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Aggregatibacter aphrophilus, a Gram negative bacterium, found in the oral cavity, causing cerebral abscesses and infective endocarditis, has been shown to be serum resistant in previous studies. Bacterial secretion systems are important for bacteria as they transfer virulence factors into other bacteria or host cells as an attack. A. aphrophilus encodes a type VI secretion system, which is a spike-like membrane protein, mainly consisting of a hemolysin co-regulated protein (Hcp). In this work, it was tested if Hcp would contribute to serum resistance of A. aphrophilus. Firstly, to assess Hcp contribution to serum resistance, a bacterial serum killing assay-method was used and data was collected from three independent experiments. Two strains of A. aphrophilus were used in the experiments: the laboratory strain HK83 and a HK83 hcp mutant strain. The results showed that Hcp provided no significant effect on serum resistance of A. aphrophilus. Secondly, optical density measurements were made for growth curve analysis, to determine if the HK83 hcp mutant strain had an impact in growth compared to HK83. The results indicated that the HK83 hcp mutant strain had a somewhat reduced growth compared to its parental strain.

Aggregatibacter aphrophilus, a Gram negative bacterium, found in the oral cavity, causing cerebral abscesses and infective endocarditis, has been shown to be serum resistant in previous studies. Bacterial secretion systems are important for bacteria as they transfer virulence factors into other bacteria or host cells as an attack. A. aphrophilus encodes a type VI secretion system, which is a spike-like membrane protein, mainly consisting of a hemolysin co-regulated protein (Hcp). In this work, it was tested if Hcp would contribute to serum resistance of A. aphrophilus. Firstly, to assess Hcp contribution to serum resistance, a bacterial serum killing assay-method was used and data was collected from three independent experiments. Two strains of A. aphrophilus were used in the experiments: the laboratory strain HK83 and a HK83 hcp mutant strain. The results showed that Hcp provided no significant effect on serum resistance of A. aphrophilus. Secondly, optical density measurements were made for growth curve analysis, to determine if the HK83 hcp mutant strain had an impact in growth compared to HK83. The results indicated that the HK83 hcp mutant strain had a somewhat reduced growth compared to its parental strain.

The life of bacteria is challenging, to endure bacteria employ a range of mechanisms to optimize their environment, including deploying the type VI secretion system (T6SS). Acting as a bacterial crossbow, this system delivers effectors responsible for subverting host cells, killing competitors and facilitating general secretion to access common goods. Due to its importance, this lethal machine has been evolutionarily maintained, disseminated and specialized to fulfil these vital functions. In fact, T6SS structural clusters are present in over 25% of Gram-negative bacteria, varying in number from one to six different genetic clusters per organism. Since its discovery in 2006, research on the T6SS has rapidly progressed, yielding remarkable breakthroughs. The identification and characterization of novel components of the T6SS, combined with biochemical and structural studies, have revealed fascinating mechanisms governing its assembly, loading, firing and disassembly processes. Recent findings have also demonstrated the efficacy of this system against fungal and Gram-positive cells, expanding its scope. Ongoing research continues to uncover an extensive and expanding repertoire of T6SS effectors, the genuine mediators of T6SS function. These studies are shedding light on new aspects of the biology of prokaryotic and eukaryotic organisms. This review provides a comprehensive overview of the T6SS, highlighting recent discoveries of its structure and the diversity of its effectors. Additionally, it injects a personal perspective on avenues for future research, aiming to deepen our understanding of this combative system.

