Engineering of specific bacteriophages for α-Synucleinopathies diagnosis
Abstract
As α-sinucleinopatias são doenças neurodegenerativas caracterizadas por depósitos anormais de agregados de α-sinucleína em neurónios ou células gliais, capazes de provocar demência e perturbações do movimento, especialmente em pessoas idosas. De acordo com a ordem de prevalência, as três principais α-sinucleinopatias são a doença de Parkinson, demência com corpos de Lewy e atrofia de múltiplos sistemas. A acumulação de α-sinucleína nos neurónios, resulta nos chamados corpos de Lewy, que são a marca patológica da doença de Parkinson e demência com corpos de Lewy, enquanto a acumulação de α-sinucleína nas células oligodendrogliais dá origem às chamadas inclusões citoplasmáticas gliais, que são a principal característica da atrofia de múltiplos sistemas. Atualmente, estas α-sinucleinopatias não têm qualquer tipo de tratamento ou diagnóstico precoce, pelo que o desenvolvimento de abordagens para diagnóstico e terapêutica são cruciais. Uma vez que a agregação de α-sinucleína é uma característica de todas estas doenças neurodegenerativas, as estratégias que tenham este biomarcador como alvo podem tornar-se terapias promissoras. No entanto, o desenvolvimento de novas estratégias de tratamento e/ou diagnóstico é limitado pela barreira hematoencefálica que reveste o sistema nervoso central. Posto isto, a presente proposta teve como principal objetivo desenvolver um sistema baseado em bacteriófagos (vírus de bactérias) capazes de atravessar a barreira hematoencefálica para diagnosticar α–sinucleinopatias, precocemente. Neste trabalho, bacteriófagos filamentosos M13, foram manipulados geneticamente para fazerem o display à superfície de péptidos (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA e 4554WN6A: KDGIVAGVKA) potencialmente capazes de reconhecerem e se ligarem à α-sinucleína. Os fagos de interesse foram obtidos com sucesso por intermédio da combinação do DNA de um fagemídeo modificado com o DNA do fago aulixiar M13KO7. De modo a avaliar a afinidade dos fagos artificiais com a região 45-54 da α-sinucleína e a caracterizar morfologicamente os fagos aquando desta interação, ensaios de caracterização foram realizados. Os resultados obtidos apenas evidenciaram que os fagos exibiam o respetivo péptido de interesse, e que a nível físico e químico apresentaram um tamanho e uma carga condizente com o que era esperado. Resumidamente, pode dizer-se que os fagos foram obtidos com sucesso, o que abre portas a novas linhas de estudo futuras. Esta ferramenta será um ponto de partida para o diagnóstico precoce e para, possivelmente, um tratamento capaz de travar a progressão destas doenças.
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UMinho | 2022 Adriana Sofia Veloso Rodrigues Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis October 2022 Adriana Sofia Veloso Rodrigues Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis
i Adriana Sofia Veloso Rodrigues Engineering Of Specific Bacteriophages For αSynucleinopathies Diagnosis Master Dissertation Master’s Degree in Biomedical Engineering Clinical Engineering Branch Project supervised by Doctor Ivone Marisa Pereira Martins October 2022
ii DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-CompartilhaIgual CC BY-NC-SA https://creativecommons.org/licenses/by-nc-sa/4.0/
iii AGRADECIMENTOS Dizem que todos nós somos um pedacinho de todas as pessoas que passam na nossa vida. De uma maneira ou de outra, as pessoas deixam a sua pegada em nós e transformam-nos sem que às vezes nos apercebamos disso. Sem dúvida que ao longo desta longa viagem passaram por mim pessoas que deixaram a sua marca, e o mínimo que lhes devo é um “obrigada”, por isso, as palavras que se seguem são dedicadas a elas. Embarquei neste projeto há um ano e com ela aprendi imenso… este parágrafo dedico-o à minha orientadora, Ivone Martins. Forneceste-me todas as bases e suporte para levar esta missão até ao fim. Deixasteme “crescer” mostrando-me que errar faz parte e que o importante é tentar e não desistir. Obrigada por tudo… “You are needed” for me. Dedico aqui também, um agradecimento ao Artur Ribeiro por todo o apoio e assistência técnica neste trabalho e ao Ricardo Pires pela assistência com o microscópio de força atómica. E porque por trás de um trabalho está sempre uma grande equipa, chegou a altura certa de agradecer a todo o grupo L-Phage. A todos eles um enorme obrigada por me terem acolhido tão bem. Queria apenas deixar um agradecimento especial àqueles que acompanharam mais de perto e que foram peças fundamentais nesta jornada. Um obrigada à Ana Brandão, ao Alex, à Cátia, ao David, à Daniela, à Jonhy, à Licinha, à Lu, à Marta, à Rita, à Rute e, por fim, um obrigada à pessoa que fez “babysitting” nas suas horas vagas, Maria, a ti o meu obrigada… vocês são o que de melhor levo desta experiência. Foi um privilégio fazer parte desta equipa. L-Phage vocês são incríveis. Às duas meninas que trilharam estes 5 anos lado a lado comigo, obrigada. Por meio de lágrimas e sorrisos juntas encontramos as saídas de todos os labirintos. Mary e Bru, vocês são muito importantes para mim… que continuemos a brilhar juntas, sempre! Para aqueles que vibram com as minhas conquistas como se fossem as deles, para aqueles que mesmo estando a quilómetros de distância, nunca se esquecem de mim; para aqueles que sempre me apoiam incondicionalmente… No vosso olhar vejo o orgulho que sentem por mim, e por isso um obrigada nunca será suficiente. Simplesmente, sou uma afortunada por pertencer à família que pertenço. O agradecimento mais significante de todos, vai para aqueles que me permitem todos os dias voar em diferentes direções. Mãe e Pai, obrigada! Obrigada por me deixarem ser a sombra mais bonita do vosso reflexo. Termino com o agradecimento àquele que vive na contradição comigo, mas que apesar de não demonstrar sei que torce por mim… amor de irmãos é isso mesmo. “The roots of work may be bitter, but the fruit is sweet” by fortune cookie, 2022
iv STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledge the Code of Ethical Conduct of University of Minho.
v Construção De Bacteriófagos Específicos Para O Diagnóstico de α-Sinucleinopatias RESUMO As α-sinucleinopatias são doenças neurodegenerativas caracterizadas por depósitos anormais de agregados de α-sinucleína em neurónios ou células gliais, capazes de provocar demência e perturbações do movimento, especialmente em pessoas idosas. De acordo com a ordem de prevalência, as três principais α-sinucleinopatias são a doença de Parkinson, demência com corpos de Lewy e atrofia de múltiplos sistemas. A acumulação de α-sinucleína nos neurónios, resulta nos chamados corpos de Lewy, que são a marca patológica da doença de Parkinson e demência com corpos de Lewy, enquanto a acumulação de α-sinucleína nas células oligodendrogliais dá origem às chamadas inclusões citoplasmáticas gliais, que são a principal característica da atrofia de múltiplos sistemas. Atualmente, estas α-sinucleinopatias não têm qualquer tipo de tratamento ou diagnóstico precoce, pelo que o desenvolvimento de abordagens para diagnóstico e terapêutica são cruciais. Uma vez que a agregação de α-sinucleína é uma característica de todas estas doenças neurodegenerativas, as estratégias que tenham este biomarcador como alvo podem tornar-se terapias promissoras. No entanto, o desenvolvimento de novas estratégias de tratamento e/ou diagnóstico é limitado pela barreira hematoencefálica que reveste o sistema nervoso central. Posto isto, a presente proposta teve como principal objetivo desenvolver um sistema baseado em bacteriófagos (vírus de bactérias) capazes de atravessar a barreira hematoencefálica para diagnosticar α–sinucleinopatias, precocemente. Neste trabalho, bacteriófagos filamentosos M13, foram manipulados geneticamente para fazerem o display à superfície de péptidos (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA e 4554WN6A: KDGIVAGVKA) potencialmente capazes de reconhecerem e se ligarem à α-sinucleína. Os fagos de interesse foram obtidos com sucesso por intermédio da combinação do DNA de um fagemídeo modificado com o DNA do fago aulixiar M13KO7. De modo a avaliar a afinidade dos fagos artificiais com a região 45-54 da α-sinucleína e a caracterizar morfologicamente os fagos aquando desta interação, ensaios de caracterização foram realizados. Os resultados obtidos apenas evidenciaram que os fagos exibiam o respetivo péptido de interesse, e que a nível físico e químico apresentaram um tamanho e uma carga condizente com o que era esperado. Resumidamente, pode dizer-se que os fagos foram obtidos com sucesso, o que abre portas a novas linhas de estudo futuras. Esta ferramenta será um ponto de partida para o diagnóstico precoce e para, possivelmente, um tratamento capaz de travar a progressão destas doenças. Palavras-chave: α-sinucleína, α-sinucleinopatias, bacteriófagos.
vi Engineering of specific bacteriophages for α-synucleinopathies diagnosis ABSTRACT α-synucleinopathies are neurodegenerative diseases that primarily affect aging people. These diseases are characterized by abnormal deposits of α-synuclein aggregates in neurons or glial cells, which cause dementia and movement disorders. In line with the order of prevalence, the three main αsynucleinopathies are Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The accumulation of α-synuclein in neurons results in the so-called Lewy bodies, the hallmark of Parkinson's disease and dementia with Lewy bodies. In contrast, the buildup of α-synuclein in oligodendroglial cells gives rise to the so-called glial cytoplasmic inclusions, which are the defining characteristic of multiple system atrophy. Currently, these α-synucleinopathies are not early diagnosed and do not have any treatment, so the development of diagnostic and therapeutic approaches is crucial. Since α-synuclein is a pathological hallmark of all these neurodegenerative diseases, the development of strategies with this biomarker as a target can be powerful therapies. However, the development of new treatment and/or diagnostic approaches is limited by the blood-brain barrier that shelters the central nervous system. Therefore, the present proposal aimed to develop a system based on bacteriophages (bacterial viruses) able to cross the blood-brain barrier to early diagnose α-synucleinopathies. In this work, filamentous bacteriophages M13 were genetically manipulated to display at the surface peptides (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA, and 4554WN6A: KDGIVAGVKA) potentially capable of recognizing and binding to α-synuclein. The phages of interest were successfully obtained by combining DNA from a modified phagemid with DNA from the helper phage M13KO7. In order to evaluate the affinity of the engineered phages for the 45-54 region of α-synuclein and to morphologically characterize the phages at the time of this interaction, characterization assays were performed. The results obtained showed that the phages exhibited the respective peptide of interest, and that at the physicochemical level presented a size and charge consistent with what was expected. In summary, it can be said that the phages were successfully obtained, which opens the door to new future lines of study. This tool will be a stepping-stone for early diagnosis and possibly a treatment capable of halting the progression of these diseases. Keywords: α-synuclein, α-synucleinopathies, bacteriophages.
