Eco and PhoBO3.1-Kpn (Table 6 Annex I) were used to amplify a 351 bp fragment from H37Rv strain. To construct pFPVSO7-int plasmid, primers ISA4-Eco and PhoBO3.1Kpn (Table 6 Annex I) were used to amplify a 350 bp segment from MBZ strain. The PCR products were inserted in pFPV27-int that had been cut with EcoRI and KpnI enzymes. Both plasmids were sequenced to verify the constructions. In this work the plasmids were electroporated into H37Rv, GC1237 and BCG strains obtaining HFPVSO5, HFPVSO7, GFPVSO5, GFPVSO7, BFPVSO5 and BFPVSO7 strains respectively. • From the IS6110 located upstream Rv2179c in GC1237 strain: To construct pFPV79c-int plasmid, primers Rv2179c-Eco and Rv2179c-Kpn (Table 6 Annex I) were used to amplify a 670 bp fragment from H37Rv strain. To construct pFPVIS79c-int plasmid, primers ISA4-Eco and Rv2179c-Kpn (Table 6 Annex I) were used to amplify a 210 bp segment from GC1237 strain. The PCR products were inserted in pFPV27-int, which had been cut with EcoRI and KpnI enzymes (Figure 18D). To verify the constructions, both plasmids were sequenced with the corresponding primers. After that, they were electroporated into H37Rv, GC1237 and BCG strains obtaining HFPV79c, HFPVIS79c, GFPV79c, GFPVIS79c BFPV79c and BFPVIS79c strains. Mª Hena r 90  Figure 1 gfp gen e gene a n GC123 7 contain s contain s pFPV79 and pF P Rv2179 c r Alonso Ezcu 1 8: gfp-pla s e . (B) pFP V n d pFPVISd a 7 strain cont a s 351 bp fr a s 350 bp f r c-int which c P VIS79c-int c gene cont a rra s mids used V dnaN-int w h a nN-int whi c a ining the p a gment ups t r agment of c ontains a 6 which con t a ining OP61 1 in this wor k h ich contain s c h contains p romoter reg t ream p ho P MBZ strai n 70 bp fragm t ains a 210 1 0. k . (A) pFPV 2 s a 913 bp f a 713 bp fr a ion of IS61 1 P gene of H n upstream ent from H3 7 bp segme n 2 7-int whic h f rom H37R v a gment ups t 1 0 (OP6110 37Rv strain phoP gen e 7 Rv strain u p n t from GC h contains th e v strain upst r t ream the d n ). (C) pFP V and pFPV e containing p stream the 1237 strain e promoterl e r eam the dn n aN gene fr o V SO5-int wh SO7-int wh OP6110. ( Rv2179c g e upstream t e ss a N o m ich ich ( D) e ne t he C (1 T h e x k a w c o s t I n p r pl o b Fi fr o pl a C M w f e a t c e ti m onstructio 8) h e replicati x plained pr e a namycin r e as cloned o mplement a t rain. n this work r eviously i n asmid wa s b taining H3 gure 19: R e o m pSO5 (1 a smid pUC 4 ell culture M H-S murin e ere grown e tal bovine s t a multipli c e lls were w a m e indicate Ch a n o f pSO 5 ve plasmi d e viously in t e sistance m into the P a tion of th e pSO7k (Fi g n this cha p s totally s e 7Rv Δpho P e plicative pl 5) and (B) 4 K was clon e and infect i e alveolar m in Dulbec c s erum (FB S c ity of infect a shed thre e d for each e a pter II: Stud y 5 k and PS O d pSO5k ( F t his chapte r m arker (Km R P stI restricti e phoP gen g ure 19B), p te r ) (Figu r e quenced P pSO7k str a asmids pS O pSO7k was e d into the P s i ons m acrophag e c o’s modifi e S ) and 4m M ion (MOI) o e times wit h e xperiment . y of the promo t O 7k plasm F igure 19 A) r , was con s R ) from the on site of e in H37R v also replic a r e 17B) w a and electr a in. O 5k and pS O constructe d s tI restrictio n e s were ob t e d Eagle m M L-glutam o f 10 bacte r h PBS, an d . t er activit y of I ids derive d ) derived f r s tructed in a plasmid p U pSO5. Th v ΔphoP o b a tive, deriv e a s constru c oporated i O 7k. (A) pS O d from pSO 7 n site of pSO 7 t ained from m edium (D M ine. Infecti o r ia per cell. d cultured i n I S6110 in diff e d from pS O r om pSO5 a previous w U C4K (Ame r is plasmid b taining H3 e d from pS c ted exact l nside H37 O 5k (adapt e 7 (18), the K 7 (18). HPA cultu r M EM) suppl o ns were p e After incu b n complete e rent location s O 5 (15) a n (15) (Figu r w ork (8). Br r sham Bios was used 7Rv Δpho P O7 (18) (d e l y as pSO Rv ΔphoP e d from (8)) i K mR marker r e collectio n emented w e rformed d b ating with b medium d u s of MTBC 91  n d pSO7 r e 17B), iefly, the c iences) for the P pSO5k e scribed 5k. The mutant s derived from the n s. Cells w ith 10% uring 4h b acteria, u ring the Mª Henar Alonso Ezcurra 92 Raw 264.7 cells were grown in RPMI 1640 medium supplemented with 10% of FBS. Infections were performed during 2h at MOI 3. After incubating with bacteria, cells were washed three times with phosphate buffered saline (PBS), and cultured in complete medium during the time indicated for each experiment. • Experiments of replication: The studies of intracellular replication of M. tuberculosis were carried out in 24-well plate. Briefly, the infections were performed at a MOI of 1 bacterium per 20 cells and 2 wells were used per strain and per day of processing. After 4h, infection was terminated by removing the overlaying medium and washing three times with PBS before adding 1ml of fresh complete culture medium per well. At 0 (4h post-infection), 4 and 7 days, the number of intracellular bacteria was evaluated by plating appropriate dilutions of lysed macrophages (0.1% triton X-100) on 7H10-ADC solid medium. After 2-4 weeks, CFUs were evaluated. • M. tuberculosis infection in vivo in mice: Intratracheal infection was performed with 100 CFU of bacteria in 50μl of PBS per mouse and 5 mice were used per strain. To deliver bacterial suspension, isoflurane anesthetized mice were orally intubated with a lachrymal olive luer-lock (UNIMED), 30mm in length and 0.6mm in diameter. Four weeks post-infection, lungs from each animal were harvested and placed in PBS for bacterial burden evaluation. To analyze bacterial replication, lungs were homogenized using GentleMacs homogeneizer (Miltenyi Biotec) and CFU counted by plating serial dilutions on 7H11-ADC solid medium. The protocol for animal handling was previously approved by University of Zaragoza Animal Ethics Committee. Isolation of RNA from mycobacteria • Extracellular conditions: M. tuberculosis strains were grown at 37ºC until the desired OD600 under aerobic conditions. The RNA from bacterial pellet was stabilized using the RNAprotect Bacteria Reagent (QIAGEN) following manufacturer’s recommendations. Cells were resuspended in 1ml acid phenol:chloroform (5:1) and 0.4ml lysis buffer (0.5% SDS, 20mM NaAc, 0.1mM EDTA) and transferred to 2ml Lysing Matrix B screw-cap tubes containing 0.1mm silica spheres (Q-BIOgene). Cells were disrupted by three 30s pulses in a FastPrep homogenizer (Q-BIOgene). After centrifugation, RNA from the supernatant was further extracted with 0.9ml chloroform:isoamyl alcohol (24:1). Total Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 93 RNA was precipitated with NaAc/isopropanol and washed with 70% ethanol. RNA pellet was treated with RNase-free DNase (Ambion), and the DNA free RNA was then further purified using and RNeasy kit (Qiagen). DNA contamination was ruled out by lack of amplification products after 35 cycles of PCR and the integrity of the RNA from the different strains were checked by gel electrophoresis on a 1% agarose gel. Purified RNA was kept at -80ºC until further use. • Intracellular conditions: Intracellular mycobacteria RNA extraction was performed with a modified protocol based on the one described by Fontan et al. (5). Briefly, 12.106 MH-S cells per flask were seeded in 150cm2 flasks. An appropriate volume of liquid mycobacteria culture was suspended in complete medium and added to each flask containing cells. After incubation, cells were washed with PBS to remove extracellular bacteria, and cultured in complete medium for 0 and 48h post-infection, when they were treated to extract intracellular mycobacteria. With this aim, cells were lysed and homogeneized during 5min adding 10ml per flask of GTC buffer (25mM sodium citrate, 4M guanidine thiocyanate, 0.5% N-lauryl sarcosine, 0.125M 2-mercaptoethanol and 0.5% Tween 80, pH 7.0). Next, samples were collected in 15ml centrifuge