The life of bacteria is challenging, to endure bacteria employ a range of mechanisms to optimize their environment, including deploying the type VI secretion system (T6SS). Acting as a bacterial crossbow, this system delivers effectors responsible for subverting host cells, killing competitors and facilitating general secretion to access common goods. Due to its importance, this lethal machine has been evolutionarily maintained, disseminated and specialized to fulfil these vital functions. In fact, T6SS structural clusters are present in over 25% of Gram-negative bacteria, varying in number from one to six different genetic clusters per organism. Since its discovery in 2006, research on the T6SS has rapidly progressed, yielding remarkable breakthroughs. The identification and characterization of novel components of the T6SS, combined with biochemical and structural studies, have revealed fascinating mechanisms governing its assembly, loading, firing and disassembly processes. Recent findings have also demonstrated the efficacy of this system against fungal and Gram-positive cells, expanding its scope. Ongoing research continues to uncover an extensive and expanding repertoire of T6SS effectors, the genuine mediators of T6SS function. These studies are shedding light on new aspects of the biology of prokaryotic and eukaryotic organisms. This review provides a comprehensive overview of the T6SS, highlighting recent discoveries of its structure and the diversity of its effectors. Additionally, it injects a personal perspective on avenues for future research, aiming to deepen our understanding of this combative system.

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii’s specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation., This work was funded by the Centre National de la Recherche Scientifique, the Aix-Marseille Université, and grants from the Agence Nationale de la Recherche (ANR-18-CE11-0023-01) and European Society of Clinical Microbiology and Infectious Diseases (ESCMID) to ED. ED is supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). YC is funded by a Doctoral school PhD fellowship from the FRM (ECO20160736014 & FDT201904008052). OK is funded by a Doctoral school PhD fellowship from DGA and Aix-Marseille University and by the FRM (01D19024292-A AID & FDT202204014851). PS post-doctoral fellowship was supported by the European Respiratory Society under the ERS Long-Term Fellowship grant agreement LTRF - 202101-00862. IFM is funded by ANR-17-CE11-0039. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Peer Reviewed, Postprint (published version)

The genes of the type VI secretion system (T6SS) from Rhizobium etli Mim1 (ReMim1) that contain possible effectors can be divided into three modules. The mutants in them indicated that they are not required for effective nodulation with beans. To analyze T6SS expression, a putative promoter region between the tssA and tssH genes was fused in both orientations to a reporter gene. Both fusions are expressed more in free living than in symbiosis. When the module-specific genes were studied using RT-qPCR, a low expression was observed in free living and in symbiosis, which was clearly lower than the structural genes. The secretion of Re78 protein from the T6SS gene cluster was dependent on the presence of an active T6SS. Furthermore, the expression of Re78 and Re79 proteins in E. coli without the ReMim1 nanosyringe revealed that these proteins behave as a toxic effector/immunity protein pair (E/I). The harmful action of Re78, whose mechanism is still unknown, would take place in the periplasmic space of the target cell. The deletion of this ReMim1 E/I pair resulted in reduced competitiveness for bean nodule occupancy and in lower survival in the presence of the wild-type strain.

The type VI secretion system (T6SS) present in many Gram-negative bacteria is a contact-dependent apparatus that can directly deliver secreted effectors or toxins into diverse neighboring cellular targets including both prokaryotic and eukaryotic organisms. Recent reverse genetics studies with T6 core gene loci have indicated the importance of functional T6SS toward overall competitive fitness in various pathogenic Xanthomonas spp. To understand the contribution of T6SS toward ecology and evolution of Xanthomonas spp., we explored the distribution of the three distinguishable T6SS clusters, i3*, i3***, and i4, in approximately 1,740 Xanthomonas genomes, along with their conservation, genetic organization, and their evolutionary patterns in this genus. Screening genomes for core genes of each T6 cluster indicated that 40% of the sequenced strains possess two T6 clusters, with combinations of i3*** and i3* or i3*** and i4. A few strains of Xanthomonas citri, Xanthomonas phaseoli, and Xanthomonas cissicola were the exception, possessing a unique combination of i3* and i4. The findings also indicated clade-specific distribution of T6SS clusters. Phylogenetic analysis demonstrated that T6SS clusters i3* and i3*** were probably acquired by the ancestor of the genus Xanthomonas, followed by gain or loss of individual clusters upon diversification into subsequent clades. T6 i4 cluster has been acquired in recent independent events by group 2 xanthomonads followed by its spread via horizontal dissemination across distinct clades across groups 1 and 2 xanthomonads. We also noted reshuffling of the entire core T6 loci, as well as T6SS spike complex components, hcp and vgrG, among different species. Our findings indicate that gain or loss events of specific T6SS clusters across Xanthomonas phylogeny have not been random.