vii INDEX RESUMO ........................................................................................................................................... v ABSTRACT ....................................................................................................................................... vi LIST OF ILLUSTRATIONS ................................................................................................................ x LIST OF TABLES ............................................................................................................................ xiii LIST OF ABBREVIATIONS AND ACRONYMS .............................................................................. xiv 1. INTRODUCTION .................................................................................................................... 18 1.1. α-Synucleinopathies ..................................................................................................... 18 a) Parkinson´s disease .................................................................................................. 18 b) Dementia with Lewy bodies ........................................................................................ 19 c) Multiple System Atrophy ............................................................................................ 20 1.2. α-Synuclein .................................................................................................................... 21 1.3. Blood-Brain Barrier ....................................................................................................... 23 1.4. Bacteriophages ............................................................................................................. 24 1.4.1. Phage Display technology .................................................................................... 25 a) M13 Phage Genetic Manipulation .................................................................................. 29 b) M13 Phage Chemical Manipulation ................................................................................ 29 c) M13 Phage Applications ................................................................................................ 30 1.5. Peptide Targeting ......................................................................................................... 32 2. MOTIVATION AND OBJECTIVES ......................................................................................... 35 3. MATERIAL AND METHODS .................................................................................................. 36 3.1. Development of phagemids ......................................................................................... 36 3.1.1. Insert synthesis ...................................................................................................... 36 3.1.2. Cloning process of the inserts into the phagemid ............................................ 38 a) Digestion Reaction ......................................................................................................... 38 b) Ligation ......................................................................................................................... 39
xiv LIST OF ABBREVIATIONS AND ACRONYMS A Aβ: Amyloid-β APS: Ammonium persulfate AS: α-Synuclein B BBB: Blood-brain barrier C CaCl2: Calcium chloride CEA: Carcinoembryonic antigen CECs: Cerebral endothelial cells CED: Convection-enhanced delivery CNS: Central nervous system CRC: Colorectal cancer D DLB: Dementia with Lewy bodies DLS: Dynamic light scattering DMPS: 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine DNA: Deoxyribonucleic acid DOX: Doxorubicin E E. coli : Escherichia coli EDTA: Ethylenediamine tetraacetic acid F FA: Folic acid
xv FDG-PET: F-fluorodeoxyglucose positron emission tomography Ff phage: Filamentous phage FR: Folate receptor H HCl: Hydrogen chloride G GCIs: Glial cytoplasmic inclusions GM-CSF: Granulocyte-macrophage colony-stimulating factor I IG region: Intergenic region IPTG: Isopropyl β-D-1-thiogalactopyranoside IEP: Isoelectric point L LB: Luria-Bertani medium LBs: Lewy bodies M MgCl2: Magnesium chloride MRI: Magnetic resonance imaging MSA: Multiple system atrophy N NAC: Non-amyloid-component NaCl: Sodium chloride P PCA: Protein-fragment complementation assay
xvi PCR: Polymerase chain reaction PD: Parkinson’s disease PBS: Phosphate buffered saline PDT: Photodynamic therapy PEG: Poly(ethylene glycol) PET: Positron emission tomography p-S129: Serine residue-129 R RNA: Ribonucleic acid S SBP: SPARC-binding peptide scFv: Single-chain variable fragments SDS-PAGE: Sodium dodecyl sulfatepolyacrylamide gel electrophoresis SEM: Standard error of the mean SNARE: Soluble N-ethylmale-imide-sensitive factor-attachment protein receptors SNCA: Synuclein-alpha gene SOC: Super optimal broth with catabolite repression SPARC: Secreted protein, acidic, and rich in cysteine SPECT: Single-photon emission computed tomography ssDNA: Single-stranded DNA STEM: Scanning Transmission Electron Microscopy T TBS: Tris-buffered saline TE: Tris-EDTA TEM: Transmission electron microscopy TEMED: Tetramethylethylenediamine TGS: TrisGlycineSDS
xvii W WT: Wild type Z ZnPc: Zinc phthalocyanine
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 18 1. INTRODUCTION 1.1. α-Synucleinopathies In the last two decades α–synuclein (AS) has come into special focus. It is considered the protein responsible for triggering certain pathologies that cause dementia and movement disorders, which have a high incidence in the population over the age of 50-65 years old. Thus, in order of prevalence, Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are neurodegenerative diseases that belong to the family of α-synucleinopathies since they are characterized by abnormal deposits of AS aggregates in neurons or glial cells [1] – [5]. AS aggregation in the neurons results in abnormal deposits called Lewy bodies (LBs). LBs are present in many regions of the brain (neocortex, forebrain, brainstem and other parts of the nervous system essentially in substantia nigra pars compacta ), leading to chemical modifications that might induce problems with thinking, behavior, mood and movement. LBs are spherical (5-25 µm in diameter) intraneural cytoplasmic inclusions, mostly consisting of AS protein aggregates surrounding their dense nucleus. Moreover, they are a pathological hallmark of PD and DLB [3], [6] – [8]. Nonetheless, they can be distinguished by the LBs distribution. LBs are localized in the mid-brain, more precisely in the substantia nigra in PD, and in cerebral cortex area in DLB [9], [10]. However, the accumulation of AS in oligodendroglial cells, present in the central nervous system (CNS), is known as glial cytoplasmic inclusions (GCIs). These inclusions, which are argyrophilic fibrillary structures composed of misfolded α-synuclein, are the most distinguishing feature of MSA [3], [11], [12]. a) Parkinson´s disease Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer´s disease, affecting more than 6 million people, being this number expected to double by 2050 [13], [14]. Statistical data show that since 1990, PD cases have almost tripled, as from 2.5 million people diagnosed with PD to a total of 6.1 million people in 2016. In 2019, more than 8.5 million individuals worldwide were living with PD, representing an even larger population than the previously reported cases of PD in 2016 [15], [16]. This α-synucleinopathy is characterized by a massive loss of dopaminergic neurons in the substantia nigra , which leads to the appearance of the four classic motor symptoms of this disease: stiffness, gait disturbance with postural instability, rest tremor and bradykinesia
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 19 (difficulty in initiating movements, which in turn are slow). The main risk factor of PD is age, but environmental factors can also be associated. In terms of gender, this disease has a 1.5–2 times higher prevalence and incidence in men than in women. In addition, women tend to demonstrate mild symptoms with a slower progression of motor disorders but have a higher rate of tremor, about 67% vs 48% in men [17]–[21]. Nowadays, only 4% of diagnoses are made in patients under 50 years of age [14]. The diagnosis of Parkinson's disease is made on the basis of clinical symptoms. Nevertheless, to rule out other conditions, magnetic resonance imaging (MRI) is often used to evaluate the anatomy and structural pathology of the brain so that patterns of structural degradation can be found to induce a correct diagnosis. In addition to MRI, other imaging studies such as single-photon emission computed tomography (SPECT) can be performed. This technique is useful for assessing the integrity of the nigrostriatal dopaminergic pathways, which in cases of PD are found to be dysfunctional. Moreover, laboratory tests can also help diagnose PD, although there are still no specific tests for this disease [20], [22]. Currently, no neurotherapy can treat, slow down and/or prevent this chronic disease. Although there is still no cure for Parkinson's disease, there are some drugs to alleviate some of the symptoms. The drugs are used to increase the level of dopamine in the brain (e.g. Levodopa), affect other neuronal chemicals (e.g. Entacapone), or help control tremor symptoms (e.g. Benzotropine) [20]. b) Dementia with Lewy bodies Worldwide, many cases of dementia are diagnosed every day. Dementia is defined as a loss of cognitive ability that negatively impacts the normal functions of people's daily routines. There are many kinds of dementia, namely dementia with Lewy bodies (DLB), a progressive disease, which means that symptoms start slowly and worsen over time. After Alzheimer´s disease, it is the second most common form of dementia accounting for 20% of dementia worldwide, mainly in the age group of 65 years and older, with males being more affected [2], [23]–[27]. At the symptomatic level, DLB causes disturbances in attention, movement, visuospatial skills, and memory recall. Patients with DLB commonly present psychiatric and behavioral dysfunctions such as sleep disturbances, hallucinations, and apathy. In cases of DLB, cognitive symptoms precede motor symptoms [20], [23]. Diagnosing DLB has been a challenge, but this is essentially done by considering the symptom characteristics. Symptomatic analysis can be supplemented with MRI to assess whether or not there is
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 20 brain atrophy, or it can be further aided by other imaging studies such as positron emission tomography (PET) and SPECT. PET is used to assess biochemical processes; thus, both PET and SPECT are used to evaluate the integrity of the nigrostriatal dopaminergic pathways to check whether or not dopamine transporter uptake is being reduced in the brain. Since in DLB it has been identified that occipital hypometabolism exists, PET can also be used to assess possible brain metabolism impairments using Ffluorodeoxyglucose (FDG), a radiopharmaceutical specific for glucose transport and metabolism. To date, there are still no laboratory tests that help diagnose DLB because no particular marker has yet been found for this disease [20], [24], [26]–[28]. Some medications, such as Levodopa are used to relieve the symptoms of DLB, but there is still no treatment that can stop disease progression [3], [20]. Even though this disease is the second most common form of neurodegenerative dementia, it is often misdiagnosed, under-researched, under-recognized, and undertreated [29]. c) Multiple System Atrophy Multiple system atrophy (MSA) is a rare, poorly characterized, fatal neurodegenerative disease with an incidence rate of 3 cases per 100,000 person-years over the age of 50 [20]. This α - synucleinopathy is highly progressive and affects multiple neurological systems including cognitive, autonomic, cerebellar and motor systems. Thus, this disease presents a wide range of symptoms, including respiratory failure, urogenital disorder, urinary incontinence, constipation, erectile dysfunction, orthostatic hypotension, respiratory stridor, sweat gland dysfunction, and motor problems similar to PD [3], [20], [30]–[32]. As with the other two α-synucleinopathies, the diagnosis is made using symptomatic tests. The use of MRI is helpful to see whether or not certain areas of the brain have characteristic atrophies of MSA. Patients with MSA are shown to have regional hypometabolism in the striatum , brainstem, and cerebellum, so performing an FDG-PET may help in the diagnosis. In contrast to DLB and PD, the use of presynaptic dopamine PET or SPECT imaging is not very helpful. The use of laboratory tests to confirm this disease does not yet exist [20]. Despite the existence of some treatments to alleviate bothersome symptoms, no effective therapy for MSA exists because the mechanism of disease progression is unknown [3], [20].