tubes and centrifuged for one hour at 4000rpm. Bacterial pellets were washed with GTC buffer and centrifuged again for one hour at 4000rpm. After that, dry bacterial pellets were treated as described above to extract mycobacterial RNA. RT-PCR Reverse transcription-PCR (RT-PCR) was carried out in two steps. RT was carried out with Expand Reverse Transcriptase (Roche) using 1μg RNA as the template and the appropriate reverse primer. Reaction mixtures were incubated at 42ºC for 90min. RT products were the subjected to PCR amplification, using TaqGold polymerase (Roche) as we previously described with the appropriate primers. Samples were analyzed by electrophoresis on a 1% agarose gel. qRT-PCR cDNA libraries from mycobacteria were constructed as follows. One μm of RNA was mixed with 25pmol of random hexanucleotides primers (Sigma) and 50U of Expand Reverse Trancriptase (Roche) in a final volume of 20μl. Reaction mixtures were incubated at 65ºC for 10min and then at 42ºC for 90min. The gene expressions were measured and normalized with respect to the levels of rrnaP1 mRNA by quantitative real-time PCR (qRT-PCR). qRT-PCR was carried out in a StepOne Plus (Applied Biosystems) instrument, using the cDNA generated by RT from 25ng of RNA as a Mª Henar Alonso Ezcurra 94 template, 1X Power SYBER green PCR master mix (Applied Biosystems), and the appropriated primers, each at a concentration of 250nM. The PCR program involved an initial denaturation step for 10min at 95ºC, followed by 40cycles at 95ºC for 15s and 60ºC for 1min. The specificity of the PCR products was confirmed by the loss of fluorescence at a single temperature, when the double-stranded DNA melted to singlestranded DNA. Protein extraction from mycobacteria • Extraction of cellular proteins: Cell-free protein extracts of mycobacteria were prepared from early log-phase cultures grown in Middlebrook 7H9-ADC-0.05% Tween-80. Cells from 50ml of culture were pelleted by centrifugation (3500rpm for 15min at 4ºC). Mycobacterial cell pellets were washed twice with PBS and then resuspended in cold PBS. Mycobacterial suspensions were transferred to tubes containing glass beads (Qbiogene) and were disrupted by mechanical traction (Fast-prep instrument) in two cycles (15s at speed 6.5m/s and 15s at 4.0m/s, respectively) cooling the samples on ice 5min between the pulses. The proteins were centrifuged at 5000rpm for 15min at 16ºC and the supernatant containing whole-cell protein extracts was filtered through a 0.22μm-pore-size low protein binding filter (Pall). • Extraction of culture filtrate proteins: Mycobacterial strains were cultured in 7H9 Middlebrook medium supplemented with dextrose in the absence of Albumin. Cultures were grown 4 weeks and then centrifuged (3500rpm for 15min at 37ºC). The supernatant was filtered through 0.22μm filter Stericup systems (Millipore). The extracellular proteins were precipitated during 1h at 4ºC with the 10% v/v of Trichlore acetic acid. After that, pellets were collected by centrifugation (4000rpm 1h at 4ºC), washed twice with cold acetone and resuspended in 250μl of PBS 1x. In both cases, proteins were quantified using the RC DC protein assay (BioRad) and stored at -80ºC. Western blot analysis Western blot analysis was carried out using standard procedures. Equivalent quantities of protein extracts were boiled (for 5min) in presence of 5μl of 150mM Tris/HCl pH 7.4, 3% SDS, 0.3mM sodium molybdate, 30mM sodium pyrophosphate, 30mM NaF, 30% glycerol, 30% mercaptoethanol and 0.06% bromophenol blue. Cellular proteins were separated by electrophoresis through a 15% polyacrylmide gel containing 0.1% SDS in Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 95 running buffer (25mM Tris, 192mM glycine, 3.4mM SDS) at constant amperage of 20mA/gel. Proteins were transferred to PVDF membranes (pre-activated with methanol) in transfer buffer (48mM Tris/HCl pH 8.3, 39mM glycine, 0.037% SDS, 20% methanol) for 1h at 20V with a semi-dry electrophoretic transfer cell (BioRad). Blotted proteins were analyzed using specific antibodies: polyclonal antibodies against phoP protein (ZEU-immunotec) (12) and monoclonal antibody against ESAT-6 protein (Abcam). The membrane was blocked with 5% skim powdered milk in buffer B (0.12M NaCl, 10mM Tris/HCl pH 8.0, 0.5% Tween-20 in PBS pH 7.4) for 30min at room temperature and mild shaking. Hereafter, the membrane was incubated with primary antibody antiPhoP (1:2000) or anti-ESAT-6 (1:5000) in buffer A (2.5% skim powdered milk in buffer B) over night. Three washes with buffer B were performed previous to the incubation with commercial anti-rabbit (in the case of PhoP) or anti-mouse (for ESAT-6) secondary antibodies (1:20,000 in buffer A) conjugated with horseradish peroxidase, for 1h at room temperature and mild shaking. Then, the membrane was washed three times with buffer B. Immunocomplex detection was performed by incubation (for 90s) with a chemiluminiscent AP substrate (ImmobilonTM western, Millipore) and subsequent exposure (for 2min) to a photographic film. Molecular weights of the detected proteins were estimated relative to the standard protein ladder. Analysis of GFP expression • Extracellular conditions: To study the expression of the GFP protein in GFP strains, the fluorescence was measured by fluorometric method (Synergy_HT, BioTek) during the bacterial growth and normalized with OD600nm. Briefly, 1ml of the mycobacterial broth cultures was collected and 200μl of the total volume were added to a 96-well plate and the GFP expression was measured at λ 485nm. After that, the OD600nm of the broth culture was measured using the rest of the volume (800μl). The obtained value of fluorescence was normalized with the OD of the culture. Fluorescence median intensity (FMI) of the different GFP strains was measured by Fluorescence activated cell sorting (FACS). Thus, 800μl of mycobacterial logarithmic cultures were centrifugated at 14000rpm 5min and the pellets were resuspended with 1ml of paraformaldehide (PFA) 4% and incubate 30min. After this time, the mixtures were centrifugated and resuspended in PBS. The cytometer used in this work was a FACSaria (BDBiosciences) and the analysis was performed with the software Weasel. Mª Henar Alonso Ezcurra 96 • Intracellular conditions: The GFP expression under intracellular conditions was studied by two methods. The first one, MH-S cells were infected with different GFP strains and H37Rv or GC1237 were used as reference. Infections were carried out in 24-well plates, where cells were seeded at 105 cells per well. Cells were collected at 0, 24, 48 and 72h post-infection and they were analyzed by FACS. Previously, cells were labeled with annexinV to discern between live and dead cells. Briefly, after washing collected cells once with AnnexinV binding buffer (ABB) (BD Biosciences), they were incubated for 15min at room temperature with annexinV APC-conjugated diluted in ABB, and then fixed with PFA 4% in calcium-containing buffer. The second one was carried out as previously describe by Brodin et al (3). Briefly, Raw cells were seeded at a density of 3x105 cells per well in 384-well plates in 50μl of medium. Adherent cells were then infected with bacterial suspensions at MOI of 3 and incubated for 2h. Cells were then washed three times with PBS and fixed with 1.5% formaldehyde for 10min, washed twice and stained with 5μg/ml DAPI dilactate (Sigma) in 0.1% triton X-100 (Sigma) in PBS. After that, confocal images were recorded on an automated fluorescent confocal microscope Opera (Evotec) using a 20X water immersion objective (NA 0.7). Subsequently, 405nm and 488nm lasers were used for excitation of DAPI and GFP, in two sequential exposures. Because the 20X magnification does not allow the acquisition of the entire 384-well plate, 6 fields within each well were recorded. For each field, two channels were recorded using two different cameras: the blue image