Pseudomonas aeruginosa is an opportunistic pathogen of great clinical impact, being the main cause of acute and chronic infections in immunosuppressed and cystic fibrosis patients. This virulence is largely due to the secretion systems possessed by the bacteria, among which the Type 6 Secretion System (T6SS) in P. aeruginosa stands out 1. The T6SS is a nanomachine that assembles inside the bacteria and injects severa effectors/toxins into target cells 2. The large number of effectors and their functional diversity play a crucial role in the virulence of the infection caused by this gram-negative bacterium. One of these effectors is the Type VI secretion system exported effector 5 (Tse5) which was described to have bacteriolytic activity although its molecular activity is still unknown 3,4. In this work we studied the molecular function of Tse5 effector. To do so, we performed growth inhibition curves to determine if it has a bacteriolytic or bacteriostatic effect. We hypothesised that the toxic domain of Tse5 (Tse5-CT) exerts its toxic activity on the membrane since its immunity protein (Tsi5) inserts into the inner bacterial membrane to neutralise the toxicity 3. To define if this toxin can increase cell permeability or disrupt membrane potential of the target bacteria, we carried out flow cytometry experiments in Pseudomonas putida. Furthermore, we designed a Tse5-CT deletion mutants with a dual reporter localize at the C-terminus that allowed to identify transmembrane regions. Our data indicate that Tse5-CT expression has a bacteriolytic effect on Pseudomonas putida cells that can be reversed by co-expression of the cognate immunity protein Tsi5. Moreover, Tse5 toxin inserts into the membrane through transmembrane and amphipathic domains and disrupts membrane potential of target cells. The results obtained in this study help to gain insight regarding the function and the mechanisms of action Tse5 effector and eventually could help to develop drugs that mimic its

The bacterium Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. The gut microbiome, or the native community of microorganisms found in the human gastrointestinal tract, is increasingly being recognized as a factor in driving susceptibility to infection, in vivo fitness, and host interactions of this pathogen. Here, we review a subset of the emerging studies in how gut microbiome structure and microbial function are able to drive V. cholerae virulence gene regulation, metabolism, and modulate host immune responses to cholera infection and vaccination. Improved mechanistic understanding of commensal-pathogen interactions offers new perspectives in the design of prophylactic and therapeutic approaches for cholera control.

The discovery of ATP-dependent proteolysis began with Lon protease in the1960s. Since that time, Lon (or LonA) has been identified as a key regulator of protein quality control and diverse cellular processes in archaea, bacteria, as well as in the mitochondria of eukaryotic cells. Despite nearly 60 years of research, the substrates of Lon and the mechanisms that dictate Lon proteolysis remain poorly understood. The work presented here focuses on understanding the mechanisms and consequences of Lon proteolysis in V. cholerae. Vibrio cholerae is the Gram-negative facultative pathogen responsible for the diarrheal disease cholera. V. cholerae remains a threat to global public health. There are estimated to be 1.3-4.0 million cases of cholera and 21,000-143,000 deaths worldwide each year. Lon plays a critical role in regulating processes important for V. cholerae's pathogenic cycle. For example, Lon regulates virulence factor production, type VI secretion system (T6SS)-dependent killing, biofilm formation, motility, c-di-GMP levels, cell division, and stress adaptation. Furthermore, V. cholerae mutants defective in Lon poorly colonize the host intestinal tract. Previous work performed by the Yildiz lab identified Lon as a repressor of the T6SS, which was a novel function attributed to prokaryotic Lon. To better understand how Lon might regulate the T6SS, I compiled the known functional and regulatory networks governing activation of the T6SS (Chapter 1). We then used whole proteome analysis to identify potential Lon targets that might explain Lon repression of the T6SS (Chapter 2). We identified TfoY as a Lon substrate and showed that Lon-dependent proteolysis of TfoY represses T6SS-dependent killing and motility. In addition, we used a combination of genetic and biochemical approaches to demonstrate that Lon binds to c-di-GMP and that c-di-GMP inhibits Lon-dependent proteolysis of TfoY. Most analyses on Lon have focused on Lon-dependent regulation in planktonic grown c