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 21 Of the three α-synucleinopathies mentioned, the first two are the most problematic, given their statistical values. However, for none of the three, there is an early diagnosis or any type of treatment that allows the cure or prevents disease progression. In this way, given that AS aggregation is a pathological hallmark of all these neurodegenerative diseases, the focus on developing strategies that target this protein aggregates may have a promising future as potential diagnostic/therapeutic approach. 1.2. α-Synuclein α-synuclein (AS) is a protein that belongs to a small family of the three synuclein proteins: αsynuclein, β-synuclein and γ-synuclein. It is composed of 140 amino acids encoded by the synuclein alpha gene (SNCA) localized on chromosome 4q21-q23, and presents a high solubility, which allows it to be easily incorporated into cell membranes. It is characterized as a natively unfolded structure and screens two different structural conformations: a disordered monomer or an α-helical multimeric shape (Figure 1). The monomers may first associate into oligomeric species (toxic forms) that subsequently progress to fibrillar aggregated forms (with β-sheet conformation), culminating in fibrillar amyloid forms, which are present in Lewy bodies. Thus, the misfolding and consequently aggregation of the monomers is a key factor in the formation and progression of the neurodegenerative diseases known as αsynucleinopathies [21], [33]. Figure 1. The α-synuclein aggregation process (adapted from [21]). Created in BioRender.com. In addition, this protein contains three distinct structural domains (Figure 2A). The N-terminal domain (residues 1–60), which is overall positively charged, has a high content of hydrophobic amino acids. It is an amphipathic lysine-rich region characterized by repetitions of the six lysine-rich highly Membrane-bound helically folded monomer Unfolded monomer ↔ Oligomer Fibrils Lewy body
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 22 conserved motifs KTK(E/Q)GV, that play an important role in lipid binding and α-helix formation. This interaction of AS with membrane lipids is becoming an increasingly attractive area for understanding the mechanisms of AS aggregation, as lipids are being identified as elements with a high preponderance in enhancing AS aggregation. The AS-lipid interaction can lead to the primary nucleation, which leads to the formation of toxic forms of AS - the oligomers, which consequently lead to the formation of fibrillar AS (Figure 2B) [13], [34]–[36]. Moreover, that region, essential between residues 45-54, has particular importance since it is in this domain where are localized all known clinical mutations that are associated with the cases of DLB and PD; therefore, these mutations are an important element to promote and increase aggregation of α-synuclein. The central hydrophobic region that is rich in hydrophobic motifs comprises the non-amyloid-component (NAC) (residues 61–95) and residues 71–82 (VTGVTAVAQKTV), crucial for AS aggregation (mainly its fibrillation). Lastly, the C-terminal region (residues 96–140), a negatively charged region, is highly acidic and proline enriched and is involved in the protein’s chaperonelike activity [8], [21], [37]–[40]. Figure 2. α-synuclein structure and aggregation process. [A] Native sequence of α-synuclein: clinical mutations (Ala30Pro, Glu46Lys, His50Gln, Gly51Asp, Ala53Thr, Ala53Val, and Ala53Glu) marked in highlighted orange; the amphipathic N-terminal region contains six imperfect lysine-rich highly conserved motif repeats, marked in purple; the central hydrophobic region is underlined and the hydrophobic region is marked in yellow; the two major phosphorylation sites (Ser87 and Ser129) are coloured in pink, the chaperone-mediated autophagy recognition sites are marked in dark blue, and nitration sites (Tyr39, Tyr125, Tyr133, and Tyr136) are shown in light blue (adapted from [21]); [B] The α-synuclein aggregation mediated by the interaction α-synuclein-lipid [34]. AS is predominantly located at the presynaptic terminals of neurons, and although its function in the body is not yet fully understood, it is believed to be involved in regulating synaptic vesicle trafficking in the healthy brain via direct interaction with lipid membranes. This function is essential for assembling the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes because these are crucial for releasing neurotransmitters at presynaptic nerve terminals, for instance, dopamine (which plays a relevant role in motivation, attention, regulating body movements, memory, and cognitive [A] M1DVFMKGLSKAKEGVVAAAEKTKQGVAEAA30GKTKEG VLYVGSKTK45EGVVHGV52AT54V55AEKTK60EQVTNV GGAVV71TGVTAVAQKTV82EGAGSIAAATGFV95KKDQLG KNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPE A140 [B]
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 23 functions). This highlights the fact that AS is a preponderant factor in neurodegeneration [8], [21], [41], [38]. The misfolding and aggregation in vitro of AS is conditioned by several conditions such as increased temperature; lipids with high solubility in aqueous solution and short hydrocarbon chains; molecular crowding; acidic pH; heparin; divalent and trivalent metal ions such as aluminum, copper (II), iron (III), cobalt (III) and manganese (II); organic solvents; and other glycosaminoglycans, polycations, pesticides and α-synuclein binding proteins. Furthermore, missense mutations (changing a nucleotide gives rise to a codon that encodes a different amino acid) and post-translational modifications of AS, like phosphorylation, ubiquitination, nitration, sumoylation, truncation, and N–terminal acetylation, are broadly implicated in the process of α-synuclein aggregation and neurotoxicity. This occurs because the accumulation of the AS aggregates in presynaptic terminals of the axons turn the cells more sensitive to a variety of toxic injuries [8], [38], [42]. The most ordinary post-translational modification is phosphorylation, as about 90% of the AS aggregates in Lewy bodies are phosphorylated on the serine residue-129 (p-S129). The ubiquitination in lysine residues (mainly at K6, K10, and K12 residues) and the nitration in tyrosine (Y39, Y125, Y133, and Y136), are the other two most common post-translational modifications [8], [38]. In this way, it is notorious that AS has a fundamental role in the development of αsynucleinopathies. 1.3. Blood-Brain Barrier Hereupon, it is crucial to create strategies for treatment and/or diagnosis of α-synucleinopathies. However, the transport of most therapeutic molecules to the brain is blocked by the existence of a physical, metabolic, transport and immunological barrier, which maintains homeostasis between the blood and the brain. This natural barrier, which protects the central nervous system (CNS), is called bloodbrain barrier (BBB) and represents a significant challenge for developing approaches for treatment and/or diagnosis of neurological diseases. BBB is a highly selective semipermeable barrier that controls the influx and efflux of different substances. This barrier only enables a specific exchange of nutrients, ions, metabolic waste products, minerals and signaling molecules between the blood and the brain. It also has the aim of protecting the brain from physiological fluctuations of the plasma and from blood-borne compounds that may interpose with the normal neurotransmission process.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 30 This introduction of functional derivatives in the coat proteins allows surface modification of the phages with various chemicals such as chromophores, fluorescent dyes, enzymes, and synthetic oligomers, playing an essential role in a panoply of applications such as bioimaging, tissue engineering and biosensors [67]. c) M13 Phage Applications Biosafety, mass manufacturing with uniform parameters at a low cost, and all the information presented here show why this phage offers potential for numerous applications in different areas [80]. As already mentioned, phages and Phage Display have been used for clinical usage. Murgas et al . demonstrated that M13 phages have a high potential as an immunotherapeutic strategy for colorectal cancer (CRC). This cancer is quite lethal, and currently, there are no effective conventional treatments. Thus, since this pathology is characterized by high expression of the carcinoembryonic antigen (CEA) that promotes the aggravation of this tumor's magnitude, this was the main target. This group of researchers, through a systemic administration of CEA-specific M13 phages (M13 phages that exhibited in the p3 protein of its capsid, single-chain variable fragments (scFv) derived from a monoclonal antibody specific for CEA), in mice with CRC showed a marked reduction in the growth of this tumor [81]. More recently, Han et al . designed a new purpose against this tumoral activity, wherein the major coat protein, p8, of the M13 phage displayed an antitumor cytokine, granulocyte-macrophage colony-stimulating factor (GMCSF), which significantly decreases tumor growth in murine. Furthermore, this approach combined with radiation improved the performance of this therapy [82]. In the current days, with the continuous increase of drugs resistance, phage therapy can be a new solution, as Lu & Collins showed. Their study demonstrated that transferring genes that decrease the SOS response of bacteria to the dysfunctions caused by antibiotics via the M13 phage weakened the bacteria making them more susceptible to antibiotics [83]. Filamentous phages can also be used as biosensors, which is helpful for visualizing the progression of diseases in such a way that relevant information can be obtained for diagnosis and treatment. This is possible, as the phage capsid contains functional groups with varying reactivity, where multiple synthetic molecules can be selectively bound to the phage scaffold. These phages are mainly presented as a nanoprobe by conjugating fluorescent molecules and targeting moieties on the capsid surface. Li et al . for example, engineered a dual-modified M13 particles able to detect human KB tumor cells by emitting fluorescence upon binding of folic acid (FA) to the tumor cells. The FA is a vitamin that plays an important role at the cellular level, and it is taken up by the folate receptor (FR) present in cells
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 31 and overexpressed in cancer. The dual-modification on the phage consisted of chemical modifications to the subunits of the p8 protein, which allowed the binding of the folic acid and fluorescent molecules [84], [85]. Gosh et al . developed M13 phages as a magnetic nanoparticle vehicle for in vivo prostate cancer imaging. In their work, the M13 phage genome was modified so that the minor capsid protein, p3, exhibited a targeting peptide with affinity for SPARC (Secreted, Acidic, Cysteine Rich Protein), a glycoprotein highly expressed in melanoma, aggressive melanoma, breast, brain, prostate, colon, and lung cancer. The peptide used is known as SPARC-binding peptide (SBP). Concerning the p8 protein, it has been chemically altered to house magnetic iron oxide nanoparticles (MNPs). Nanoparticles are promising contrast agents for dynamic molecular imaging due to their intrinsic optical, electrical and magnetic properties, so MNPs were used to detect specific tumors via dark contrast magnetic resonance imaging. The results of this strategy suggested that the use of M13 compared to nanoparticles, that were directly functionalized with target peptides, was more effective for amplified targeting in vitro and was able to obtain better dark contrast imaging in vivo , as each SPARC target molecule delivered a large number of nanoparticles to the cells [86]. Moreover, Gosh et al ., in 2012, used the M13 phage as a promising platform for prostate tumor cell imaging and drug delivery. They genetically manipulated the M13 phage so that the p3 protein was fused to SPB. In addition, the phage was also genetically and chemically functionalized so that the drug doxorubicin (DOX), commonly used in chemotherapeutic sections, was coupled to the p8 protein subunits, and also so that fluorescent molecules (streptavidin Alexa Fluor® 488 Alexa) were presented on the surface of the p9 protein to detect the prostate cancer cells. This whole phage complex has been demonstrated in vitro to be a potential monitoring tool due to the acquired fluorescence. Besides that, this phage approach was proved to have therapeutic potential as it was able to decrease not only the viability of the cancer cells but also reduced the concentration of the applied drug, as it is a targeted drug delivery [87], [88]. Liposomes are artificial phospholipid vesicles characterized by easy degradation, little or no side effects, high loading capacity and good biocompatibility, making them suitable drug carriers. However, this type of drug delivery is not stable in the biological system, so Ngweniform et al . developed a phage M13-liposome complex to optimize the performance of these nanomolecules. Through electrostatic interactions, triggered by the negatively charged peptide displayed on the majority protein of the protein coat, the liposomes were coupled to the phage. Inside, the liposomes were loaded with zinc phthalocyanine (ZnPc), a drug tested as a photosensitizer for photodynamic therapy (PDT). This