for nuclei channel (Figure 20A) and the green image for GFP (Figure 20B). Each image was then processed using dedicated in-house image analysis software (IM 3.0). To determine the number of macrophages and delineate their outlines, cell centers were positioned using an algorithm that identifies the pixels with a maximum local intensity in the blue image. Cells that were too small based on cell radius area enclosed were discarded. The result of this step was the acquisition of the average cell surface that is the number of cells and their spatial localization (Figure 20D). To quantify the bacterial load, a similar procedure was carried out with green image (Figure 20B). In this case, the GFP-intensity of the different strains was one of the important parameters and for that, a threshold for GFP signal was determined with GFP-control strains. As DAPI stain did not allow us to define the contour of the cell, a macrophage was considered infected if labeled bacterium objects were distant of less than 5 pixels from the cell nucleus (Figure 20F). Proximity is a parameter manually set-up by the user given the fact that one cell has only one nucleus and a macrophage was labeled as infected if there were at least three contiguous green pixels overlapping the cell surface. Once images were processes Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 103 Transcripts from IS6110 to aroG and from Rv2180c to aroG were detected in GC1237 strain and in H37Rv strain, respectively To determine if a transcript extended from IS6110 or Rv2180c into downstream genes Rv2179c and aroG, RNA from broth cultures of GC1237 and H37Rv strains was extracted and RT-PCR was performed as previously described in this chapter. We obtained amplification products from Rv2180c to aroG in H37Rv strain (Figure 25A and C). These results indicate that the three genes are cotranscribed in an operon. In the case of GC1237 strain, amplification products were obtained from 3’ end of IS6110 to aroG suggesting that the 3’ end of IS6110, Rv2179c and aroG genes are cotranscribed (Figure 25B and D). All the RT-PCR products were sequenced and the blast results verified their specificity. Figure 25: RT-PCR analysis of Rv2179c region from M. tuberculosis H37Rv and GC1237 strains. (A). Schematic diagram of Rv2179c region in H37Rv strain. (B) Schematic diagram of Rv2179c region in GC1237 strain. In both diagrams, the primers used for RT-PCR and the sizes of the fragments obtained with each pair of primers are indicated. The direction of transcription for Rv2179c is indicated by arrows. (C) RT-PCR analysis of this region in H37Rv. (D) RT-PCR analysis of this region in GC1237. In both RT-PCR the combination of primers is indicated above each set of reaction. Each set of three reaction consist of a positive control PCR assay with genomic DNA as the template (+), an RT-PCR (*), and a negative control assay without reverse transcriptase (-). The aroG gene is an essential gene involved in the biosynthesis of chorismate precursor of the three aromatic amino acids in M. tuberculosis (14). There are no available data about the function of the protein encoded by Rv2179c gene. In fact, according to Tuberculist database, the product of this gene is a conserved essential hypothetical protein and it is conserved in different mycobacterial strains (11). From our results we can hypothesize that the overexpression of this gene would be Mª Hena r 104  advant a macrop These r results d IS6110 intrace • E To disc e presen c plasmi d the reg i constru HFPV2 7 GFP e x techniq u than th fluorom of GF P Theref o Rv217 9 Figure evolutio n growth n and HF P r Alonso Ezcu a geous fo r hages. r esults indi c d o not excl u is acting llular con d E xtracellu l e rn that Rv c e of IS61 d s FPV79c i on upstre a cted and t r 7 was use d x pression w u es showe d e observe d et r y during P in this c o re, this res 9 c gene. 26: GFP e x n of the fluo r n ormalized w P VIS79c str a rra r GC1237 c ated that t h u de the po s as a pro m d itions l ar conditi o 2179c upr e 10 and no and FPVI S a m Rv2179 c r ansformed d as GFP-c o as measur e d that the f d in HFPV 7 the growth c ase was ult suggest s x pression i r escence of H w ith OD600nm. a ins was me a at least h e three g e s sibility of i n m oter of R o ns e gulation o b t to the di S 79c conta i c in H37R v into H37R v o ntrol strain e d both by f f luorescen c 7 9c strain curve indi c independe n s that in G C n HFPV79 c H FPV27, H F (B) Fluores c a sured by fl o in certain e nes are c o n dependen t R v2179c g e b served in G fferent ge n i ning the p v strain or i n v obtaining . f luorometry c e of HFP V (Figure 2 6 c ated that t h n t of bact e C 1237 strai n c and HFP V F PV79c and c ence medi a o w cytometr y environm e o transcribe d t promoters e ne both u G C1237 str n etic back g romoterles s n GC1237 HFPV79c and flow c y V IS79c stra 6 A-B). Mor e h e effect of e rial grow t n , IS6110 i s V IS79c stra HFPVIS79c a n intensity ( F y at logarith m e nts such d in an ope r for each g e u nder extr a ain was re a g round, the s gfp gene strain, res p and HFPV I y tometry. R in is about e over, dat a IS6110 on t t h phase ( s acting as ins. (A) Ti m strains duri n F MI) of HFP V m ic phase. as infecti r on, but th e e ne. a cellular a n a lly due to t recombin a preceded p ectively w e I S79c strai n R esults of b o 5-fold hig h a obtained t he regulati ( Figure 26 A a promote r m e course t n g the bacte r V 27, HFPV 7 ng e se n d t he a nt by e re n s. o th h er by on A ). r of  t he r ial 7 9c T o pl s t a n hi d e Fi B F ( B Fi fl u n o p h o confirm t h asmids we t udied duri n n d 28A-B s gher than e pendence gure 27: G F F PV27, BFP B ) FMI of the gure 28: G u orescence o rmalized wi t h ase. Ch a h at this pro m re transfor m n g the grow t s how that t h that in th on the gen F P express V79c and B F three strain s G FP expres s of GFPV2 7 t h OD600nm. ( a pter II: Stud y m oter activ m ed in GC 1 t h curve b o h e GFP ex p e mutants etic backgr o ion in BFP V F PVIS79c s t s measured s ion in G F 7 , BFPV79c ( B) FMI of t h y of the promo t ity was not 1 237 and B o th by fluor o p ression o f without I S o und of IS 6 V 79c and B t rains during by flow cyto m F PV79c an d and BFP V h e three strai t er activit y of I exclusive i B CG strains o metry and f the muta n S 6110. Thi s 6 110. B FPVIS79c s the bacteria m etry at log a d GFPVIS7 9 V IS79c strai ns measure d I S6110 in diff e nside H37 R and the G flow cytom e n ts with IS 6 s result c o s trains. (A) l growth nor m a rithmic pha s 9 c strains. ns during t d by flow cy t e rent location s R v strain, t h FP expres s e try. Figur e 6 110 is abo o rroborates GFP fluore s m alized with s e. (A) Evoluti o t he bacteri a t ometry at lo s of MTBC 105  h ese two s ion was e s 27A-B ut 2-fold the no  s cence of OD600nm.  o n of the a l growth garithmic Mª Hena r 106  • I Murine differen to excl consid e hand, c the inf e negativ e infecte d the con under e cells te n contras t Figure 2 (A) Hist o and R2 with HF respect perform e r Alonso Ezcu I ntracellul a immortaliz e t GFP exp r ude non-i n e ring thei r F ells contai n e cted ones e cells, in o d with HFP V trol at all ti m e xtracellula r n ded to inc r t , in the ab s 2 9: GFP ex p o grams of H represents t PV79c and to FMI valu e d. rra a r conditio e d alveola r r essing stra n fected cel F MI as neg a n ed in the r e (R2 in Fi g o rder to an a V IS79c str a m es studie d r condition s r ease durin s ence of IS 6 p ression in 37Rv and H t he fluoresc e HFPVIS79 c es of HFP V ns r macroph a ins and inf e ls, it was a tive fluore s e gion with a g ure 29 A ). a lyze only l a in showed d . These r e s . Further m g the expe r 6 110 this e v HFPV79c a FPVIS79c i n e nt cells. (B ) c strains. T h V 27 strain. F a ges (MHS e cted cells F also infe c s cence lev e a higher flu o Moreover , ive cells. A a