The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coll. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function. IMPORTANCE The type VI secretion system (T6SS) is an effective weapon used by bacteria to outgrow or kill competitors. It can be used by endogenous commensal microbiota to prevent invasion by pathogens or by pathogens to overcome resident flora and successfully colonize a host or a specific environmental niche. The T6SS is a key contributor to this continuous arms race between organisms as it delivers a multitude of toxins directed at essential processes, such as nucleic acid synthesis and replication, cell wall and

The bacterium Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. The gut microbiome, or the native community of microorganisms found in the human gastrointestinal tract, is increasingly being recognized as a factor in driving susceptibility to infection, in vivo fitness, and host interactions of this pathogen. Here, we review a subset of the emerging studies in how gut microbiome structure and microbial function are able to drive V. cholerae virulence gene regulation, metabolism, and modulate host immune responses to cholera infection and vaccination. Improved mechanistic understanding of commensal-pathogen interactions offers new perspectives in the design of prophylactic and therapeutic approaches for cholera control.

The discovery of ATP-dependent proteolysis began with Lon protease in the1960s. Since that time, Lon (or LonA) has been identified as a key regulator of protein quality control and diverse cellular processes in archaea, bacteria, as well as in the mitochondria of eukaryotic cells. Despite nearly 60 years of research, the substrates of Lon and the mechanisms that dictate Lon proteolysis remain poorly understood. The work presented here focuses on understanding the mechanisms and consequences of Lon proteolysis in V. cholerae. Vibrio cholerae is the Gram-negative facultative pathogen responsible for the diarrheal disease cholera. V. cholerae remains a threat to global public health. There are estimated to be 1.3-4.0 million cases of cholera and 21,000-143,000 deaths worldwide each year. Lon plays a critical role in regulating processes important for V. cholerae's pathogenic cycle. For example, Lon regulates virulence factor production, type VI secretion system (T6SS)-dependent killing, biofilm formation, motility, c-di-GMP levels, cell division, and stress adaptation. Furthermore, V. cholerae mutants defective in Lon poorly colonize the host intestinal tract. Previous work performed by the Yildiz lab identified Lon as a repressor of the T6SS, which was a novel function attributed to prokaryotic Lon. To better understand how Lon might regulate the T6SS, I compiled the known functional and regulatory networks governing activation of the T6SS (Chapter 1). We then used whole proteome analysis to identify potential Lon targets that might explain Lon repression of the T6SS (Chapter 2). We identified TfoY as a Lon substrate and showed that Lon-dependent proteolysis of TfoY represses T6SS-dependent killing and motility. In addition, we used a combination of genetic and biochemical approaches to demonstrate that Lon binds to c-di-GMP and that c-di-GMP inhibits Lon-dependent proteolysis of TfoY. Most analyses on Lon have focused on Lon-dependent regulation in planktonic grown c

Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to out-compete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.

Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. For its survival advantage, the bacterium adapts to a motile or sessile lifestyle when infecting the host. The active type III secretion system in motile bacterial cells enables a cytotoxic effect on the host. In contrast, an operative type VI secretion in sessile bacterial cells embedded in biofilms helps survive against bacterial competitors. P. aeruginosa switches between the lifestyles through regulatory pathways, including Gac-Rsm and secondary messengers like c-di-GMP. In this thesis, different molecular techniques were applied to understand the regulatory pathways of H1-T6SS, and the newly identified modulators were characterized. The results revealed the RNA binding protein RtcB as a switch controller of a motile and sessile bacterial lifestyle. Wildtype PAO1 in in-vitro conditions, armed with an active T3SS and inactive T6SS. In contrast, PAO1(∆retS) bears an active T6SS and repressed T3SS. Deletion of rtcB led to simultaneous expression of T3SS and T6SS in both PAO1 and PAO1(∆retS). Furthermore, the deletion of rtcB increased the biofilm formation in PAO1(∆rtcB) and restored the motility of PAO1(∆rtcB∆retS). The killing assay showed that H1-T6SS was activated in PAO1(∆rtcB) and could compete against Escherichia coli. Transcriptome analysis was performed for PAO1, PAO1(∆rtcB), PAO1(∆retS), and PAO1(∆rtcB∆retS). It revealed 370 genes of PAO1(∆rtcB) and 1030 genes in PAO1(∆rtcB∆retS) were differentially expressed. That includes genes encoding various virulence factors and the four types of secretion systems. Quantification of c-di-GMP showed elevated levels in PAO1(∆rtcB), which contributed to the altered phenotype and characteristics of PAO1(∆rtcB). The long-chain-fatty-acid—CoA ligase FadD1 was also identified as a new player in H1-T6SS regulation in this thesis. It deactivated the expression of

The bacterial type VI secretion system (T6SS) system is a contractile secretion apparatus that delivers proteins to neighboring bacterial or eukaryotic cells. Antibacterial effectors are mostly toxins that inhibit the growth of other species and help to dominate the niche. A broad variety of these toxins cause cell lysis of the prey cell by disrupting the cell envelope. Other effectors are delivered into the cytoplasm where they affect DNA integrity, cell division or exhaust energy resources. The modular nature of T6SS machinery allows different means of recruitment of toxic effectors to secreted inner tube and spike components that act as carriers. Toxic effectors can be translationally fused to the secreted components or interact with them through specialized structural domains. These interactions can also be assisted by dedicated chaperone proteins. Moreover, conserved sequence motifs in effector-associated domains are subject to genetic rearrangements and therefore engage in the diversification of the arsenal of toxic effectors. This review discusses the diversity of T6SS secreted toxins and presents current knowledge about their loading on the T6SS machinery., info:eu-repo/semantics/published

Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. For its survival advantage, the bacterium adapts to a motile or sessile lifestyle when infecting the host. The active type III secretion system in motile bacterial cells enables a cytotoxic effect on the host. In contrast, an operative type VI secretion in sessile bacterial cells embedded in biofilms helps survive against bacterial competitors. P. aeruginosa switches between the lifestyles through regulatory pathways, including Gac-Rsm and secondary messengers like c-di-GMP. In this thesis, different molecular techniques were applied to understand the regulatory pathways of H1-T6SS, and the newly identified modulators were characterized. The results revealed the RNA binding protein RtcB as a switch controller of a motile and sessile bacterial lifestyle. Wildtype PAO1 in in-vitro conditions, armed with an active T3SS and inactive T6SS. In contrast, PAO1(∆retS) bears an active T6SS and repressed T3SS. Deletion of rtcB led to simultaneous expression of T3SS and T6SS in both PAO1 and PAO1(∆retS). Furthermore, the deletion of rtcB increased the biofilm formation in PAO1(∆rtcB) and restored the motility of PAO1(∆rtcB∆retS). The killing assay showed that H1-T6SS was activated in PAO1(∆rtcB) and could compete against Escherichia coli. Transcriptome analysis was performed for PAO1, PAO1(∆rtcB), PAO1(∆retS), and PAO1(∆rtcB∆retS). It revealed 370 genes of PAO1(∆rtcB) and 1030 genes in PAO1(∆rtcB∆retS) were differentially expressed. That includes genes encoding various virulence factors and the four types of secretion systems. Quantification of c-di-GMP showed elevated levels in PAO1(∆rtcB), which contributed to the altered phenotype and characteristics of PAO1(∆rtcB). The long-chain-fatty-acid—CoA ligase FadD1 was also identified as a new player in H1-T6SS regulation in this thesis. It deactivated the expression of

Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.

Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.

Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.