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 32 nanostructure has been shown to have the potential to be used to targeted drug delivery in PDT and for reaching specific cells [89], [90]. Those works showed different examples where the M13 filamentous phage is used as a platform. Thus, this phage can be applied as an innovative strategy to diagnose/treat α-synucleinopathies. Being this purpose powered by the works of Hemi Dimant and their colleague, that described the natural capacity of phage M13 to disaggregate α-synuclein aggregates in vitro . They observed that when this phage is incubated with an α-synuclein fibril solution, fibril disaggregation occurred through a possible interaction of the filamentous phages to a hydrophobic stretch in the central hydrophobic region spanning amino acids 73–85 [37], [91]–[93]. This ability is an exciting and relevant skill for this work. 1.5. Peptide Targeting Directly targeting the aggregation process is an appealing therapeutic strategy. Hence, the interest in using peptides in this area, especially when it comes to neurodegenerative research, has been growing. This happens because peptides have a high potential to prevent fibrillar formation, as they are able to block the surface protein-protein interactions formed during the aggregation process, which is not achieved by most of the small molecules that constitute the commercial drugs. Moreover, peptides have a higher specificity of targeting, which leads to a reduction in undesirable side effects [94]. In 2015, Cheruvara et al . designed a multiplexed intracellular protein-fragment complementation assay (PCA) library screening system based on the native AS sequence 45-54, which is described as the region of AS that contains missense mutations implicated in the aggregation process. Using this sequence as a template, they created a 209,952-member peptide library with ten amino acids in length that spans residues 45–54 (KEGVVHGVAT). From this library, the peptide 4554W (K1DGIVN6GVKA) has been identified as a potential candidate that showed in in vitro assays reduced AS fibril formation and cytotoxicity [95]. Recently, Torpey and their colleagues, based on the work of Cheruvara et al ., reported that 4554W peptide can bind to partially or wholly aggregated AS species, preventing further fibrillation and disaggregating pre-formed AS fibrils resulting in shorter fibrils. Additionally, they did not observe any association of the peptide with monomeric AS, which means that the 4554W peptide ((KDGIVNGVKA)) does not interfere with the native function of AS [94]. Then, Meade et al . studied the role of the interaction between AS-lipid in the primary nucleation since primary nucleation is the first step of aggregation. Their
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 33 experiment used 1,2-dimyristoyl-sn-glycerol-3-phospho-L-serine (DMPS) vesicles as a phospholipid model, since they are an important element in dopaminergic synaptic vesicles, which lead to AS membrane binding, and consequently an increase of local concentration of AS that accelerates primary nucleation. The results indicated that 4554W inhibits lipid-induced primary nucleation of AS in vitro . Therefore, the 4554W has a crucial role in the primary nucleation, which is important because the primary nucleation is considered a critical step for the neurotoxicity associated with AS and a desired point for inhibitor function [96]. More recently, Meade et al . published a work where they optimized the peptide 4554W. They performed a complete alanine scan analysis (each residue of 4554W peptide was replaced for an alanine). Indeed, they verified that the substitution N6A corresponding to the peptide 4554WN6A (K1DGIVA6GVKA) exhibited a significant increase in peptide efficacy in inhibiting lipid-induced primary nucleation, which suggests that the other residues of the original peptide have an important function in the inhibitory effect of the aggregation pathway. Intending to reduce the size of the 4554WN6A peptide to the smallest functional unit required in the activity, they found that truncation at the N-terminus of the peptide, which resulted in the deletion of the K amino acid from position 1, gave rise to the 4654WN6A peptide (DGIVA6GVKA). This new peptide evidenced a significant increase in reducing the number of toxic oligomeric species in lipid membranes, blocking AS aggregation and associated cytotoxicity in vitro , which means that the K residue is indispensable. This study also evidenced that peptides 4554W, 4554WN6A and 4654WN6A, being antagonists of AS aggregation and associated toxicity, do not directly prevent the binding of AS to lipids, which is a desirable factor since the association of AS to small synaptic neurotransmitter vesicles should not be disrupted, so not to interfere with the normal activity of the dopaminergic vesicle fusion and recycling process [34]. In May of this year, Meade and colleagues used PCA methodology to screen the 45-54 peptide library, but this time against five of the characteristic clinical mutations in this 45-54 region (A30P, E46K, H50Q, G51D, and A53T), with the goal of identifying a peptide that would be effective not only against the familial AS variant from which it was selected, but also against wild-type (WT) AS. The screening identified five peptides: A30PW (EEGVIDGIAA), E46KW (KEGVVNVVKA), H50QW (ENGVVNGVAT), G51DW (EDGVVDGVDA), and A53TW (EDAVVNGVAA), all of which had common residues at some places but differed at others. All reduced aggregation of the relevant target, and some were shown to reduce aggregation when incubated with other variants. The G51DW peptide demonstrated the better activity among these peptides. In addition to the new peptides identified, the previously optimized peptide, 4554WN6A, was re-performed in this study, concluding that it is not only highly effective against WT AS
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis INTRODUCTION 34 but also against several mutant forms. In the cell toxicity studies, however, where 4554WN6A excelled in recovering toxicity, G51DW had the reverse impact and appeared to increase toxicity for some AS variants. This highlights the promising nature of peptide 4554WN6A for diagnostic and therapeutic applications [97]. Based on all the aforementioned, it can be inferred that the construction of a phage particle expressing peptide ligands capable of identifying AS aggregates paves the way for developing a novel approach for early diagnosis, prevention, and/or treatment of α-synucleinopathies.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MOTIVATION AND OBJECTIVES 35 2. MOTIVATION AND OBJECTIVES α-synucleinopathies affect millions of people worldwide. The development of new strategies for diagnosis and/or treatment of these neurodegenerative diseases is urgent to prevent its onset and possibly halt/inhibit disease progression. Achieving this feat will be tantamount to returning people with these diseases to normal lives. However, the progress of new approaches is limited by the BBB that houses the CNS, and currently, there is no effective approach developed to early diagnose nor treat αsynucleinopathies. Therefore, the present proposal aims to develop a phage-based system capable of detecting the α-synuclein aggregates by shuttling α-synuclein specific peptides across the BBB able to recognize and bind α-synuclein aggregates in the brain. Hereupon, this project involves three main objectives: i) the development of a phagemid containing the DNA sequence of α-synuclein specific peptides, with high binding potential to α-synuclein aggregates; ii) the development of engineered phages containing the peptide(s) of interest; and iii) characterization assays incubating the engineered phages with α-synuclein to assess their binding capacity to the 45-54 region of α-synuclein, and check the morphology that phages and protein adopt when they bind.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 36 3. MATERIAL AND METHODS This project includes three main tasks: synthesis of the phagemids containing the peptides of interest, engineered M13 phage production, and characterization assays. 3.1. Development of phagemids This task involves developing three phagemids containing the DNA sequence of α-synuclein specific peptides (4554: KEGVVHGVAT, 4554W: KDGIVNGVKA, and 4554WN6A: KDGIVAGVKA). The peptides 4554W and 4554WN6A have been reported in the literature to have high potential affinity for αsynuclein. These peptides were generated based on the sequence of the 45-54 AS region. However, they differ from the native sequence. Thus, in order to have a peptide that functions as a control in relation to the other two, the 4554 peptide was designed in this work. The sequence of 4554 peptide corresponding to the native region 45-54 of AS, without any modification. 3.1.1. Insert synthesis In this work, three peptides (4554, 4554W, 4554N6A) will be displayed on the surface of the M13 phage, namely fused to protein 3. The first step is the synthesis of the peptides of interest. This process was based following the Rangel method: annealing of two primers with 100 % complementarity was performed, followed by a heating and cooling protocol, to obtain the inserts [98]. For that, it was first necessary to design the primers (reverse and forward) for each peptide. The amino acid sequences of the peptides of interest were converted to nucleotide sequences using the specific codons for each amino acid, to obtain the primers. It should be noted that, in addition to the sequence for each peptide of interest, each primer also includes an enzyme restriction site for a posterior cloning process. The sequences of the primers are represented in Table 1.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 37 Table 1. Primers used to obtain the sequences of the desired peptides: In blue the restriction site of the SacI enzyme; in green the SalI enzyme restriction site and in yellow the peptides of interest. Primers Sequence 5’-3’ Base Pairs (bp) Peptide Sequence 4554 (Foward) TATATAGAGCTCAAGGAGGGCGTGGTGCACGGCGTGGCCACCCGTCGACTATATA 55 KEGVVHGVAT 4554 (Reverse) TATATAGTCGACGGGTGGCCACGCCGTGCACCACGCCCTCCTTGAGCTCTATATA 4554W (Foward) TATATAGAGCTCAAGGACGGCATCGTGAACGGCGTGAAGGCCCGTCGACTATATA 55 KDGIVNGVKA 4554W (Reverse) TATATAGTCGACGGGCCTTCACGCCGTTCACGATGCCGTCCTTGAGCTCTATATA 4554WN6A (Foward) TATATAGAGCTCAAGGACGGCATCGTGGCCGGCGTGAAGGCCCGTCGACTATATA 55 KDGIVAGVKA 4554WN6A (Reverse) TATATAGTCGACGGGCCTTCACGCCGGCCACGATGCCGTCCTTGAGCTCTATATA In this reaction, the individual primers of each desired insert (produced by STAB VIDA, Portugal) were able to anneal, therefore creating a double-stranded DNA product containing the insert of the specific sequence. All primers were resuspended in nuclease-free water to a final concentration of 100 µM. The annealing process was performed by adding equimolar amounts of each primer (20 µL of each respective primer at a concentration of 10 µM). Then, 10 µL of Trishydrochloride (HCl) at 10 mM, pH8, was added to a final volume of 50 µL. The annealing was carried out following the conditions described in Table 2, in a MyCyclerTM Thermal Cycler (Bio-Rad Laboratories, Inc.). Then, the concentration was determined using a NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Scientific) and the inserts were stored at -20 °C until usage. Table 2. Annealing conditions. Step Temperature (°C) Time (min) 1 93 3 2 80 20 3 75 20 4 70 20 5 65 20 6 40 60 7 4 ∞