clear inc e sults are i n m ore, GFP r iment, bei n v ent failed t a nd HFPVIS 7 n fected cells ) Time cour s h e percent a F igure show s S cell line) F MI was m e c ted with n e l (R1 in F i o rescence l e , data wer e A s it is obs e rease of th e n agreeme n expression n g this rise t o happen ( 7 9c strains . R1 indicat e s e of FMI of a ge of incre s a represe n were infe c e asured by n on fluore s i gure 29 A ). e vel were e e referred e rved in Fi g e ir FMI val u n t with the o of HFPVI S more dram a Figure 29B in murine m e s the non-fl u cells gated ment was c n tative expe r c ted with t FACS. Th u s cent H37 R On the ot h e stablished to annexi n g ure 29B c e u es regardi o btained d a S 79c-infec t a tic at 72h. ). m acrophag e u orescent c e in R2, infec t c alculated w r iment of th r t he u s, R v, h er as n Ve lls ng a ta t ed In e s. e lls t ed w ith r ee Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 107 A previous work showed a reinforcement of IS6110 promoter activity under intracellular conditions (16). However, in that work authors studied the expression of different genes and the presence or not of IS6110 upstream in two different strains. Nevertheless, authors did not discern the possibility of a regulation due to the different genetic background of the studied strains. Our results clearly show that the promoter activity of IS6110 on Rv2179c gene is only related to disposition of the sequence with respect to this gene. As part of the three genes forming an operon we analyzed the expression of aroG gene by qRT-PCR, but we could not observe significant increment of this gene in GC1237 strain compared with H37Rv strain. This result does not exclude the possibility of another regulation of aroG gene. Mª Henar Alonso Ezcurra 108 3. The effect of IS6110 in phoP of the location of MBZ strain Soto et al. demonstrated that the presence of IS6110 insertion sequence 75 bp upstream the phoP gene in the MDR M. bovis B strain causes an increment in its transcription (18). This fact could have an important consequence due to this gene is an important transcriptional regulator. To depth in the study of the promoter activity of IS6110 in phoP of MBZ strain the plasmids pFPVSO5, pFPVSO7 containing the promoterless gfp gene preceded by the region upstream phoP in H37Rv strain or in MBZ strain, respectively were used. The plasmids were then transformed in H37Rv strain obtaining HFPVSO5 and HFPVSO7 strains and the RNA at logarithmic and stationary phase were obtained. The GFP expression of HFPVSO7 strain was measured by qRT-PCR and compared with that in HFPVSO5 strain. Figure 30 shows that the GFP expression of the mutant which contains IS6110 is 3.5 (logarithmic phase) to 8 (stationary phase) times higher than that in HFPVSO5 strain. Figure 30: Relative quantification (RQ) of GFP in HFPVSO5 and HFPVSO7 strains. The GFP expression (normalized with rrnaP1 levels) of both strains was measured by qRT-PCR at logarithmic and stationary phases. In both cases the expression was referred to GFP levels of HFPV27 strain. The results are a mean of three independent experiments. Once the overexpression of gfp gene in HFPVSO7 strain was confirmed by qRT-PCR (Figure 30), this overexpression was analyzed in three different genetic backgrounds. For that issue, these two gfp-plasmids were transformed into two more strains: the clinical isolate GC1237 and M. bovis BCG and the GFP expression during the growth curve was studied in the three groups of strains both by fluorometry and flow cytometry. As controls, HFPV27, GFPV27 and BFPV27 strains were used. Similar high increment in the GFP expression was observed when IS6110 was located upstream of g f s t Fi o f s t B F ( C f p gene. R e t rains is ab o gure 31: C o f the GFP e t rains mea s F PV27 (as a C ) GFPVSO 5 Ch a e sults of b o o ut 4 to 5 f o o mparison o e xpression s ured by fl o control) str a 5 , GFPVSO 7 a pter II: Stud y o th techni q o ld higher t h o f GFP exp r during the o w cytomet r a ins. (B) HF P 7 and GFPV 2 y of the promo t q ues show e h an the obs e r ession of t growth cu r r y at logari t P VSO5, HF P 2 7 (as a con t t er activit y of I e d that the e rved in F P he three gr o r ve and FM t hmic phas e P VSO7 and H t rol) strains. I S6110 in diff e fluorescen P VSO5 stra i o ups of GF I value of t e . (A) BFP V H FPV27 (as e rent location s ce of the F i ns (Figure P-strains. E t he three g r V SO5, BFPV a control) st s of MTBC 109  F PVSO7 31). E volution r oups of SO7 and r ains and Mª Henar Alonso Ezcurra 110 This result indicates that, independently of the genetic background, IS6110 acts as a promoter in the studied region in different mycobacteria of M. tuberculosis complex causing a similar increment of expression of the upstream gene. Furthermore, this result corroborates the data obtained with the natural insertion of IS6110 upstream the Rv2179c gene indicating that the increment of the expression caused for IS6110 when it is acting as a promoter is regardless of the region. The next step was to analyze whether the behavior of the promoter activity of this sequence in this location was also independent of the strain or genetic background inside the cell so, two different macrophages cell lines, MHS and Raw, were infected with the GFP-expressing H37Rv and GC1237 strains, and the fluorescence was analyzed at different times with two methods, by cytometry and by confocal microscope. In this case we do not infect with BCG-GFP strains due to the lack of replication inside the host. MHS Infected cells FMI were measured by FACS. Thus, to exclude non-infected cells, we also infected with non-fluorescent H37Rv and GC1237, considering their FMI as negative fluorescence level. Data was referred to annexinV-negative cells, in order to analyze only live cells. Moreover, the FMI levels were normalized with respect to promoterless gfp strains. As it is observed in Figure 32A-B cells infected with HFPVSO7 and GFPVSO7 strains respectively, shows a clear increase of their FMI values regarding the control (HFPVSO5 and GFPVSO5 strains) at all times studied. These results are in agreement with the obtained data in extracellular conditions. The increment observed in HFPVSO7 strain was slightly higher than that in GFPVSO7 strain but the tendency of both mutants is exactly the same. This result confirms the promoter activity of IS6110 in this location under intracellular conditions. Fi m H F H F o f c a e x T h m c o fi x s t s o s p c o w f o t h n o G gure 32: G F m urine macr o F PVSO7 str F PV27 strai n f cells, infe c a lculated wit x periment of h e promot e m ethod. In t o nfocal mi c x ation, ima g t udied time s o ftware. Fir p atial positi o o ntour of th ere distant o und to be i h e GFP le v o rmalized w FPV27). Ch a F P expressi o o phages ( M ains. The p e n . The resul t c ted with G F h respect t o three perfor e r activity o t his case, t c roscope. F g es were c s (Figures st, the nuc l o n in non i n e cell, a m a of less tha i nfected af t v els insid e w ith respe a pter II: Stud y o n in HFPV S M HS). (A) Ti e rcentage o f t is a mean o F PVSO5 an d o FMI value s med. o f IS6110 i n t he intensi t or that iss u c ollected w 33A and 3 4 l ear stain D n fected we l a crophage w n 5 pixels f t er 2h (Fig u e the mac r ct to the y of the promo t S O5/HFPV S me course o f increment w o f three inde p d GFPVSO 7 s of GFPV2 7 n these G F t y of infect i u e, RAW c e ith the co n 4 A). After t D API was u l ls. Becaus e w as consid f rom the c e u res 33B a n r ophages ( levels of p t er activit y of I S O7 and GF P o f FMI of c e w as calcula t p endent exp 7 strains. T h 7 strain. Th e F P-strains w i on of GF P e lls were i n focal micr o t hat, imag e u sed to def i e this stain ered infect e e ll nucleus. n d 34B). T h ( Figures 3 3 p romoterle s I S6110 in diff e P VSO5/GFP e lls, infected t ed with res p eriments. ( B h e percenta g e graphic sh o w as also st P strains w n fected an d o scope Op e e analysis w i ne macrop did not all o e d if labele d Around 90 % h e intensity 3 C and 3 4 s s gfp str a e rent location s VSO7 strai n with HFPV p ect to FMI v B ) Time cour s g e of incre m o ws a repr e udied by a as measur d after stai n e ra (Evote c w as done i n hages nu m o w us to d e d bacteriu m % of the c e of infectio n 4 C) and t h a ins (HFP V s of MTBC 111  n s inside S O5 and v alues of s e of FMI m ent was e sentative second ed by a n ing and c ) at the n IM 3.0 m ber and e fine the m objects e lls were n means h at was V 27 and Mª Hena r 112  Figure 3 (A) Re p strains a (C) Inte infectio n indicate r Alonso Ezcu 3 3: Intensit y p resentative a nd analyze d nsity of inf e n of HFPV27 the standar d rra y of infectio n pictures of d at: 0, 24, 4 e ction of H F strain. Res u d deviations o n of HFPV2 7 RAW cells 8 and 72h p F PVSO5 an d u lts are the m o f the mean s 7 , HFPVSO 5 infected wit h p os t -infectio n d HFPVSO 7 m eans of fo u s . 