The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy.

The immunogenic potential of bacterial flagella for Salmonella-mediated tumor therapy Sebastian Felgner1,2,#, Imke Spöring1, Vinay Pawar2,4, Dino Kocijancic5, Matthias Preusse2, Christine Falk3, Manfred Rohde4, Susanne Häussler2, Siegfried Weiss5,&, Marc Erhardt1,6,& 1 Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany 3 Institute of Transplant Immunology, Medical School Hannover, Hannover, Germany 4 Central Facilities for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany 5 Institute of Immunology, Medical School Hannover, Hannover, Germany 6 Institute of Bacterial Physiology, Humboldt University, Berlin, Germany NOVELTY AND IMPACT: Balancing safety and therapeutic efficacy remains a major challenge for the rational design of bacteria for immunotherapy applications. We demonstrated that manipulating the spatiotemporal regulation of flagella synthesis can confer this balance in Salmonella. Unexpectedly, abrogating flagella synthesis at an early stage resulted in outer membrane vesicle formation, which enhanced the immunogenic properties. These engineered Salmonella strains might have the potential to serve as vector platform for various therapies ranging from immunization to cancer therapy. KEYWORDS: Salmonella Typhimurium, host-pathogen interaction, flagella, luminex, bacteriamediated tumor therapy ARTICLE CATEGORY: Cancer therapy and prevention Accepted Article This article is protected by copyright. All rights reserved. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ijc.32807 2 # Address correspondence to Sebastian Felgner, [email protected], HelmholtzCentre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany, Phone: + 49 531 6181 3133 & Contributed equally to this work ABBREVIATIONS: PAMP = pathogen-associated molecular pattern, LPS = lipopolysaccharide, TLR = toll-like receptor, PPR = pattern-recognition receptor, UK-1 = Universal Killer 1, Wt = wild-type, MOI = multiplicity of infection, LDH = lactate dehydrogenase, IL = Interleukin, TNF-α = tumor necrosis factor α, hpi = hours post infection, dpi = days post infection, OMV = outer membrane vesicle, CT26 = murine colon carcinoma, RenCa = renal adenocarcinoma, ELISA = enzyme-linked immunosorbent assay, BMDM = bone-morrow derived macrophages Abstract: 150 words Words: 5152 Accepted Article This article is protected by copyright. All rights reserved. 3 ABSTRACT: Genetically engineered Salmonella Typhimurium are potent vectors for prophylactic and therapeutic measures against pathogens as well as cancer. This is based on the potent adjuvanticity that supports strong immune responses. The physiology of Salmonella is well understood. It simplifies engineering of both enhanced immune-stimulatory properties as well as safety features, thus, resulting in an appropriate balance between attenuation and efficacy for clinical applications. A major virulence factor of Salmonella is the flagellum. It is also a strong pathogen-associated molecular pattern recognized by extraand intracellular receptors of immune cells of the host. At the same time, it represents a serious metabolic burden. Accordingly, the bacteria evolved tight regulatory mechanisms that control flagella synthesis in vivo. Here, we systematically investigated the immunogenicity and adjuvant properties of various flagella mutants of Salmonella in vitro and in a mouse cancer model in vivo. We found that mutants lacking the flagellum-specific ATPase FliHIJ or the inner membrane ring FliF displayed the greatest stimulatory capacity and strongest anti-tumor effects, while remaining safe in vivo. Scanning electron microscopy revealed the presence of outer membrane vesicles in the ΔfliF and ΔfliHIJ mutants. Finally, the combination of the ΔfliF and ΔfliHIJ mutations with our previously described attenuated and immunogenic background strain SF102 displayed strong efficacy against the highly resistant cancer cell line RenCa. We thus conclude that manipulating flagella biosynthesis has great potential for the construction of highly efficacious and versatile Salmonella vector strains. Accepted Article This article is protected by copyright. All rights reserved. 