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 38 3.1.2. Cloning process of the inserts into the phagemid Once the synthesis of the inserts is completed, the next task was the ligation between peptides (inserts) and vector (phagemid pETDuet-1 previously cloned with the gene 3 of M13). This task comprises the steps depicted in Figure 6. Figure 6. Schematic representation of the digestion: ligation protocol (created with Snapgene software). a) Digestion Reaction Before proceeding to the cloning process, it was first necessary to digest the inserts and the vector with the restriction enzymes, SacI (Thermo Scientific, Lot 00544923) and SalI (Thermo Scientific, Lot 00433634). The vector used was based in the commercial plasmid pETDuet-1 (Addgene, 71146). This plasmid has been previously manipulated in order to contain part of a phage genome, in this case, the gene 3 responsible for codifying the M13 phage minor coat protein 3 and the signal sequence peIB, which was previously added for promoting phage protein translocation through the bacterial membrane and assembly in phage particles (Figure 7). Figure 7. Geral scheme of the main regions of pETDuet-1 phagemid. Created in BioRender.com. [a] DIGESTION [b] LIGATION
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 39 The digestion of the inserts and vector is essential because it will allow the cloning procedure. Moreover, the enzymes used are unique cutters that form complementary sticky ends in the vector and inserts and consequently enable the insert to be introduced immediately before gene 3. The vector DNA used for the digestion had been isolated previously with the ZR BAC DNA Miniprep kit (ZYMO) following the manufacturer's instructions and was stored at -20ºC. The inserts and vector DNA were digested following the conditions of Table 3 (note that different conditions were tested and that the optimized condition was the one selected). Then, the samples were incubated at 37 °C for 2 hours, followed by a 15 minutes enzyme inactivation step at 65 °C. Table 3. Reaction conditions for vector and insert digestion. b) Ligation Subsequently to the digestion process, the following step was to clone the digested inserts into the digested vector. In this reaction, the T4 DNA ligase enzyme was used (NewEngland BioLabs® Inc, M0202S). This enzyme joins the cohesive end terminals of double-stranded DNA by catalyzing the creation of a phosphodiester link between juxtaposed 5' phosphate and 3' hydroxyl terminals. Therefore, the ligation was carried out following the condition on Table 4, using a 3:1 insert molar excess to 20-100 ng vector ratio. First, the reaction was mixed and incubated at 16 °C overnight. Then, the T4 DNA ligase was inactivated at 65 °C for 10 minutes. Subsequently, the ligation product was kept at -20 °C until bacterial transformation. Note that different conditions were tested and that the optimized condition was the one selected. Vector Insert DNA 1500 ng 1000 ng SacI 1 µL SalI 1 µL 10x FastDigest buffer (Thermo Scientific, Lot 00440534) 2 µL Nuclease free water Up to 20 µL
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 46 master mix shown in Table 7. Then, the PCR reaction for the sample was performed in MyCyclerTM Thermal Cycler (Bio-Rad Laboratories, Inc.) according to the conditions present in Table 6. The correct length of the PCR product was confirmed by loading 6 µL of the PCR product in a 2.5 % agarose gel. After gel confirmation, the PCR product was cleaned and purified using the DNA Clean & ConcentratorTM - 5 kit (ZYMO) following the manufacturer's instructions, and the concentration of the DNA was quantified in NanoDrop One Microvolume UV-Vis Spectrophotometers equipment (Thermo Scientific). Table 7. PCR protocol. Lastly, samples were prepared with 500 ng of cleaned PCR product, 3 μl of reverse primer (#7, see ANNEX I) at a concentration of 10 mM, which amplifies the region of interest of the gene 3, to check the presence of peptide sequences of interest, and water to a final volume of 10 μL was achieved. The samples were sequenced by Eurofins. 3.3. Characterization assays Once the bacteriophages that exhibit the peptides of interest were successfully engineered: i) phage 4554 (phage F1) that displays the KEGVVHGVAT peptide sequence (corresponding to the native region), ii) phage 4554W (phage F2) that displays the KDGIVNGVKA peptide sequence, and iii) phage 4554WN6A (phage F3) that displays the KDGIVAGVKA peptide sequence, all the conditions were met to move on to the step that provided information on the performance of the developed phages. To this end, monitored characterization assays were performed, where commercial α-synuclein (1MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTK45EGVVHGVAT54VAEKTK60), (RayBiotech, Inc), with a molecular weight of 6.1 kDa containing the N-terminal amphipathic domain (Figure 9), was incubated with each of the phages displaying the different peptides. These assays aimed to provide insights into the interactions between the different peptides and the target, i.e., to interpret their ability to bind to AS and whether the peptides actually interact with the 45-54 region of AS. Consequently, the Components PCR tube 10 μM Forward Primer (#41/#42/#43) 1 µL 10 μM Reverse Primer (#25) 1 µL Xpert Fast Master Mix (Ref: QE15, Grisp) 12.5 µL DNA 100 – 200 ng H2O free nuclease Up to 25 µL
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 47 performance of each peptide exhibited by phage M13 was evaluated and compared with each other. In addition, these assays were intended to supply information about the characteristics of the artificial phages produced. Figure 9. A schematic representation of the I-TASSER protocol for protein structure [99]–[101]. 3.3.1. AS sample The AS used for the experiments was a commercial protein resuspended in 50 µL of a solution of 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 to a final concentration of 1 mg/mL. 3.3.2. Phage samples preparation Before proceeding with the experiments, it was necessary to prepare aliquots of each phage at a concentration of 1 x 109 PFU/mL resuspended in the same buffer as that of the protein (20 mM Tris-HCl + 100 mM NaCl, pH= 7.5). Thus, in 2 mL eppendorf tubes, 2 mL of each phage was added from its respective stock solution stored at 4 °C (Table 8). Then, the samples were centrifuged at 1000 rpm for 20 min at 4 °C and the supernatant was thoroughly discarded. The resultant pellet from each sample was resuspended in 2 mL of buffer to a concentration of 1 x 109 PFU/mL. All samples were stored at 4 °C until further use. α-Synuclein (1-60 region)
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 48 Table 8. Phages concentrations in stock. 3.3.3. Dynamic light scattering (DLS) measurements The size distribution and the Zpotential of the phages (M13 (control), F1, F2, and F3) and the phages incubated with AS at a final concentration of 6.25 µg/mL (M13+AS, F1+AS, F2+AS, and F3+AS) was measured by DLS, using Zetasizer Nano ZS (Malvern Instruments) at 25 °C. In an appropriate cell DTS0012 or DTS1070, depending on the parameter that is being measured, size or Zpotential, respectively, 800 µL of the prepared sample were added. The sample was then placed in the equipment and read in triplicate, being the results described as mean ± standard deviation. It is worth noting that the phage samples were tested under four conditions: no incubation, 5 minutes, 24and 48-hours incubation at 36.6 °C. On the other hand, the samples containing phage + AS were only exposed to the last three conditions. Note that the selected incubation temperature was 36.6 ºC, as this is the mice's body temperature [102]. The tests were simulated with this temperature with a view to possible future in vivo tests. 3.3.4. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) In order to ascertain which of the engineered phages had a greater interaction with the protein, the free protein (which did not bind to the phage) in each sample was eluted and run on an SDS-PAGE gel. a) Preparation of the samples To recover the protein in solution, all the samples used in the previous procedure were reused and subjected to a spin down. Then the content of each sample was transferred to new 1.5 mL Eppendorf tubes and 100 µL of Coomassie blue dye (ThermoScientific) was added to each sample. Subsequently, the samples were vortexed vigorously and were left to incubate at room temperature. After 30 minutes of incubation the samples were centrifuged for 10 minutes at 17000 rpm so that the proteins present in solution and stained with blue would precipitate. The resulting supernatant was removed, and the pellet was placed to dry at 39 ºC and stored at room temperature until future use. All stored samples were Phage Peptide Displayed Stock Concentration (PFU/mL) M13 ------------ 3.6 x 1010 F1 4554 2.2 x 1010 F2 4554W 1 x 1010 F3 4554WN6A 7 x 109
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 49 resuspended in 20 µL of 1 x TrisGlycineSDS (TGS) buffer (Nzytech) and left to incubate for 2 - 3 hours. Then only the content of the samples containing phage and AS was aspirated and transferred to the respective Amicon® Ultra 0.5 mL column (Merck). In order that the free protein in solution was eluted, the samples were centrifuged for 15 minutes at 13300 rpm and the supernatant was collected in new 1.5 mL Eppendorf tubes. b) Preparation of the SDS-PAGE (15 % (w/v) acrylamide) gel Before beginning the gel preparation, the electrophoresis system's glasses were washed with distilled water, dried, and cleaned with alcohol to ensure that all the grease has been removed. Then, after assembling the gel support, the resolving gel at 15% (that allows the separation of proteins based on their molecular weight) and the stacking gel (that aligns all the protein samples loaded in the gel, so that they can enter the resolving gel at the same time), were prepared according to the specifications described in Table 9. Briefly, the resolving gel was first prepared and pipetted immediately onto the gel carrier. This was sealed by a thin line of distilled water, which was removed once the resolving gel polymerized. Following that, the stacking gel was prepared and promptly pipetted on top of the resolving gel, till the upper limit of the glasses was reached. Finally, the comb was injected quickly after pipetting to minimize retention and/or bubble formation, and the gel was allowed to polymerize. It should be emphasized that APS (Ammonium Persulfate) and TEMED (tetramethylethylenediamine) are polymerizing agents, so they were the last reagents to be introduced when preparing the gels. Table 9. SDS – PAGE. Reagents Resolving gel (15 %) Stacking gel (6 %) H2O free nuclease 2.8 mL 2.9 mL 40 % Acrylamide/Bis – Acrylamide 3 mL 0.750 mL 1.5 M TrisHCl, pH= 8.8 2 mL -------------- 0.5 M TrisHCl, pH= 6.8 -------------- 1.25 mL 10% SDS 80 µL 50 µL 20 % APS 80 µL 50 µL Total 8 mL 8 mL c) Application of the samples to be loaded onto the gel The samples present in Table 10 were loaded onto the gel. However, it was first necessary to prepare the samples for analysis, so to 8 µL of each sample, 2 µL of 5 x SDSPAGE Sample Loading Buffer (Nzytech) was added. Next, 10 µL of each prepared sample was loaded into the respective well of