5 and HFPV h HFPV27, n . (B) Infecti o 7 strains n o u r independ e SO7 strain s HFPVSO5 a o n rate at 0h o rmalized w i e nt experim e s in Raw cel a nd HFPVS O post-infecti o i th intensity e nts; error b a ls: O 7 o n. of a rs Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 119 Conclusions • The insertion sequence IS6110 located upstream dnaN gene in M. tuberculosis Beijing strain GC1237, has minimal effect on its gene expression both, in broth and in intracellular conditions. • The copy of IS6110 located upstream Rv2179c gene in M. tuberculosis GC1237 is acting as a mobile promoter both in broth and inside macrophages. Furthermore, this IS-promoter activity was checked in three different mycobacteria and the results indicated that this activity is independent of the genetic background. • Rv2180c, Rv2179c and aroG genes are cotranscribed in H37Rv strain and the 3’ end of IS6110 (OP6110), Rv2179c and aroG genes are cotranscribed in GC1237 strain indicating the existence of an operon. • The overexpression of gfp gene in HFPVSO7, which contain the region upstream phoP gene of MBZ (including the OP6110), was confirmed by qRT-PCR and the high GFP expression of this strain was confirmed by cytometry and fluorometry in broth. These results demonstrated the promoter activity of IS6110 in this location. • A high expression of GFP in HFPVSO7 and with similar profile was observed inside two different cell lines and by two different methods as flow cytometry and confocal microscope indicating the robustness of these results. • H37Rv ΔphoP pSO5k and H37Rv ΔphoP pSO7k strains presented similar transcriptional levels of phoP gene and similar PhoP protein expression. Furthermore, both strains presented similar replication inside the cell. • The colony morphology of H37Rv ΔphoP pSO7k strain is smaller than that in H37Rv ΔphoP pSO5k strain however; the bacterial growth in broth of H37Rv ΔphoP pSO7k strain was similar to that in H37Rv ΔphoP pSO5k strain. • No difference on replication in mice was observed between H37Rv ΔphoP pSO5k and H37Rv ΔphoP pSO7k strain. Mª Henar Alonso Ezcurra 120 References 1. Barker, L. P., D. M. Brooks, and P. L. Small. 1998. The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein. Mol Microbiol 29:1167-77. 2. Beggs, M. L., K. D. Eisenach, and M. D. Cave. 2000. Mapping of IS6110 insertion sites in two epidemic strains of Mycobacterium tuberculosis. J Clin Microbiol 38:2923-8. 3. Brodin, P., Y. Poquet, F. Levillain, I. Peguillet, G. Larrouy-Maumus, M. Gilleron, F. Ewann, T. Christophe, D. Fenistein, J. Jang, M. S. Jang, S. J. Park, J. Rauzier, J. P. Carralot, R. Shrimpton, A. Genovesio, J. A. Gonzalo-Asensio, G. Puzo, C. Martin, R. Brosch, G. R. Stewart, B. Gicquel, and O. Neyrolles. High content phenotypic cellbased visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling. PLoS Pathog 6. 4. Casart, Y., L. Turcios, I. Florez, R. Jaspe, E. Guerrero, J. de Waard, D. Aguilar, R. Hernandez-Pando, and L. Salazar. 2008. IS6110 in oriC affects the morphology and growth of Mycobacterium tuberculosis and attenuates virulence in mice. Tuberculosis (Edinb) 88:545-52. 5. Fontan, P., V. Aris, S. Ghanny, P. Soteropoulos, and I. Smith. 2008. Global transcriptional profile of Mycobacterium tuberculosis during THP-1 human macrophage infection. Infect Immun 76:717-25. 6. Frigui, W., D. Bottai, L. Majlessi, M. Monot, E. Josselin, P. Brodin, T. Garnier, B. Gicquel, C. Martin, C. Leclerc, S. T. Cole, and R. Brosch. 2008. Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog 4:e33. 7. Ghanekar, K., A. McBride, O. Dellagostin, S. Thorne, R. Mooney, and J. McFadden. 1999. Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a microaerobic environment. Mol Microbiol 33:982-93. 8. Gonzalo Asensio, J., C. Maia, N. L. Ferrer, N. Barilone, F. Laval, C. Y. Soto, N. Winter, M. Daffe, B. Gicquel, C. Martin, and M. Jackson. 2006. The virulenceassociated two-component PhoP-PhoR system controls the biosynthesis of polyketidederived lipids in Mycobacterium tuberculosis. J Biol Chem 281:1313-6. 9. Jacobs, W. R., Jr., G. V. Kalpana, J. D. Cirillo, L. Pascopella, S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol 204:537-55. 10. Mahillon, J., and M. Chandler. 1998. Insertion sequences. Microbiol Mol Biol Rev 62:725-74. 11. Marmiesse, M., P. Brodin, C. Buchrieser, C. Gutierrez, N. Simoes, V. Vincent, P. Glaser, S. T. Cole, and R. Brosch. 2004. Macro-array and bioinformatic analyses reveal mycobacterial 'core' genes, variation in the ESAT-6 gene family and new phylogenetic markers for the Mycobacterium tuberculosis complex. Microbiology 150:483-96. Chapter II: Study of the promoter activity of IS6110 in different locations of MTBC 121 12. Martin, C., A. Williams, R. Hernandez-Pando, P. J. Cardona, E. Gormley, Y. Bordat, C. Y. Soto, S. O. Clark, G. J. Hatch, D. Aguilar, V. Ausina, and B. Gicquel. 2006. The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs. Vaccine 24:3408-19. 13. Otal, I., A. B. Gomez, K. Kremer, P. de Haas, M. J. Garcia, C. Martin, and D. van Soolingen. 2008. Mapping of IS6110 insertion sites in Mycobacterium bovis isolates in relation to adaptation from the animal to human host. Vet Microbiol 129:333-41. 14. Parish, T., and N. G. Stoker. 2002. The common aromatic amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis. Microbiology 148:3069-77. 15. Perez, E., S. Samper, Y. Bordas, C. Guilhot, B. Gicquel, and C. Martin. 2001. An essential role for phoP in Mycobacterium tuberculosis virulence. Mol Microbiol 41:17987. 16. Safi, H., P. F. Barnes, D. L. Lakey, H. Shams, B. Samten, R. Vankayalapati, and S. T. Howard. 2004. IS6110 functions as a mobile, monocyte-activated promoter in Mycobacterium tuberculosis. Mol Microbiol 52:999-1012. 17. Schurch, A. C., K. Kremer, A. Kiers, O. Daviena, M. J. Boeree, R. J. Siezen, N. H. Smith, and D. van Soolingen. The tempo and mode of molecular evolution of Mycobacterium tuberculosis at patient-to-patient scale. Infect Genet Evol 10:108-14. 18. Soto, C. Y., M. C. Menendez, E. Perez, S. Samper, A. B. Gomez, M. J. Garcia, and C. Martin. 2004. IS6110 mediates increased transcription of the phoP virulence gene in a multidrug-resistant clinical isolate responsible for tuberculosis outbreaks. J Clin Microbiol 42:212-9. 19. Turcios, L., Y. Casart, I. Florez, J. de Waard, and L. Salazar. 2009. Characterization of IS6110 insertions in the dnaA-dnaN intergenic region of Mycobacterium tuberculosis clinical isolates. Clin Microbiol Infect 15:200-3. 20. Wards, B. J., and D. M. Collins. 1996. Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol Lett 145:101-5. Chapter III                                Chapter III 125 Introduction The insertion sequence IS6110 was isolated from a Mycobacterium tuberculosis (MTB) cosmid library as a repetitive sequence and was found to be specific to mycobacteria belonging to the MTBC (35). MTB strains typically contain multiple copies of this element (up to 25 copies per genome), although strains with only a single copy or no copies have been identified (14). In contrast, M. bovis usually have a low copy number such as M. bovis BCG which has either one or two copies (13). Although the genome of M. tuberculosis is highly homogeneous, strains are highly polymorphic with respect to copy number and location of IS6110. This sequence does not have a known target or consensus sequence; it has been found within ORFs and intergenic regions. However, M. tuberculosis and M. bovis strains with only one copy of this sequence show that it is inserted conservatively into one of array of 36-bp directly repeated sequences (16). Another study shows the possible conserved sites of insertion in strains of M. tuberculosis with less than seven copies (12) and also other hot-spots of IS6110 have been shown such as the ipl loci (11), the phospholipase C regions (17, 39) and the oriC (20). M. tuberculosis low copy number strains (LCS) and high copy number strains (HCS) M. tuberculosis strains with less than six copies of IS6110 are usually referred as low copy number strains (LCS) in the literature. Several studies reported the presence of LCS from regions as India, Vietnam or Tanzania (3, 27, 31). Furthermore, the 66% of the M. tuberculosis strains isolated in Tiruvallur, South India presented a single IS6110 (32). High copy number strains (HCS), with six or more copies of IS6110, were reported by a greater number of papers. One study from Brazil, reported that 93.6% of M. tuberculosis strains had at least six copies ranging from 1 to 18 (34). In San Francisco, of 1,326 isolates investigated, 90% had six o more copies and only two isolates had no copies of IS6110 (42). A majority (96.2%) of the 183 strains fingerprinted from Kampala were HCS, the number of IS6110 copies ranged from 1 to 20 (2). Chauhan et al. analyzed 308 isolates of M. tuberculosis, from different parts of India and 56% of the isolates showed HCS of IS6110 (9). Both groups of strains, LCS and HCS, can originate outbreaks at similar proportion. The Beijing family is one of the lineages with the highest number of copies of IS6110. There are controversies among the factors that contribute to the success of this family. On the one hand, it is well known the wide distribution of Beijing strain 210 in the United States in 1995 (42) and its MDR clone, W, which caused disease in more than 350 patients in New York City (5, 6) and as we mention previously (Chapter 1), the Mª Henar Alonso Ezcurra 126 clinical isolate GC1237 has been responsible of epidemic outbreaks since it appeared in 1993 (7). However, one study conducted in Cape Town (South Africa) found no significant association between the M. tuberculosis genotype and transmissibility within the household (21). Besides, there are outbreaks reported caused by LCS, as was the extensive transmission of M. tuberculosis in a rural population with minimal risk factors for TB. This strain was designated as CDC1551 and the fingerprint showed 4 copies of IS6110 (38). LCS versus HCS and IS6110 locations Several reports have strongly suggested that the severity and clinical manifestations of tuberculosis depend on the immunogenicity and pathogenicity of the infecting M. tuberculosis strain. In this regard the IS6110 sequence varies in number and position within the genome generating a high level of DNA polymorphism among strains. Interestingly, the location of IS6110 in M. bovis isolates from endogenous reactivation cases from elderly people were studied in comparison to the bovine M. bovis strains, concluding that the presence of more copies in human strains could be related to the adaptation from the animal to the human host (25). On the other hand, a highly preferred site of insertion of IS6110 was detected and designated as “DK1” (Rv0403c) in M. tuberculosis LCS but the prevalence of this site decreases sharply in HCS, suggesting a separate lineage for the HCS and the LCS (12). This contrasts with M bovis analysed without copy inserted at the same genomic position that M tuberculosis strains (25). This agreed with the idea that LCS of M. tuberculosis and M. bovis evolved separately after the progenitor acquired IS6110 at the DR region. IS6110 has been also checked as a tool to analyze the evolution of members of the MTBC. In such a case, transposition may have influence on the evolution of the strains, the parental strains should carry low copy number and the descendant, more evolved, would carry high copy number. One example that theoretically support that consideration are the Beijing lineage, members of this family are HCS of IS6110 (between 15-25 copies per genome) and have shown high prevalence and high transmissibility (22). These characteristics could be seen as selective advantages of bacteria to its main purpose: infect humans (15). Chapter III 127 Objectives • To locate the common points of insertion of IS6110 in Beijing strains. • To carry out a scrutiny of points of insertion of IS6110 in available literature and in the DNA sequence databases. • To generate a representative primer collection of Beijing-IS6110 points of insertion. • To identify and analyze the common sites of IS6110 in non-Beijing strains with respect to Beijing strains, being both groups HCS. Mª Henar Alonso Ezcurra 128 Material and methods Bacterial selected strains, culture media, growth conditions and isolation of mycobacteria genomic DNA Sixty-one M. tuberculosis clinical isolates were used in this work. The 61 isolates comprised 17 Beijing and 44 non-Beijing strains. Among the 17 Beijing strains 8 (NHN5, HM77, HM903, HM764, 990172, W4, N4 and CAM22) were previously selected as representative of this genotype using several typing methods from Europe. These 8 strains were selected as they share 80% or more of identity with Beijing strains from Shanghai area, China. The rest of the strains, each representing a different cluster, were selected for its high number copies of IS6110 and were collected from Hospital Universitario Miguel Servet (HMS), Hospital Clínico Universitario LozanoBlesa (HCU) from Zaragoza and Hospital General San Jorge from Huesca. Finally, M. tuberculosis H37Rv and M. tuberculosis GC1237 were used as control strains. An internal control, HMS 1301, was included in the study due to its RFLP is identical to GC1237 strain. Mycobacterial strains were grown at 37ºC in Middlebrook 7H9 broth supplemented with ADC and 0.05% Tween 80 and genomic DNA of all of them was isolated as it was described in Chapter 1. Table 4 summarizes the clinical isolates used in this work and figure 39 shows the RFLP, the spoligotyping and the family of all of them. Location of the copies of IS6110 insertion sequence in the eight Beijing strains selected as representative strains of this genotype The study of the localization of the copies of IS6110 in the eight representative Beijing strains (NHN5, HM77, HM903, HM764, 990172, W4, N4 and CAM22) was carried out by two methods, both based on PCR. A first research was conducted by LMPCR as previously described in Chapter 1. Briefly, LMPCR was performed with SalI enzyme and the digestions were then subjected to PCR with the primers SalgD and ISA1 or ISA3 (Table 2 of Annex I). The amplified products were treated and sequenced and when a match was found, additional primers were designed and used with the eight strains to verify the point of insertion. These primers amplify the completed sequence of IS6110 and approximately 300bp of both flanking sequences. Secondly, it was performed PCRs with specific primers, previously designed for amplifying the locations of IS6110 in 210, W and GC1237 strains (Chapter 1). H37Rv and GC1237 were used as control strains in both cases. All this primers are collected in Table 5 of Annex I. Chapter III 135 characteristic of ancestral sublineages