4 INTRODUCTION Despite the exponential growth of biomedical knowledge over the last decades, we are still facing health conditions that are not controllable. Thus, present biomedical research is called upon to provide solutions to this dilemma. In face of the demographic changes within the world population these problems become even more acute with cancer being one of the most pressing problems. Consequently, scientists need to develop novel and/or more effective strategies for vaccines and immunotherapies. Search for proper protective antigens is required for the success of such immune interventions but at the same time efficacious and safe adjuvants are required for such strategies. Employment of pathogens as platforms for many of such approaches is therefore a straightforward possibility. These microorganisms are able to act as potent carriers because they elicit strong immune reactions i.e. they exhibit strong adjuvant properties. Viruses are often employed in this context 1. However, bacteria have also successfully been tested. For instance, Salmonella spp. are known to elicit strong cellular and humoral immune activities which underscore their potential as an effective live carrier 2–4. Salmonella spp. is a pathogen. It may elicit live threatening disease in the host. Hence, their pathogenic properties have to be attenuated to ensure safe application. In this case, attenuation and immune-stimulation needs to be well in balance to guarantee safety and efficacy 5,6. This represents the basic problem of live bacterial carriers. Immune-recognition, immune-stimulation as well as immune-evasion of Salmonella are closely connected to the availability of pathogen associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS) or flagella 7. Responses to LPS, as an agonist of the Toll-like receptor 4 (TLR-4) and causative agent of sepsis are intensively investigated 8. LPS has been established as one of driving forces for adjuvanticity 5. These experiments included generation of conditionally attenuated Salmonella carrier strains. A regulatory connection between LPS modifications and Accepted Article This article is protected by copyright. All rights reserved. 5 flagella synthesis via the RflP/ClpXP pathway has been suggested 9. In addition, the impairment of both these two important PAMPs - LPS and flagellum - directly led to a loss of therapeutic potency. Apparently, such molecules are essential assets of the adjuvanticity of the Salmonella vector. However, the contribution of the flagellum to the establishment of a successful infection and its involvement in an efficacious immune stimulation is less well explored. On the one hand, flagellar motility contributes to pathogenesis by promoting bacteria-host interactions, adherence and invasion of host cells 10,11. On the other hand, as soon as Salmonella reaches its anatomical destination in the body, flagella synthesis is a serious burden for the microorganism. Downregulation to avoid immune recognition by Pattern-Recognition-Receptors (PPR), like extracellular TLR-5 or intracellular caspase-1 is essential for bacterial survival in vivo 12,13. This evasion mechanism already indicates that bacteria constitutively expressing flagella might elicit strong immune activation. The flagellum is a sophisticated macromolecular apparatus composed of several thousand copies of approximately 25 different proteins. It can be classified into three main parts: i) a basal body embedded in the cytoplasmic membrane that traverses the periplasm and cell wall up to the outer membrane (the engine), ii) a long external filament (the propeller) and iii) a flexible, curved structure known as the “hook”, which connects the basal body with the rigid filament 14. S. Typhimurium usually encodes two antigenically distinct filament proteins (the flagellins FliC or FljB), whose mutually exclusive expression is regulated by phase switching 15. Of note, strains constitutively expressing the phase-2 flagellin protein FljB were found to be more potent in targeting tumors in a murine tumor model compared to strains expressing phase-1 FliC 16. In line with these observations, Eom and colleagues observed an enhanced adjuvanticity of Salmonella that co-expressed both flagellin proteins FliC and FljB 17. These results demonstrate that a modulation of synthesis and assembly of flagella might allow to engineer appropriately modified bacterial Accepted Article This article is protected by copyright. All rights reserved. 