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 50 the gel, as well as 5 µL of the PageRulerTM Unstained Broad Range Protein Ladder (ThermoScientific, 01135959). The electrophoresis was carried out in 1 x TGS buffer, and the power supply was automated for 30 minutes at 80 V, until the samples pass through the concentrator gel and are completely in the separator gel. Subsequently, the voltage was increased to 120 V for 40 minutes. Table 10. Samples loaded on SDS-PAGE gel Sample Description 1 Recovered protein from M13 phage sample 2 Eluted protein from sample with M13 and AS 3 Recovered protein from F1 phage sample 4 Eluted protein from sample with F1 and AS 5 Only AS Protein (at a final concentration of 6.25 µg/ mL) 6 Recovered protein from F2 phage sample 7 Eluted protein from sample with F2 and AS 8 Recovered protein from F3 phage sample 9 Eluted protein from sample with F3 and AS d) Protein Staining - BlueSafe protein stain: After the electrophoresis process, the gel was carefully removed from the electrophoresis system and washed with distilled water. It was then placed in the staining solution, BlueSafe protein stain (Nzytech, MB15201), overnight. The next day, the staining solution was discarded, and the revealed bands were analyzed. Finally, the gel was preserved in distilled water. - Silver stain for protein gel: For the purpose of greater detection of banding patterns formed after the electrophoresis process the staining can be done in silver nitrate. Silver staining provides a high degree of peptide coverage while maintaining excellent sensitivity (in the very low nanogram range). Briefly, the staining process sequentially consists of protein fixation, sensitization, washing, silver impregnation and finally image development [103], [104].Thus, following the protocol for silver staining the gel was first rinsed in distilled water. After 10 minutes, the distilled water was removed, and sodium thiosulfate at 0.2 g/L (VWR Prolabo Chemicals, 16I154125) was added to the gel and left in contact with the gel for 1 minute. Then, the sodium thiosulfate solution was removed, and the gel was washed 2 x 20 seconds in distilled water. The distilled water was removed, and the gel was impregnated in a silver nitrate solution at 2 g/L (VWR
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 51 Prolabo Chemicals, 13C250013) during 30 minutes. Subsequently, the silver nitrate was removed, and the gel was rinsed with distilled water for 10 seconds. Thereinafter, the distilled water was discarded and the developer solution (0.7 mL/ L formaldehyde 3738 % w/w stabilized with methanol PA-ACS (Panreac, 131328.1211), 30 g/ L sodium carbonate anhydrous (Panreac, 4D013888) and 10 mg/ L sodium thiosulphate) was added 2 x 3 minutes and the gel was shaken carefully until the desired color was achieved. The developer solution was removed, and the gel was incubated with the stop solution (50 g/L Tris-Base (Fisher BioReagentsTM, 153464) and 2.5 % acetic acid glacial (Fisher BioReagentsTM, 1733740) for 1 minute. Finally, the protocol ended by washing the gel in distilled water and the developed bands were analyzed. 3.3.5. Scanning Transmission Electron Microscopy (STEM) The morphological analyses of the 8 samples described in the Table 11 were performed in an Ultra-high resolution Field Emission Gun Scanning Electron Microscopy (FEG-SEM), NOVA 200 Nano SEM, FEI Company. Before analyses, samples were installed in Cu-C grids (AGAR S160-4), by immersion in the solution containing the phages or phages and AS. Then the samples were analyzed with an acceleration voltage of 15 kV, using a scanning transmission electron detector (STEM). The image files were examined and converted into TIF format. Table 11. Samples analyzed in STEM technique Sample Description Conditions M13 Sample containing M13 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 Without incubation F1 Sample containing F1 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F2 Sample containing F2 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F3 Sample containing F3 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 M13 + AS Sample containing M13 at 1 x 109 PFU/mL and AS at 6.25 µg/ mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 With 5 minutes of incubation at 36.6 ºC F1 + AS Sample containing F1 at 1 x 109 PFU/mL and AS at 6.25 µg/ mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F2 + AS Sample containing F2 at 1 x 109 PFU/mL and AS at 6.25 µg / mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F3 + AS Sample containing F3 at 1 x 109 PFU/mL and AS at 6.25 µg / mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 3.3.6. Atomic Force Microscopy (AFM) AFM was performed to characterize the morphology and structure of the modified phages. In this experiment, seven samples were analyzed (Table 12). Samples were imaged in noncontact mode using
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 52 a NanoWizard® 3 BioScience AFM (JPK Instruments). The AFM data were obtained under different conditions. The samples AS, M13, F2, and F3 were directly analyzed from the prepared fresh samples. Nevertheless, the samples M13+AS, F2+AS, and F3+AS once prepared were analyzed after 24 hours of incubation at 36.6 °C. It should also be stressed that phage F1 was not analyzed due to the time limitation. Table 12. Samples analyzed in AFM. Sample Description AS Sample containing AS at 0.00625 mg/ mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 M13 Sample containing M13 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F2 Sample containing F2 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F3 Sample containing F3 at 1 x 109 PFU/mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 M13 + AS Sample containing M13 at 1 x 109 PFU/mL and AS at 6.25 µg/ mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F2 + AS Sample containing F2 at 1 x 109 PFU/mL and AS at 6.25 µg / mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 F3 + AS Sample containing F3 at 1 x 109 PFU/mL and AS at 6.25 µg / mL resuspended in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5 In short, a drop of 15 µL from each sample was dropcast onto a freshly mica discs (TED PELLA, INC.) in dry conditions. The excess was removed. Following adsorption of the samples (2 minutes) at room temperature, the samples were dried using a stream of nitrogen gas. Then, samples were immediately analyzed by AFM. The images were subsequently acquired in tapping mode with ACTA-SS50 probe (T: 4.0 µm; L: 125 µm; W: 30 µm; f0: 270.7 kHz; k: 22.187 N/m) at a scan rate of 1.0 Hz and a resolution of 512 x 512 pixels. All images were obtained at room temperature. The image files were examined using JPK data processing software and converted into TIF format. 3.3.7. Statistical Analysis Statistical analysis and graphical presentations were performed using GraphPad Prism 8 software (Version 8.0.1 for Windows, GraphPad Software, San Diego, CA, USA). Before any statistical analysis, each condition was tested for normality with the Shapiro-Wilk test. For comparison of one or two normally distributed samples, t-test was performed. If non-normally distributed, Mann-Whitney test was used. To test differences between more than two normally distributed samples, the ANOVA test was applied. In case of at least one of the samples is non-normally distributed, the Kruskal-Wallis test was performed. p
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis MATERIAL AND METHODS 53 values of less than 0.05 indicated statistical significance. Error bars indicate standard error of the mean (SEM. 4. RESULTS AND DISCUSSION In this thesis, the main focus was on the development of phages that display at p3, peptides that recognize AS in characterization experiments. To this end, three essential AS-specific phages were engineered: i) phage F1, which was used as a control to prove the higher efficacy of the other engineered phages (F2 and F3), ii) phage F2, and iii) phage F3. Of note, the phage M13KE (empty phage) was also used as a negative control in all experiments. Thus, we used these peptide sequences to generate modified phages with the ability to identify AS clusters. 4.1. Insert Synthesis To acquire the three inserts (4554, 4554W, and 4554WN6A) that codify the peptides of interest, a 100% complementarity technique was used to anneal two primers, the forward and reverse primers of each insert. The inserts obtained from this PCR reaction should be an expected size around 55 bp (see Table 1). To verify if the insert synthesis was successful, the resulting PCR product of each insert was visualized on a 3 % agarose gel (Figure 10). Of note, the predicted conformational structure of the peptides was simulated using the I-TASSER software and this information can be found in ANNEX II.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 54 Figure 10. Insert size confirmation (samples were loaded on a 3 % agarose gel): [A] Representation of GRS Low Range Ladder; [B] Results of the electrophoresis process for each insert – L) GRS Low Ranger Ladder, 1) 4554 insert, 2) 4554W insert, 3) 4554WN6A insert. Taking into consideration the size of the designed primers (Table 1), a band with a size of 55 bp should appear. Looking at the three gels in the Figure 10 and comparing the resulting bands with the GRS Low Ranger Ladder, it could be seen that all inserts showed the expected length (≈ 55 bp), as they exhibited a band slightly above the 50 bp ladder band, which was expected. After this confirmation, the DNA concentration of all samples was determined using a NanoDrop One Microvolume UV-Vis Spectrophotometers (Thermo Scientific), and the results are shown in Table 13. Table 13. DNA quantification of the inserts and purity ratios. Sample [DNA] ng/µL Ratio A260/A280 Ratio A260/A230 4554 202.3 1.82 1.10 4554W 177.9 1.81 1.27 4554WN6A 213.1 1.84 1.22 Note: The A260/A280 ratio is commonly used to assess the purity of DNA or RNA. A260/A280 ratios for pure DNA should be around 1.8 (and for RNA should be around 2). A lower ratio suggests the presence of proteins, phenol, or other contaminants that absorb strongly at or near 280 nm. High A260/A280 purity ratios may indicate RNA contamination in the sample of DNA, indicating a problem with the extraction protocol. The A260/A230 is used to measure the purity of nucleic acids. The presence of organic contaminants such as phenol, carbohydrates, chaotropic salts, and other aromatic compounds is indicated by the A260/A230 ratio. Samples with A260/A230 ratios less than 1.8 are thought to contain a significant amount of these contaminants. The A260/A230 ratio in a pure sample should be around 1.8 - 2.2. A high A260/A230 ratio could be the result of performing a blank measurement on a dirty pedestal or using an ineffective blank measurement solution [105], [106]. 50 300 100 700 L 2 25 300 100 50 25 700 c) d) L 3 25 50 75 300 L 1 700 [A] [B]
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 55 The results showed that the annealing process occurred with success because were obtained an adequate DNA concentration for the samples. Relatively, to observance ratios A260/A280, it can be said that the purity level of all samples was acceptable since the ratios meet around the reference value (≈ 1.8). However, the ratios A260/A230, indicated the presence of organic contaminants likely salts and phenols in the samples, since the ratios 1.10, 1.27, and 1.22 are below the 1.8 ≤ A260/A230 ≤ 2.2 (standard values). This could be due to the base solution (Tris-HCl pH = 8) used in the annealing primers process because, in its composition, this component contains salts, which can have such interference in the ratios A260/A230. It should be noted that although the samples presented this contamination, it did not represent an obstacle. Thus, the samples were used in the steps that followed, the digestion. 4.2. Phagemid cloning system As stated in the topic 1.4.1 the use of the phagemid system is very applied in phage display system because they offer more flexibility and independence for the phage packaged during the amplification phase, allowing the insertion of the entire peptide sequence [61]. Therefore, to produce the phage display particles of interest, firstly was necessary to conjugate the DNA of the peptides of interest with the phagemid vector, in this case, the modified pETDuet-1 plasmid. Display of the peptides of interest in p3 is achievable, but only if the gene sequence of each insert is inserted between the signal sequence, pelB, and the upstream codon of gene 3 [58], [68]. In order to give a better design of the phagemid cloning system, the SnapGene software (www.snapgene.com) was utilized to execute in silico construction of the plasmid including the inserts of interest (Figure 11).