The Gram-negative bacterium Vibrio cholerae is responsible for the severe diarrheal pandemic disease cholera, representing a major global public health concern. This pathogen transitions from aquatic reservoirs into epidemics in human populations, and has evolved numerous mechanisms to sense this transition in order to appropriately regulate its gene expression for infection. At the intersection of pathogen and host in the gastrointestinal tract lies the community of native gut microbes, the gut microbiome. It is increasingly clear that the diversity of species and biochemical activities within the gut microbiome represents a driver of infection outcome, through their ability to manipulate the signals used by V. cholerae to regulate virulence and fitness in vivo. A better mechanistic understanding of how commensal microbial action interacts with V. cholerae pathogenesis may lead to novel prophylactic and therapeutic interventions for cholera. Here, we review a subset of this burgeoning field of research.

Bacteria evolved multiple strategies to survive and develop optimal fitness in their ecological niche. They deployed protein secretion systems for robust and efficient delivery of antibacterial toxins into their target cells, therefore inhibiting their growth or killing them. To maximize antagonism, recipient factors on target cells can be recognized or hijacked to enhance the entry or toxicity of these toxins. To date, knowledge regarding recipient susceptibility (RS) factors and their mode of action is mostly originating from studies on the type Vb secretion system that is also known as the contact-dependent inhibition (CDI) system. Yet, recent studies on the type VI secretion system (T6SS), and the CDI by glycine-zipper protein (Cdz) system, also reported the emerging roles of RS factors in interbacterial competition. Here, we review these RS factors and their mechanistic impact in increasing susceptibility of recipient cells in response to CDI, T6SS, and Cdz. Past and future strategies for identifying novel RS factors are also discussed, which will help in understanding the interplay between attacker and prey upon secretion system-dependent competition. Understanding these mechanisms would also provide insights for developing novel antibacterial strategies to antagonize aggressive bacteria-killing pathogens.

Bacteria evolved multiple strategies to survive and develop optimal fitness in their ecological niche. They deployed protein secretion systems for robust and efficient delivery of antibacterial toxins into their target cells, therefore inhibiting their growth or killing them. To maximize antagonism, recipient factors on target cells can be recognized or hijacked to enhance the entry or toxicity of these toxins. To date, knowledge regarding recipient susceptibility (RS) factors and their mode of action is mostly originating from studies on the type Vb secretion system that is also known as the contact-dependent inhibition (CDI) system. Yet, recent studies on the type VI secretion system (T6SS), and the CDI by glycine-zipper protein (Cdz) system, also reported the emerging roles of RS factors in interbacterial competition. Here, we review these RS factors and their mechanistic impact in increasing susceptibility of recipient cells in response to CDI, T6SS, and Cdz. Past and future strategies for identifying novel RS factors are also discussed, which will help in understanding the interplay between attacker and prey upon secretion system-dependent competition. Understanding these mechanisms would also provide insights for developing novel antibacterial strategies to antagonize aggressive bacteria-killing pathogens.

The Gram-negative bacterium Vibrio cholerae is responsible for the severe diarrheal pandemic disease cholera, representing a major global public health concern. This pathogen transitions from aquatic reservoirs into epidemics in human populations, and has evolved numerous mechanisms to sense this transition in order to appropriately regulate its gene expression for infection. At the intersection of pathogen and host in the gastrointestinal tract lies the community of native gut microbes, the gut microbiome. It is increasingly clear that the diversity of species and biochemical activities within the gut microbiome represents a driver of infection outcome, through their ability to manipulate the signals used by V. cholerae to regulate virulence and fitness in vivo. A better mechanistic understanding of how commensal microbial action interacts with V. cholerae pathogenesis may lead to novel prophylactic and therapeutic interventions for cholera. Here, we review a subset of this burgeoning field of research.