6 vector strains for therapeutic applications. Recent results confirm the importance of FlaB flagella for successful Salmonella based cancer therapy 18. Furthermore, motility, chemotaxis and the presence of flagella as antigen have been shown to be important for tumor therapy as well 18–21. Thus, manipulations of the various flagellum sub-structures may positively influence the performance of the therapeutic strains. In the present study, we aimed to systematically unravel the connection between the presence of flagella components and immune stimulatory potency. We hypothesized that manipulating the spatiotemporal onset of flagella synthesis or of various steps in flagellar assembly might represent a valid strategy to increase the adjuvant power of Salmonella vector strains without increasing their pathogenicity. Thus, we investigated three groups of flagella mutants (Figure 1): i) flagellin phase locked mutants (FliC-ON, FljB-ON, FliC-ON & FljB-ON), ii) non-filamentous mutants (∆flgK, ∆fliF, ∆fliHIJ) and flagella overproduction mutants (∆rflP, ∆rflP ∆flgM and ∆rflP ∆flgM ∆rflM). In summary, our results highlight the importance of a controlled spatiotemporal regulation of flagella synthesis during host-pathogen interactions. Salmonella mutants lacking the inner membrane ring and basal body component FliF or the flagellum-specific ATPase complex FliHIJ displayed the highest therapeutic efficacy in murine tumor models despite the fact that they are unable to assemble flagella. We thus believe that the flagellum of Salmonella represents an ideal target for immunomodulatory modifications and it might be possible to generate optimized safe vector strains with improved adjuvant properties for prophylaxis and therapy. Accepted Article This article is protected by copyright. All rights reserved. 7 MATERIALS AND METHODS Ethics statement: All animal experiments were performed according to guidelines of the German Law for Animal Protection and with permission of the local ethics committee and the local authority LAVES (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) under permission number 33.9-42502-04-12/0713, 33.9-42502-04-13/1122 and 33.9-42502-04-13/1191. Strains and preparation of inoculum: Bacterial strains are shown in Table S1. Strain construction was done by P22 phage transduction or λ-red recombination 22. Salmonella strains were grown overnight and sub-cultured to mid-log phase in LB media at 37 °C. The bacteria were washed twice and adjusted to the desired OD600 in pyrogen free PBS. Plating served as control. Cell lines and primary cells: Immortalized RAW264.7 macrophages (RRID:CVCL_0493) were used for invasion assays and obtained from Raschke et al. 23 (Salk Institute, San Diego, USA). CT26 tumor cells (RRID:CVCL_7524, obtained from: Brattain et al. 24, Comprehensive Cancer Center, University of Alabama, USA) and RenCa tumor cells (RRID:CVCL_2174, obtained from: Wells et al. 25, Tumor Biology Center, Freiburg, Germany) were used for the subcutaneous murine tumor model. All cell cultures used in this study were tested and confirmed as mycoplasma-free. Bone-marrow derived macrophages (BMDMs) were isolated from the femur of BALB/c mice and differentiated using 20% (v/v) L929 (RRID:CVCL_0462, obtained from: Monner et al. 26, National Research Center for Biotechnology, Braunschweig, Germany) conditioned medium in RPMI containing 10% FCS. All cells were maintained at 37 °C, 5% CO2 and 90% rel. humidity. Motility assay: The motility of the Salmonella strains was assessed on semi-solid agar plates containing 0.3% (wt/vol) agar by inoculating 2 µl of a bacterial overnight culture into the agar and incubated at 37 °C. The swarm diameter was measured after 4 h incubation. Flagella immunostaining: Salmonella strains grown to mid-log growth phase were fixed on Llysine coated microscopy slides using formaldehyde (cf= 2%, v/v) and glutaraldehyde (cf = 0.2%, Accepted Article This article is protected by copyright. All rights reserved. 8 v/v). Flagellum staining was accomplished using polyclonal rabbit anti-FliC (Difco) as primary and anti-rabbit Alexa Fluor-488 as secondary antibody and the bacteria were stained with DAPI (SigmaAldrich). Images were taken using an Axio Observer microscope equipped with an Axiocam HR camera (Zeiss) at 100x magnification and analyzed with ImageJ. Invasion assays: RAW 264.7 and BMDM mycoplasma-free cells were used for the phagocytic uptake and intracellular replication. The assay was performed as described before 27 using MOIs of 1 and 10. CFUs were determined by plating of serial dilutions and compared to the corresponding parental strains. Murine tumor model: Six-week old BALB/c mice (Janvier) were intradermally inoculated with 5*105 syngeneic CT26 or 2*106 RenCa tumor cells in the right flank. Tumor development was monitored using caliper measurements. Upon reaching a tumor volume of approx. 