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 62 Looking on the results obtained in the electrophoresis gel, it was possible to observe that the amplification region presented a band with the expected size (≈ 323 bp) since the samples should present a band slightly above the 300 bp ladder band, which was verified. As the samples presented a band with the expected size in the gel, each PCR product was cleaned and purified in order to prepare a sample for sequencing in to confirm the correct nucleotide sequence on each phage genome. The cleaning was performed using the DNA Clean & ConcentratorTM - 5 kit (ZYMO) following the manufacturer's instructions. Afterwards this cleaning process, the concentration of the DNA for each sample was quantified in NanoDrop One Microvolume UV-Vis Spectrophotometers equipment (Thermo Scientific), and the results are present in Table 17. Table 17. DNA quantification of the clean PCR product and purity ratios. Samples [DNA] ng/ µL Ratio A260/A280 Ratio A260/A230 F1 Phage DNAclean PCR product 107.7 1.87 2.18 F2 Phage DNAclean PCR product 93 1.87 2.17 F3 Phage DNAclean PCR product 64.3 1.85 1.36 The results exhibited in the table showed that extraction occurred with the success. Regarding the absorbance ratio A260/A280 it can be said that the purity level of all samples was acceptable since it is around the reference value, ≈ 1.8. With concerning the A260/A230 ratio, it can be stated that the values of 25 150 300 400 L 1 2 50 150 300 400 L 2 25 50 150 300 400 L 3 ① Figure 13. Results of the electrophoresis gel for the PCR products (samples were loaded on a 2.5 % agarose gel): ① Representation of GRS Low Range Ladder; LGRS Low Range Ladder; 1PCR product containing the amplification of gene zone 3 from phage DNA that harbours the sequence of interest of peptide 4554; 2PCR product containing the amplification of gene zone 3 from phage DNA that harbours the sequence of interest of peptide 4554W; 3PCR product containing the amplification of gene zone 3 from phage DNA that harbours the sequence of interest of peptide 4554WN6A.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 63 the A260/A230 absorbance ratio is considered acceptable for the two first samples, as the ratios are within the 1.8 ≤ A260/A230 ≤ 2.2 reference values, which means that the samples are free from contamination by organic compounds such as salts and phenols. Nevertheless, the same was not verified for the sample of the clean PCR product, corresponding to the DNA of phage F3. The ratio A260/A230 this sample indicated the presence of organic contaminants likely salts and phenols, since the ratio 1.36, is below the standard values. This may be due to the buffers used in the kit because these are composed of salts and phenols. This contamination may also have resulted from incomplete evaporation of ethanol, which is applied in one of the steps of using the kit. In both cases, this contamination did not represent any hurdle to proceed, and therefore all samples were sent for sequencing. The sequencing results were analyzed in the SnapGene software (Table 18). Through a close look at the results, it was possible to observe that the sequence of the peptide 4554WN6A was fully inserted into the phage genome. Regarding the sequencing results for the DNA of phages F1 and F2 containing the peptide sequence 4554 and 4554W, respectively, by using the SnapGene software it was possible to verify that there was no match with the sequences of interest. A detailed analysis using the "Align" tool between the results obtained and the sequence of interest allowed to detect that the sequences of interest from both samples were present. Nonetheless, these did not coincide with the original sequence, as they presented insertions. From the chromatogram data of each sample (see ANNEX V) it was possible to observe that the extra base pairs corresponded to undefined peaks or to peaks overlapping with the main peak (contamination), which ended up interfering with the clear reading of the data. In view of all the analysis performed, it was agreed that the sequences of interest were present and that the fact that the original sequence of the inserts of interest was not detected was due to errors associated with the sequencing process. Thus, it was confirmed the presence of the correct 4554, 4554W, and 4554WN6A peptide sequences in the respective DNA phage sample.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 64 Table 18. Sequencing results of DNA phages. In underline the region the sequence of the peptide of interest, and in in red erased the additional insertions in nucleotide sequence of the peptides of interest. Phage Insert of Interest Reverse Peptide Sequence (5´- 3’) Nucleotide Sequence from sequencing (5’-3’) F1 4554 GGTGGCCACGCCGTGCAC CACGCCCTCCTT …TAATTCTTTCTCACAGTAAGCTCCGTCGTACGG GTGGG157CCACGCCGTGCACCACGCT176CCTCCTT CTAATCTAC F2 4554W GGCCTTCACGCCGTTCACG ATGCCGTCCTT …TCGACGGGCCTTT161CACGCCGTTCACGA A176 T177 TGCCGTCCTTAAATCA F3 4554WN6A GGCCTTCACGCCGGCCAC GATGCCGTCCTT …GAAGCTTAGTCGACGGGCCTTCACGCCGGCCA CGATGCCGTCCTTAAACGACTGTCGGCGTGCTTG … 4.4. Characterization assays The characterization assays performed aimed to detect whether engineered phages displaying the peptides of interest (4554W and 4554WN6A), considered in the literature to be able to interact with AS in the 45-54 region, were indeed able to recognize AS [34], [94]–[97]. Furthermore, these assays were intended to morphologically characterize the synthetic phages produced. 4.4.1. Physicochemical characterization of the phages particles and phages particles incubated with AS For a phage concentration of 1 x 109 PFU/mL and an AS concentration of 6.25 µg/mL, phage particles (M13, F1, F2, and F3) and phage particles incubated with AS (M13+AS, F1+AS, F2+AS, and F3+AS) were characterized at the level of their size distribution (Z-average) and Zeta potential under different conditions. The results from this first test, using the Zetasizer Nano ZS equipment, are illustrated in Figure 14 and Figure 15. According to the results present in the Figure 14, it was possible to observe that phages without incubation, with the exception of phage F1(≈ 446,43 nm), presented the expected size since the size distribution obtained for phage was in line with the literature for phage M13 (≈ 800 - 1000 nm) [58], [68]. Besides, phages F2 (≈ 805.07 nm) and F3 (≈ 811.65 nm), which display peptides 4554W and 4554WN6A, respectively, showed a larger size compared to phage M13 (790 nm), which also was expected. Only phage F1 presented a smaller size than expected. As already pointed out phages F1, F2 and F3 displayed peptides at the p3 protein end. However, it should be remarkably noted, that the
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 65 peptides of phages F2 and F3 (4554W: KDGIVN6GVKA and 4554WN6A: KDGIVA6GVKA, respectively) are quite similar to each other differing only in the amino acid at position 6, whereas the peptide of phage F1 presents an amino acid sequence with more dissimilarities (KEGVVHGVAT). This particularity highlighted here can justify the size presented by phage F1. In other words, peptide 4554 may be interfering with the overall phage architecture, hence the phage is smaller in size. Considering the data for samples incubated for 5 min at 36.6°C (condition t1) it was possible to observe that there were no significant changes between the non-incubated and incubated phage conditions. Furthermore, when comparing samples containing only phage with samples containing both phage and AS, it was notable that there was no substantial change in the size of phages when incubated with AS. After 24 hours of incubation (condition t2), a decrease in the size distribution of the samples containing only phage was noticeable, whereas the samples containing both phage and AS, with the exception of sample M13+AS, showed no size fluctuations relative to the t1 condition. Finally, it was verified that the samples, excluding the sample F3, practically stabilized their size after 48 hours (condition t3). A close look at the set of inferences drawn from these data raises the hypothesis that phages exhibiting a peptide in their p3, have established some kind of interaction with AS and that this interaction does not influence the physical characteristics of the phage, as throughout the incubation time the phage has conserved its size. Nevertheless, for this hypothesis to be corroborated other types of assays were carried out. It should be underscored that these values are only an estimate and may not be an approximation of reality, since for the measurement of the sample sizes the phages were considered protein material as a whole. In addition, the equipment gives a spherical approximation of the size.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 66 Figure 14. Characterization of phage particles and phage particles incubated with AS prepared in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5. The results plotted show means SD of n= 8 samples. Size distribution over time (t0: samples without incubation; t1: samples with 0.083 h (5 min) of incubation at 36.6 °C; t2: samples with 24h of incubation at 36.6 °C; t2: samples with 48h of incubation at 36.6 °C). Statistical significance of the data was determined by t-test and two-way ANOVA (*p ≤ 0.05 and ****p ≤ 0.0001). Statistical analysis between the samples to each time point was terminated by two-way ANOVA (data not shown). Regarding the Z-potential results (Figure 15), their analysis showed that nearly all samples exhibited a uniform loading pattern over time. In the t0 condition (no incubation at 36.6ºC), where only the charge of the samples with isolated phages was evaluated, it was visible that the phages that displayed a peptide at the end of the p3 protein (F1, F2, F3) showed a more negative charge than the phage M13 (control), which indicates that the peptide attached to the p3 protein conferred a more negative charge to its respective phage. The peptides exhibited may be altering the phage structure, promoting a greater exposure of the negative amino acids on the phage surface, which induces this increase in charge. In the remaining conditions and comparing the results obtained, it is concluded that the incubation of the phages with protein does not interfere with the stability of the phage charge, and that regardless of the incubation time, the Z-potential remained practically unchanged. The sample with individual phage M13 presented the greatest oscillations, with a significant increase in charge (in absolute value) during the course of time. It should be noted that it was expected that the phages would show a negative charge given that the outer layer of the phage protein shell is mainly occupied by the negative N-terminal region of the p8 protein, which is highly rich in acidic amino acid residues that interact with the solvent and endow the F3 (4554WN6A) 0 200 400 600 800 1000 t0 t1 t2 t3 Z-avarage (nm) Time (h) M13 M13+AS F3 F3+AS F2 (4554W) 0 200 400 600 800 1000 t0 t1 t2 t3 Z-avarage (nm) Time (h) M13 M13+AS F2 F2+AS t0 t1 t2 t3 0 50 100 150 200 250 500 1000 1500 Z-avarage (nm) M13 M13+AS F1 F1+AS F2 F2+AS F3 F3+AS Time (h) ***** **** **** **** **** **** Samples: t0 t1 t2 t3 -30 -20 -10 0 Zeta (mV) M13 M13+AS F1 F1+AS F2 F2+AS F3 F3+AS Time (h) **** **** **** ** * *** **** ** * Samples: [A] [B] F1 (4554) 0 200 400 600 800 1000 t0 t1 t2 t3 Z-avarage (nm) Time (h) M13 M13+AS F1 F1+AS