All bacteria must compete for growth niches and other limited environmental resources. These existential battles are waged at several levels, but one common strategy entails the transfer of growth-inhibitory protein toxins between competing cells. These antibacterial effectors are invariably encoded with immunity proteins that protect cells from intoxication by neighboring siblings. Several effector classes have been described, each designed to breach the cell envelope of target bacteria. Although effector architectures and export pathways tend to be clade specific, phylogenetically distant species often deploy closely related toxin domains. Thus, diverse competition systems are linked through a common reservoir of toxin-immunity pairs that is shared via horizontal gene transfer. These toxin-immunity protein pairs are extraordinarily diverse in sequence, and this polymorphism underpins an important mechanism of self/nonself discrimination in bacteria. This review focuses on the structures, functions, and delivery mechanisms of polymorphic toxin effectors that mediate bacterial competition.

Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.

Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus Francisella, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of Francisella were estimated and compared to those of two other facultative intracellular pathogens, Legionella and Piscirickettsia. Francisella had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for Legionella and Piscirickettsia respectively, while the pan-genome of Legionella was more than twice the size of that of the other two genera. Also, the composition of the Francisella Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. In silico approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus Francisella and for the identification of putative T6SS virulence traits.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S. Gallinarum G9 and its ΔSPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 ΔSPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

Salmonella Pathogenicity Islands 19 (SPI19) encodes a type VI secretion system (T6SS). SPI19 is only present in few serovars of S. enterica, including the host-adapted serovar S. Dublin and the host-specific serovar S. Gallinarum. The role of the SPI19 encoded T6SS in virulence in these serovar is not fully understood. Here we show that during infection of mice, a SPI19/T6SS deleted strain of S. Dublin 2229 was less virulent than the wild type strain after oral challenge, but not after IP challenge. The mutant strain also competed significantly poorer than the wild type strain when co-cultured with strains of E. coli, suggesting that this T6SS plays a role in pathogenicity by killing competing bacteria in the intestine. No significant difference was found between wild type S. Gallinarum G9 and its ΔSPI19/T6SS mutant in infection, whether chicken were challenged orally or by the IP route, and the S. Gallinarum G9 ΔSPI19/T6SS strain competed equally well as the wild type strain against strains of E. coli. However, contrary to what was observed with S. Dublin, the wild type G9 strains was significantly more cytotoxic to monocyte derived primary macrophages from hens than the mutant, suggesting that SPI19/T6SS in S. Gallinarum mediates killing of eukaryotic cells. The lack of significant importance of SPI19/T6SS after oral and systemic challenge of chicken was confirmed by knocking out SPI19 in a second strain, J91. Together the results suggest that the T6SS encoded from SPI19 have different roles in the two serovars and that it is a virulence-factor after oral challenge of mice in S. Dublin, while we cannot confirm previous results that SPI19/T6SS influence virulence significantly in S. Gallinarum.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

Bacteria live in complex communities and must compete with their neighborsfor resources. They have therefore evolved multiple different competition systems inorder to improve their fitness in dense, complex environments. The type VI secretionsystem (T6SS) is a Gram-negative weapon used to inhibit the growth of neighboringcells. It does this by delivering toxic effector proteins directly into neighbors using aspeargun-like apparatus. A spear-shaped protein complex is propelled out of theT6SS-expressing cell, and this spear punctures a neighboring cell to deliver its toxicpayload. Toxic effector proteins are bound either covalently or non-covalently to thissecreted complex, and these toxins can target both periplasmic or cytoplasmicsubstrates, as well as membranes. Kin protect themselves from these effectors byexpressing cognate immunity proteins to block effector activity.This thesis explores several aspects of the T6SS in Enterobacter cloacae ATCC13047. In Chapter 1, I provide a general introduction to the T6SS and covers topicssuch as the structure and assembly of the apparatus, regulation of T6SS loci andeffectors, and effector diversity. I then describe the genetics of the T6SS in E. cloacaeand explore what effectors are deployed in this system in Chapter 2. In Chapter 3, Ifocus on one particular effector deployed by this system, the type VI secretion systemlipase effector, Tle, and investigate its intriguing reliance on its cognate immunityprotein, Tli, for toxicity. Next, Chapter 4 discusses the role of rearrangement hotspotix(Rhs) proteins in the assembly of the T6SS apparatus. Chapter 5 then looks at therole of the Rhs accessory protein effector-associated gene with Rhs (EagR). Finally, Isummarize my findings and discuss open questions in the field in Chapter 6.