150 mm³, the mice were injected intravenously into the tail vein with 5*106 Salmonella. Therapeutic efficacy: Tumor development was monitored using caliper measurements for as long as tumors persisted or until confronted with a humane endpoint in terms of exceedingly large tumor size (~ 1 cm³) or morbidity. Body weight as general health indicator was monitored using a scale. A loss of body weight below 80% of the original body weight was incentive to euthanize a subject. TNF-α ELISA measurement: Supernatant samples of cultivated macrophages were taken 6 h post infection. The TNF-α ELISA MaxTM Standard Kit (Biolegend) was used to determine the TNF-α level according to the manufacturer’s manual. Three different biological replicates were analyzed and a PBS treated group served as negative control. Cytokine, chemokine and growth factor detection in supernatants and sera: Cytokine, chemokine and growth factor concentrations in supernatants of 264.7 RAW macrophages cells (6 hpi) or sera (1.5 hpi, 6 hpi and 24 hpi) were quantified by the Luminex-based multiplex technique according to the manufacturer’s instructions (Bio-Rad, USA). Standard curves and concentrations were Accepted Article This article is protected by copyright. All rights reserved. 9 calculated with Bio-Plex Manager 6.0, the detection sensitivity of all proteins was between 1 pg/ml and 40 µg/ml. RNA isolation and sequencing: RNA isolation was performed as described previously 16. Library preparation of planktonic cultures grown to mid-log phase was done using the ScriptSeqTM v2 RNA-Seq Library Prep Kit (Illumina) and the vendor’s protocol. Sequence reads were mapped to the genome sequence of the reference strain Salmonella enterica subsp. enterica serovar Typhimurium UK-1 (Genbank, CP002614.1) using bowtie2 28. Differential gene expression was calculated using robust generalized linear models and the quasi-likelihood F-test of the R package edgeR 29. Genes were considered differentially expressed if the fold change expression was significantly greater than 2 (edgeR function glmTreat) with a false-discovery rate (FDR) cutoff of 0.05. Multidimensional scaling plots (MDS) were visualized using ggplot2 30. Genome sequencing and SNP calling: Genomic DNA was extracted from planktonic overnight cultures using the DNeasy Blood & Tissue kit (Qiagen) and sequenced using Illumina HiSeq. Single nucleotide polymorphism (SNP) analysis of DNA sequencing data was carried out using samtools mpileup and python 31. Statistics: Significance between two groups was determined using the nonparametric MannWhitney test, while one-way analysis of variance (ANOVA) with Bonferroni posttest was used to compare two or more groups. Significance levels of p < 0.05, p < 0.01, or p < 0.001 were denoted with asterisks: *, **, and ***, respectively. Data availability: All raw and processed sequencing data have been submitted to GEO (GSE116623; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE116623). All raw and processed genome sequencing data have been submitted to SRA (SRP153811, https://www.ncbi.nlm.nih.gov/sra/SRP153811). Accepted Article This article is protected by copyright. All rights reserved. 16 that may be responsible for this particular phenotype. We employed scanning electron microscopy to search for alterations at the cell surface or bacterial shape (Figure 6). Interestingly, this analysis revealed the presence of outer membrane vesicles (OMV) for SF102 + ΔfliF and SF102 + ΔfliHIJ mutants while all the other mutants did not exhibit such structures. Importantly, the combination of all features (e.g. ΔlpxR ΔpagP ΔpagL - ΔaroA - ΔfliF or ΔfliHIJ) was required to promote OMV formation (Supplementary Figure S6). Salmonella derivatives that lack at least one of these properties did not display any OMVs under our conditions. As OMVs include high amounts of LPS and are known to contain flagellar proteins 44, thus, the increased immunogenic potential may very well be attributed to these structures. Another possibility