within the Beijing genotype (19). Following this classification, these eight strains are ancestral as the absence of IS6110 in this region. The majority of the IS6110 located copies were due to transposition mechanism The flanking regions of each copy of IS6110 of the eight Beijing strains were analyzed and with the exception of three locations of this element (indicated with minus in Table 5), the presence of direct repeats (DR) was detected (Table 5). The absence of DRs in two of the other three locations, RD152 and RD207, is probably a consequence of recombination between two adjacent copies of IS6110 (36) as it was observed loss of these genomic regions. The last of the three IS6110 elements without DR, which was localized between Rv0794c:Rv0797, was identical to GC1237 (explained in Chapter 1) and could be explained by reorganization of this region. This event could be characteristic of Beijing genotype. As already mentioned in Chapter 1, due to the high number of copies of IS6110 per strain, it was possible to observe copies flanked by DR and other copies without DR indicating that the probability of rearrangement process between copies rises when the number of those increases. Mª Henar Alonso Ezcurra 136 Table 5: Locations of IS6110 of the eight representative Beijing strains, part 1 Genes Points of insertion Direct Repeats 210 W GC1237 Rv0001(dnaA):Rv0002(dna N ) 1594 ATT + + + Rv0794c:Rv0797 (IS1547)889072 - + Rv0840c (pip) 937116 ACG + Rv1135c (PPE16) 1262963 AGC + + Rv1358:Rv1359 1530161 GCG Rv1359 1530203 CTC Rv1371 1543972 GAGG + + + Rv1469 (ctp D ) 1657016 CGT + + + Rv1527c (pks5) 1724406 CCGA Rv1528c (papA4) 1729409 CTT Rv1542c(glb N ):Rv1543 1745059 ACC Rv1754c 1986638 / + + Rv1754c-Rv1765c (RD152) 19866381998625 - + Rv1798:Rv1799 (lppT) 2038898 / + Rv1800 (PPE28):Rv1801 (PPE29) 2041741 GCG Rv1917c (PPE34) 2163392 TTA ++ 2163649 TCC + 2167310 / + + Rv2016 2263619 TCC 2263627 AGG + + + 2263778 CCT Rv2019 2266167 GAC MT2080 (Mb2047c):MT2081 (Mb2048c) 22687262268693 GAA + Rv2077A:Rv2078 2334666 AGG 2334686 AGG + Rv2104c:Rv2107 (PE22) 2366897 CAC + - / + Rv2107 (PE22):Rv2108 (PPE36) 2367679 CGA + Rv2180c 2442348 AGC + Rv2282c 2555718 AAT Rv2286c 2559506 ATC + Rv2332 (me z ) 2605438 ATT Rv2349c (plc C ) 2627829 CAG Rv2352c (PPE38) 2634022 / + + Rv2353c (PPE39):Rv2356c (PPE40) 26355922637688 CCG + Rv2356c (PPE40):Rv2357c(gly S ) 2639612 Rv2813-Rv2820c (RD207) 3127927 - + + + Rv2957 3310163 GTT Rv3018A (PE27A):Rv3019c (esx R ) 3378553 GCC + + + Rv3019c (esx R ): Rv3020c (esx S ) 3379027 CTG + + Rv3125c (PPE49) 3491592 CTC Rv3128c 3493908 CGA + + Rv3128c:Rv3129 - / + Rv3178:Rv3179 3547342 ATG + 3549199 CGG + + Rv3184:Rv3187 - / + + Rv3190c 3555513 CGC Rv3324A:Rv3327 (IS1547)3711737 GGC + + + Rv3382c (lytB1) 3796689 TGG Rv3383c (idsB) 3797823 ATC + + + Rv3427c (IS1532):Rv3428c (IS1532) 3844681 CGGG + + + 3844737 CCCG + Chapter III 137 Table 5: Locations of IS6110 of the eight representative Beijing strains, part 2 Strains NHN5 HM764 990172 W4 N4 HM903 HM77 CAM22 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Mª Henar Alonso Ezcurra 138 Five of the eight Beijing strains have a possible IS-promoter The orientation and distance of each copy of IS6110 to the neighbouring genes was analyzed and in addition to the locations of IS6110 described in Chapter 1 where could act as a promoter, 5 new copies were located with the same orientation and close enough to neighbouring genes to act as a mobile promoter (Table 6). Table 6: New locations of IS6110 of the eight representative Beijing strains where IS6110 could act as a mobile promoter Strain Genes of IS6110 Distances to neighbouring genes NHN5 Rv1358:Rv1359 12 bp upstream Rv1359 HM764 PPE40:glyS 77 bp upstream PPE40 990172 Rv2077A:Rv2078 92 bp upstream Rv2077A W4 Rv1542c:Rv1543 5 bp upstream Rv1543 N4 Rv2016 370 bp upstream Rv2017 It is probably that the IS6110 located upstream Rv1359 gene in NHN5 and the copy located upstream Rv1543 in W4 do not act as a mobile promoter due to the distance to these gene is too short however, to confirm this, the effect of this element in these genes should be studied. Finally, it is noteworthy that these IS6110 were observed only in one strain and all of them were different from each indicating the genomic variability that IS6110 generates among strains of a same genotype. Randomly distribution of IS6110 in reference sequenced strains of GeneBank The analysis of the points of insertion of IS6110 of 43 reference sequenced strains of GenBank was carried out comparing their sequenced fragments with H37Rv genome. All the genomic locations of IS6110 of these strains were plotted on the H37Rv genome map (Figure 41). The IS-locations were grouped in two circles; the first one corresponds to Beijing strains and another one to non-Beijing strains. In this DNA plotter the locations of IS6110 of GC1237 (red circle) and of H37Rv (green circle) were also included. A randomly distribution of this element throughout the genome was observed among these strains (Figure 41). However, the presence of numerous preferential integration loci of IS6110 were also detected (some examples are indicated with black arrows in Figure 41). Beijing and no-Beijing strains presented copies of IS6110 in these hot-spots corroborating that preferential integration regions are common among M. tuberculosis Chapter III 139 strains. In all the strains this element was found at an identical position within the DR region (with the exception of the Beijing strains which presented their characteristic deletion: RD207, so, characteristic point). Moreover, in all Beijing strains it was observed an identical copy of this element between dnaA:dnaN genes. In this region but in a different point, a copy of IS6110 in a non-Beijing strain (SUMu003) was observed indicating that the presence of an IS6110 in this intergenic region is characteristic of Beijing genotype but not exclusive of this family. This result is in agreement with studies which show different locations of IS6110 between these two genes (8, 20, 37). The detection of Beijing strain amplifying this entire intergenic region is a useful tool but it is necessary another genomic feature of this genotype if not, the result could be a Beijing false positive. Figure 41: Graphic representation of point of insertion of IS6110 in M. tuberculosis genome with Artemis DNA plotter. Distribution of insertion loci identified with the sequence analysis of genomic database in this work. The first circle (green) represents the genomic locations of IS6110 in H37Rv. The second concentric circle represents the distribution of this sequence in non-Beijing strains. The third circle represents the locations of IS6110 in Beijing strains and the fourth concentric circle (red) correspond to the genomic points of insertion of IS6110 in GC1237 strain. All the IS-locations were plotted on H37Rv genome. The hot-spots: IS1547, plcD region, DR region and dnaA:dnaN region are indicated by arrows. In addition, the unique IS6110 of GC1237 (upstream Rv2180c) is indicated. Mª Henar Alonso Ezcurra 140 The copy of IS6110 located upstream Rv2180c gene in GC1237 was also indicated in the M. tuberculosis map with an arrow (Figure 41) and, as it is observed, there was no strain with this location confirming again that this location is unique of this strain. It is noteworthy that this sequence was found inserted more often in some genomic regions, as the one from 1800000 to 2700000 (coordinates referred to H37Rv genomic map, Figure 41); whereas other regions lacked the presence of IS6110 as happens in the region around the OriC. Probably, these areas could be more abundant in essential genes. In fact, although an IS6110 transposes in these regions, if they are essential, the outcome of this event will not be observed. These findings are in agreement with previous studies of chromosomal distribution of IS6110 (29, 30, 41). It has been