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 67 phage with a low isoelectric point (IEP) [107], [108]. Paolo et al . proved that the charge of phage M13 came predominantly from the charges of residues constituting the majority coat protein, p8, and experimentally determined that phage M13 had an IEP of 4.05 ± 0.065 [107]. Thus, since all samples were prepared in a buffer with pH= 7.5 and given that the charge of proteins is related to pH and IEP, it was expected that the phage-containing samples would be negatively charged because pH > IEP [109]. Figure 15. Characterization of phage particles and phage particles incubated with AS prepared in 20 mM Tris-HCl + 100 mM NaCl, pH= 7.5. The results plotted show means SD of n= 8 samples. Zeta-potential over time (t0: samples without incubation; t1: samples with 0.083 h (5 min) of incubation at 36.6 °C; t2: samples with 24h of incubation at 36.6 °C; t2: samples with 48h of incubation at 36.6 °C). Statistical significance of the data was determined by t-test and two-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p≤ 0.001, and ****p ≤ 0.0001). Statistical analysis between the samples to each time point was terminated by two-way ANOVA (data not shown). F3 (4554WN6A) -18 -16 -14 -12 -10 -8 -6 -4 -2 0 t0 t1 t2 t3 Zeta (mV) Time (h) M13 M13+AS F3 F3+AS F2 (4554W) -18 -16 -14 -12 -10 -8 -6 -4 -2 0t0 t1 t2 t3 Zeta (mV) Time (h) M13 M13+AS F2 F2+AS F1 (4554) -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0t0 t1 t2 t3 Zeta (mV) Time (h) M13 M13+AS F1 F1+AS t0 t1 t2 t3 -30 -20 -10 0 Zeta (mV) M13 M13+AS F1 F1+AS F2 F2+AS F3 F3+AS Time (h) **** **** **** ** * *** **** ** * Samples:
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 68 4.4.2. Protein electrophoresis interpretation As previously mentioned, the results from the analysis with the Zetasizer Nano ZS equipment are not sufficient to validate an interaction between the designed phages and the target of interest. To confirm and evaluate the phage recognition capacity, the samples used in the previous assay were submitted to an SDS-PAGE analysis. This procedure aimed, by means of the intensity of the bands obtained, to verify which of the phages showed greater binding to AS. Thus, based on the starting point that all samples containing phage and AS had the same concentration of protein, recovering the free protein present in these samples and loading it into the gel would result in responses. The more intense the AS band (6.1 kDa) for a given sample the lower the binding capacity between the phage and the AS. Unfortunately, as seen in Figure 16, the SDS-PAGE process did not occur successfully. No clear, well-defined bands were observed on the gel, rendering interpretation of the results impossible. Although two staining methods were used, and despite the fact that the silver nitrate stain was more sensitive and highlighted new bands that were not visualized in the gel when stained with BlueSafe, the bands were more smeared. This made reading the results even more difficult. Samples 1, 3, 6 and 8 were used as control for samples 2, 4, 7 and 9, respectively. Even though the lanes of these samples showed bands, they were not well defined. In addition, in samples 2, 4, 7 and 9 similar band patterns to the respective control samples were visualized, when only bands between the 5 kDa and 10 kDa bands corresponding to the molecular weight of the AS monomers (6.1 kDa) were supposed to be observed. The presence of all these bands means that the elution of only free protein did not occur successfully. It is noteworthy that the ladder band pattern was not well defined and sample 5 (containing only AS), used as a positive control showed rather faint and undefined bands. In addition, this also did not show the 6.1 kDa band, which was expected. This evidence would invalidate any kind of illation.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 69 Figure 16. SDS-PAGE gel: ① Representation of PageRulerTM Unstained Broad Range Protein Ladder; [A] Gel stained with BlueSafe and [B] Gel stained with silver nitrate (LPageRulerTM Unstained Broad Range Protein Ladder; 1sample with recovered protein from M13 phage sample; 2sample with eluted protein from sample with M13 and AS; 3sample with recovered protein from F1 phage sample; 4sample with eluted protein from sample with F1 and AS; 5sample with AS Protein from stock; 6sample with recovered protein from F2 phage sample; 7sample with eluted protein from sample with F2 and AS; 8sample with recovered protein from F3 phage sample; 9sample with eluted protein from sample with F3 and AS). Finally, it remains to be mentioned that from these results no conclusion about the binding capacity between phages and AS was obtained. Consequently, no conclusion about the performance of each peptide was drawn. The absence of results can be explained by the insufficient migration time of the proteins in the gel, which caused no defined bands to be obtained in the gel, and by the lack of denaturation of the sample proteins. Before being loaded into the gel, all samples should have undergone a denaturation step at 95°C for 5 minutes so that the samples would show a band on the gel corresponding to the native form of the protein and not also show a range of bands corresponding to oligomer species, which then end up interfering with data reading. In addition to these two factors, the columns used for the protein elution may not be in good preservation conditions, which may have conditioned their performance in the elution step. In summary, the SDS-PAGE procedure should be repeated again considering all the conditions listed here. 15 ① 1 2 3 4 L 5 6 7 8 9 250 20 10 L 15 1 2 3 4 L 5 6 7 8 9 250 20 10 L [A] [B] 5
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 70 4.4.3. Characterization of AS-specific phages by STEM In ordered to obtain information about the morphology of the individual phages and phages incubated with AS, the 8 samples shown in Table 11 were visualized by STEM, as shown in Figure 17. From the SEM images obtained, it was not possible to determine the phage morphology and how it is affected upon incubation with AS. Consequently, no conclusions about the binding capacity of the engineered phages could be drawn from this assay. The low resolution provided by the equipment and the existence of salts from the solution used in the preparation of the samples were preponderant factors that made it impossible to capture detailed and clear images. Thus, it was agreed that a new technique with higher resolution, such as transmission electron microscopy (TEM), should be used to analyze the samples at the nanometer scale. Furthermore, to avoid that the content of interest of the sample is masked by salts, a new buffer, containing less or no salts in its composition, should also be used. 5 minutes of incubation at 36.6 °C F1+AS 1 µm F2+AS 1 µm M13+ASControl 1 µm F3+AS 1 µm Without incubation F2 (4554W) 1 µm F1 (4554) 4 µm F3 (4554WN6A) 1 µm M13Control 1 µm Figure 17. STEM results. STEM images of samples with phages (M13, F1, F2, and F3) and samples with phages incubated with AS (M13+AS, F1+AS, F2+AS, and F3+AS), with and without a 5 minutes incubation at 36,6ºC.
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis RESULTS AND DISCUSSION 71 4.4.4. Characterization of AS-specific phages by AFM Visual characterization of the AS-specific phages was also performed by AFM. In this assay only the morphology and size of phages F2 and F3, which exhibit the peptides with high potential to recognize AS, 4554W and 4554WN6A, respectively, was compared with the wild-type phage M13. The AFM images are shown in Figure 18. Figure 18. AFM characterization results: [A] AFM image of AS sample, [B] AFM images of samples with individual phages (M13, F2, and F3) without incubation; [C] AFM images of samples with phage and AS (M13+AS, F2+AS, and F3+AS) with 24 h of incubation at 36.6 ºC. One of the features of phage M13, as already mentioned, is that it is a type of filamentous virus that has a long cylindrical shape of about 800-1000 nm. Looking at the Figure 18[B] image of the individual phages, it was found that phages M13, F2 and F3 presented the expected morphology and
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Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis ANNEXES 85 8. ANNEXES 8.1. ANNEX IPrimers Sequences The primers sequences used in PCR process and in the samples sent to sequencing. Table 19. Detailed information about the primers used. Primer Sequence 5’-3’ Information Target/ Template #30 (Foward) ATCGATCTCGATCCCGCGAA Primers used to sequence or to confirm the insertion of the sequences of interest into the plasmid Annealing with primer 6 to prove insertion of AS inserts into the petDuet #6 (Reverse) CTAGTTATTGCTCAGCGGT Primers used to confirm the insertion of the sequences of interest into the plasmid Annealing with primer 30 to prove insertion of AS inserts into the petDuet #25 (Reverse) CCGCCACCCTCAGAGCCACCACCCTCATTTTCAGG Primers used in the PCR process to amplify the region of interest of phage DNA that includes the sequence of the insert of interest To confirm correct cloning after infection with M13 Helper Phage. Pair with 23, 24 and 41. #41 (Foward) AAGGACGGCATCGTGGCCGGCGTGAAGGCC Primers used in the PCR process to amplify the region of interest of phage DNA that includes the sequence of the 4554WN6A insert To confirm correct cloning after infection with M13 Helper Phage. Pair with 25. #42 (Foward) AAGGACGGCATCGTGAACGGCGTGAAGGCC Primers used in the PCR process to amplify the region of interest of phage DNA that includes the sequence of the 4554W insert To confirm correct cloning after infection with M13 Helper Phage. Pair with 25. #43 (Foward) AAGGAGGGCGTGGTGCACGGCGTGGCCACC Primers used in the PCR process to amplify the region of interest of phage DNA that includes the sequence of the 4554 insert To confirm correct cloning after infection with M13 Helper Phage. Pair with 25. #7 (Reverse) CCCTCATAGTTAGCGTAACG Primers used in the samples sent to sequencing To sequence or amplify the M13KE gene 3 after peptide insertion
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis ANNEXES 86 8.2. ANNEX IIPredicted conformational structure of the peptides The conformational structure data provided by the I-TASSER software: Figure 19. A schematic representation of the I-TASSER protocol for p structure [99]–[101].
Engineering Of Specific Bacteriophages For α-Synucleinopathies Diagnosis ANNEXES 87 8.3. ANNEX IIIExpected Size of PCR products by SnapGene software The action, PCR, of the SnapGene software was used to performed in silico the expected size of PCR products: Figure 21. Expected size of PCR product for amplification of gene 3 zone containing the insert sequences: #30 – foward primer and #6 – reverse primer. product_PCR_#PetDUet_g3_pelB#primers6-30 1840 bp Figure 20. Expected size of PCR product for amplification of gene 3 zone with primers #30 and #6 (negative control - empty phagemid): #30 – foward primer and #6 – reverse primer. Peptide of interest gene product_PCR_#PetDUet_g3_pelB_insert#primers6-30 1858 bp