for the observed OMV production was due to an independent, unintentional mutation that may have accumulated during the strain constructions. Therefore, we sequenced the genome of the ΔfliF mutant and its parental strains as it exhibited the most pronounced OMV production (Table S3). No apparent SNPs or additional deletions were detected in the SF102 + ΔfliF strain that may have contributed to the OMV phenotype. Therefore, the genomic or transcriptional origin of the enhanced OMV formation remains unanswered. ΔfliHIJ and ΔfliF mutants display advanced anti-tumor properties: Next, we evaluated whether the strongly induced cytokine pattern and production of OMVs correlated with the therapeutic efficacy of the flagella mutant strains in a murine tumor model. Of note, tumor colonization itself appears not to be affected by the modifications of the bacterial flagella as shown previously 33 and confirmed by plating (data not shown). CT26 tumor-bearing mice were infected intravenously with 5*106 bacteria and the body weight changes as indicator for the health burden of therapy as well as the therapeutic efficacy was assessed (Figure 7). Upon bacterial application, all infected mice survived the therapy and weight loss was around 10% at most. This can be considered as minor Accepted Article This article is protected by copyright. All rights reserved. 17 (Figure 7A). The regulatory mutants SF102 + ΔflgK and SF102 + ΔrflP ΔflgM ΔrflM appeared to be highly attenuated as the body weight recovered very fast upon an initial drop. In contrast, the structural flagella mutants induced a persistent weight loss that only slowly recovered after 6 days. However, these structural mutants SF102 + ΔfliF and SF102 + ΔfliHIJ displayed the most pronounced anti-tumor effect. The bacteria cleared all the CT26 tumors within 8 or 4 days, respectively (Figure 7B and Supplementary Figure S7 for individual tumor development). In addition, the parental strain SF102 and its ΔflhDC derivative were able to clear 80% of the analyzed tumors (5/6). In contrast, the hyper-flagellated strain SF102 + ΔrflP ΔflgM ΔrflM and the assembly mutant SF102 + ΔflgK appeared to be over-attenuated. The tumor-clearing capacity was even lower than that of the parental SF102 strain. In summary, the ΔfliHIJ mutant strain exhibited the strongest potential to clear CT26 tumors. However, the ΔfliF strain exhibited the best balance of safety and efficacy. The mice recovered fastest from effects of the infection when exposed to these bacteria. Combination of SF102+ΔfliF or SF102+ΔfliHIJ strongly affect RenCa tumors: Although the cause of the strong production of OMVs in the SF102 + ΔfliF or SF102 + ΔfliHIJ strains remains unknown, these structures likely contribute to the improved immunogenicity. We therefore wondered how strongly the therapeutic potency of these strains was improved. Thus, we tested these mutants against the highly resistant cancer cell line RenCa (Figure 7C). As shown before, the LPS mutants only retarded the growth RenCa shortly 5. In contrast, employing SF102 + ΔfliF and SF102 + ΔfliHIJ for therapy dramatically improved the anti-tumor response and significantly prolonged the survival of the RenCa bearing mice. Thus, the unique combination of mutations and specifically the introduction of the gene deletions ΔfliF or ΔfliHIJ affecting flagella assembly and regulation resulted in anti-cancer strains of exceptional therapeutic potency. Accepted Article This article is protected by copyright. All rights reserved. 18 DISCUSSION Salmonella exerts unique direct interactions with the host cell via specialized secretion systems, effector proteins or PAMPs. Thus, Salmonella displays great potential as a highly versatile targeted delivery system for vaccination and cancer immune therapies 45–48. Flagella represent one of the major PAMPs of these bacteria. Hence, in the present study we aimed to investigate the effects of the structure, spatio-temporal regulation and synthesis of the bacterial flagellum during hostpathogen interactions and in therapy. To cover a broad range of flagellar phenotypes, we investigated the behavior of eleven different Salmonella variants mutated in various regulatory and structural components of the flagellum. For instance, deleting the hin recombinase responsible for flagellin phase variation in Salmonella allowed to engineer flagellin phase-locked mutants