included a detailed table in Annex II with all the points of insertion of IS6110 of the analyzed reference strains of GeneBank. A primer collection which amplify locations of IS6110 in MTBC strains was generated in this study Available literature (1, 4, 10-12, 18, 20, 23-25, 28-30, 33, 39-41) was also reviewed and when a location of IS6110 in a Beijing strain was not included in the amplified regions of the primer collection new pair of primers were designs to amplify the area. In addition, as one of the objectives was to detect whether the preferred site of insertion of IS6110 in LCS were present or absent in the selected HCS, the DK regions (12) were analyzed and when necessary, primers were designed and included in the oligonucleotide collection. Moreover, the primers used to amplify preferred locations of IS6110 in M. bovis with human host (25) were included to check whether these locations are also detected in the selected M. tuberculosis clinical isolates as the host in both cases is the same. Finally, as one of the interesting IS6110 was the located upstream phoP gene in MBZ strain and it was quiet studied in Chapter 2, the primers BCG2A and BCG2B which amplify the promoter region of this gene (33) were included with the aim of checking the frequency with which IS6110 is inserted in this region. Figure 42 shows the distribution of the amplified regions with this collection and a detailed table has been included in Annex I (Table 5 of this Annex). Chapter III 141 Figure 42: Distribution of the genomic regions which can be amplified with the primer collection generated in this work (Table 5 of Annex I) plotted in M. tuberculosis H37Rv genome. Mapping IS6110 locations in 61 clinical isolates of M. tuberculosis With the aim of obtaining a global view of the distribution of IS6110 throughout the genome of M. tuberculosis and compare them between Beijing and non-Beijing strains, a scrutiny of points of insertion of IS6110 was performed with 61 clinical isolates of M. tuberculosis. These strains were selected for its high copy number of IS6110 (HCS). Seventeen were Beijing strains and, as it has been explained above, eight of them were previously selected as representatives of this genotype. HMS1301, which presents identical RFLP as GC1237, was used as internal control and H37Rv and GC1237 strains were used as external controls. The study was carried out by PCR using all the primer collection (Figure 42 and Table 5 of Annex I) and when a match was found, the fragment was sequenced with primers directed outwards (Table 5 of Annex I). By analysis of the sequences, it was obtained the point of insertion, the DR, the orientation and the distance to neighbouring genes of each copy of IS6110 (Table 7). After this analysis, all the locations were plotted in the H37Rv genomic map and represented with DNA plotter in two different circles, the red one corresponds to points of insertion of IS6110 in Beijing strains and the blue one to non-Beijing strains. The locations of IS6110 in GC1237 were included in a separated circle (green) as reference points of insertion (Figure 43). Mª Henar Alonso Ezcurra 142 A total of 160 different points of insertion of IS6110 were obtained (Table 7) with the primer collection (Table 5 of Annex I). It is stood out that several genes or intergenic regions were observed interrupted by this element in different points and in both orientations. Considering this fact, a total of 81 different regions were disrupted or altered by IS6110 (Table 7). By this method, the common locations of IS6110 of these strains were located; the fact that the number of copies of IS6110 located in the group of non-Beijing strains is much lower (1 to 4) than the group of Beijing strains (at least 10) may indicate that Beijing genotype evolved separately from a common precursor at an early stage. Thus, the parental Beijing strains should carry low copy number of IS6110 (common with other lineages) and the descendants, more evolved, would carry high copy number. Copies of IS6110 were detected randomly around the genome of these strains with some exceptions. One of them is the region around OriC, which lacks this element, corroborating the importance of keeping this region intact. In both groups of strains (Beijing and non-Beijing), this element was observed inserted in the general hot-spots such as ipl loci or plc regions confirming again that IS6110 has preferential sites. All the strains presented one copy of IS6110 in the DR region and two copies were obtained in one of the Beijing strains (CAM22) as explained above in this Chapter. The 90.9% of the non-Beijing strains presented the same point of insertion in this region; however, it is noteworthy that in the remaining non-Beijing strains this copy is located in the DR region but in another point, different even among these four strains. The insertion of IS6110 in the possible IS6110 Beijing-hot-spot RD152 was also observed in non-Beijing strains (plcD region) indicating that this region is not exclusive of Beijing genotype (Figure 43 and Table 7). However, comparing these IS6110 points of insertion, it was observed that only Beijing strains presented the deletion of the region of difference RD152. IS-locations within Rv1371, Rv2016, ctpD, idsB genes and between the two IS1532, were only observed in Beijing strains (Figure 43 and 44 and Table 7) indicating again that these regions could be specific hot-spots of Beijing genotype (IS6110 Beijing-hot-spots). These locations could be the key of some advantages of the Beijing genotype and its study should be taken into account in further research. According to Fomukong et al. (12) and after the analysis done in this study, among the different Beijing strains analysed until now, no IS6110 has been detected in the DK1 locus (mmpS1 gene). However, near to the 15% of the group non-Beijing strains presented a copy of IS6110 in this gene (Table 7). Specifying more, 6 of them presented an IS6110 at the exact DK1 point of insertion (12). This point is the preferred site of IS6110 in LCS and the authors defend the idea that its prevalence decreases in Chapter III 143 HCS suggesting a separate lineage for HCS and LCS (12). The fact that the 15% of the studied HCS strains present this copy could indicate that the increment in number of copies of IS6110 is the result of the transposition of another IS6110 of their genomes. Figure 43: Distribution of IS6110 thorough the M. tuberculosis genome of the 61 clinical isolates. The obtained locations of IS6110 of each strain were plotted in M. tuberculosis H37Rv genome and represented in three concentric circles. The blue one corresponds to the ISlocations in non-Beijing strains. The red one represents the IS-locations in Beijing strains and the green one corresponds to the locations of IS6110 in GC1237 strain. The general hot-spots of M. tuberculosis are indicated by blue arrows and with black arrows it has indicated the possible specific hot-spots of Beijing genotype. Mª Henar Alonso Ezcurra 144 Figure 44: IS6110 distribution of the 17 Beijing strains. The IS6110 obtained locations in the 17 Beijing strains were plotted in M. tuberculosis H37Rv genome and represented each one in a concentric circle. The IS6110 locations of GC1237 were included as control. The general hotspots are indicated by blue arrows and with black arrows the IS6110-Beijing-hot-spots. Two of the sixty-one strains presented a copy of IS6110 in the promoter region of phoP gene (Table 7). One copy was located 132 bp upstream phoP gene (HSJ067 Beijing strain) but in opposite orientation to phoP, and the other one was located 196 bp upstream this gene (HMS2405 non-Beijing strain) and in the same orientation as phoP gene. Although this point is different of the present in MBZ, this last strain could be a possible candidate for studying the effect of IS6110 in phoP gene in a sensible M. tuberculosis strain.