that expressed either FliC or FljB 49. In order to generate a mutant that co-expressed both filament proteins, we additionally deleted the negative regulator of fliC translation, FljA, in a FljB-ON background 50. Under these conditions, every individual flagellum presumably consists of a mixture of both proteins. The variant exhibited reduced motility although the number of flagella per cell was normal. It might be possible that the structural differences between both flagellin types decrease the efficiency of flagella function, e.g. by impairing filament bundle formation. In comparison to the Wt, the FliC-ON phase-locked mutant did not exhibit any superior immunogenic behavior in vitro and in vivo. In contrast, immune activation by the FljB-locked strain was increased during the early stages of infection. This correlated with previous observations that Salmonella expressing FljB exhibit higher adjuvant potential 17,51. This might also explain why the strain SF102, published previously, represents a highly immunogenic therapeutic vector. It predominately expresses FljB 16. The beneficial effect of FljB-only expression vanishes at the later stages of infection. Accepted Article This article is protected by copyright. All rights reserved. 19 Salmonella in the mammalian host is known to down-regulate flagella expression to avoid recognition by TLR-5 or caspase-1 13,52. To counteract this escape mechanism, we investigated the role of regulators of flagella synthesis: rflP, flgM and rflM 38,53,54. As expected, strains bearing deletions of either gene resulted in a significantly increased flagellation status and enhanced flagellin production. Interestingly, this overproduction affected bacterial growth in vitro. Furthermore, when exposing the overproducing triple mutant ΔrflP ΔflgM ΔrflM to macrophages, we detected increased levels of LDH in the supernatants. This suggested that the hyper-flagellation might induce intracellular caspase-1 resulting in pyroptosis and macrophage cell death 55. Bacterial death would consequently be the result from exposure to gentamycin in the medium. In vivo, such flagella-overproducing strains would not be able to hide from immune recognition to establish their intracellular niche. This is consistent with in vivo efficacy of this strain. It appeared over-attenuated. The mice recovered already 4 dpi and the tumor clearing efficacy was only slightly above 50%. Thus, hyper-flagellation of the Salmonella vector could represent a promising strategy to increase vaccine adjuvanticity but may not be a potent strategy for tumor therapeutic applications. As hyper-flagellation did not appear to be beneficial due to the metabolic burden and/or the extensive activation of pyroptosis, we next investigated the ΔflgK mutant, which is able to secrete monomeric flagellin, but is unable to assemble flagellar filaments 56. In vitro, the ΔflgK mutant induced a strong IL-6 and TNF-α response. This may indicate a strong stimulation of the TLR-5 receptor due to the enhanced levels of monomeric flagellin 57. However, this strong phenotype disappeared in vivo. Possibly, soluble flagellin was diluted or digested in the blood or the tissue and did not reach concentrations sufficient to stimulate TLR-5 or intracellular receptors like NOD-like receptors or NLRP3. This also correlated with the low efficacy in the anti-tumor response. Therefore, a ΔflgK deletion may not represent a good choice when attempting to optimize a vector strain for therapy. Accepted Article This article is protected by copyright. All rights reserved. 20 Finally, we tested strains without extracellular flagella. We deleted the gene coding for FliF, which forms the MS-ring of the flagellar basal body in the inner membrane 37, or FliHIJ, which is an ATPase complex involved in the export of flagella building blocks 58. Both strains neither expressed nor secreted flagellin and thus were non-motile. Interestingly, both strains were highly immunogenic in vitro and in vivo. In an otherwise Wt background, the ΔfliF mutation induced secretion of high levels of pro-inflammatory cytokines like TNF-α or IL-6. Once the mutation was transferred onto our immunogenic background strain UK-1, the ΔfliHIJ mutation displayed the most pronounced cytokine response. As FliC is an important PAMP that confers immunogenicity, this finding was surprising since these mutants are not able to express flagellin and therefore lack the flagellar filament. Importantly, in our therapeutic model the SF102 + ΔfliF and SF102 + ΔfliHIJ strains were able to clear all CT26 tumors within 8 or 4 days, respectively. All mice survived the therapy. Therefore, these strains exhibit an optimal balance of safety and efficacy. As putative reason for their superiority, we made the serendipitous discovery that a substantial amount of OMVs was produced by these mutants. Although the mechanism of OMV production still remains elusive, we believe that they significantly contribute to the superior therapeutic efficacy of these strains. The increased levels of pro-inflammatory cytokines may derive from the LPS that predominately forms the vesicles or the putative cargo of the OMVs that might include bacterial RNA and DNA 59. In addition, recent proteome studies have shown that OMVs can carry flagellar proteins 44. The MS-ring made of FliF and the ATPase complex FliHIJ are important components of the flagella export apparatus that secretes flagellar building blocks from the intracellular to the extracellular space. The deletion of either the Class 2 genes fliF or fliHIJ results in defective flagellar basal bodies and therefore accumulate flagellar building blocks in the cytoplasm. Thus, the enhanced OMV production might be a strategy of the cell to remove excessive flagellar proteins. This Accepted Article This article is protected by copyright. All rights reserved. 21 hypothesis is supported by the fact that no negative regulator controlling flagella synthesis at the level of Class 2 gene expression was found to be significantly regulated. Besides their immune-stimulatory capacity, the presence of OMVs could be further exploited as delivery system in future studies 60,61. Therefore, using deletions of the Class 2 genes fliF and fliHIJ in combination with other immunomodulatory mutations like the SF102 background could turn Salmonella into a very effective delivery vector platform. Various immunogenic cargos or therapeutic agents may be transported directly into tumor cells or immune cells of the host. Taken together, the correct spatiotemporal regulation of flagella synthesis during host-pathogen interactions exerts direct impact on the therapeutic efficacy of Salmonella vector strains. While mutants over-expressing flagella were too sensitive to the immune system of the host, the mutations ∆fliF and ∆fliHIJ appeared to be highly efficacious in our murine tumor models. Increased immunogenic properties were conferred by these mutations. Especially the unique production of high numbers of OMVs induced by mutations of fliF and fliHIJ might allow to enhance intrinsic therapeutic features of the bacteria and render Salmonella into an efficacious delivery platform. Such Salmonella-based vectors might represent potent strains for cancer therapy, which is evidenced by the substantial growth retardation of the very resilient RenCa tumors, which have been highly resistant to this type of therapy thus far. Accepted Article This article is protected by copyright. All rights reserved. 22 ACKNOWLEDGEMENTS Our gratitude is extended to Regina Lesch and Nadine Körner for their expert technical assistance. Moreover, we thank Roy Curtiss III for providing parental strains of Salmonella, along with expert advice concerning strain design. The study was supported in part by the Niedersächsische Krebsgesellschaft, the Deutsche Krebshilfe, the Bundesministerium für Bildung und Forschung (BMBF) via the INDIGO program, Hannover Biomedical Research School (HBRS) (all to SW), a Lichtenberg Fellowship from the Niedersächsiche Ministerium für Wissenschaft und Kultur (MWK) (to SF), an Exploration Grant of the Boehringer Ingelheim Foundation (BIS) (to SF and ME) and the Helmholtz Association Young Investigator grant no. VH-NG-932 (to ME). AUTHOR CONTRIBUTIONS S.F., I.S., V.P., D.K., C.F. and M.R. performed and analyzed the experiments. M.P. and S.H. were responsible for the sequencing analyses. S.H., S.W. and M.E. provided ideas and critical comments. S.F., S.W. and M.E. wrote the manuscript. S.W. and M.E. directed the project. CONFLICT OF INTEREST The authors declare that there is no conflict of interest. Accepted Article This article is protected by copyright. All rights reserved. 23 REFERENCES 1. Bournazos S, Ravetch J V. 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