Bacteria are widespread on Earth. They play important roles in shaping the global ecosystems as well as influencing human health. In nature, different bacterial species often coexist in a common environment. The interactions of them are diverse and often lead to intricate spatial-temporal dynamics. Quantitative experimental measurements as well as mathematical modeling could help us better understand the mechanisms behind such dynamics. In this dissertation, I discuss several spatial-temporal structures for multi-strain bacterial systems and different modeling strategies are used to help explore the mechanisms of different spatial-temporal patterns. A key question regarding a system of different bacterial species is how coexistence is maintained, considering that different bacterial species often have different growth rates and they can even kill each other sometimes. In Chapter Two, I discuss a novel theoretical framework for robust coexistence and pattern formation in bacterial mixtures with contact-dependent killing. In Chapter Three, I present a beautiful spatial flower-like pattern in two-species bacterial systems from experiments and build two different models, a discrete interface model and a phase-field model, to elucidate how differential motility and mechanical interactions between bacterial species can create complex spatial structures. In Chapter Four, I introduce the agent-based modeling, which simulates each individual bacterial cell in large populations. Two examples of applications of such agent-based modeling in microfluidic environments are described. All these chapters combine to highlight the effort towards understanding the spatial-temporal dynamics of multi-strain microbial systems quantitatively.

Bacteria live in complex communities and must compete with their neighborsfor resources. They have therefore evolved multiple different competition systems inorder to improve their fitness in dense, complex environments. The type VI secretionsystem (T6SS) is a Gram-negative weapon used to inhibit the growth of neighboringcells. It does this by delivering toxic effector proteins directly into neighbors using aspeargun-like apparatus. A spear-shaped protein complex is propelled out of theT6SS-expressing cell, and this spear punctures a neighboring cell to deliver its toxicpayload. Toxic effector proteins are bound either covalently or non-covalently to thissecreted complex, and these toxins can target both periplasmic or cytoplasmicsubstrates, as well as membranes. Kin protect themselves from these effectors byexpressing cognate immunity proteins to block effector activity.This thesis explores several aspects of the T6SS in Enterobacter cloacae ATCC13047. In Chapter 1, I provide a general introduction to the T6SS and covers topicssuch as the structure and assembly of the apparatus, regulation of T6SS loci andeffectors, and effector diversity. I then describe the genetics of the T6SS in E. cloacaeand explore what effectors are deployed in this system in Chapter 2. In Chapter 3, Ifocus on one particular effector deployed by this system, the type VI secretion systemlipase effector, Tle, and investigate its intriguing reliance on its cognate immunityprotein, Tli, for toxicity. Next, Chapter 4 discusses the role of rearrangement hotspotix(Rhs) proteins in the assembly of the T6SS apparatus. Chapter 5 then looks at therole of the Rhs accessory protein effector-associated gene with Rhs (EagR). Finally, Isummarize my findings and discuss open questions in the field in Chapter 6.

Bacteria are widespread on Earth. They play important roles in shaping the global ecosystems as well as influencing human health. In nature, different bacterial species often coexist in a common environment. The interactions of them are diverse and often lead to intricate spatial-temporal dynamics. Quantitative experimental measurements as well as mathematical modeling could help us better understand the mechanisms behind such dynamics. In this dissertation, I discuss several spatial-temporal structures for multi-strain bacterial systems and different modeling strategies are used to help explore the mechanisms of different spatial-temporal patterns. A key question regarding a system of different bacterial species is how coexistence is maintained, considering that different bacterial species often have different growth rates and they can even kill each other sometimes. In Chapter Two, I discuss a novel theoretical framework for robust coexistence and pattern formation in bacterial mixtures with contact-dependent killing. In Chapter Three, I present a beautiful spatial flower-like pattern in two-species bacterial systems from experiments and build two different models, a discrete interface model and a phase-field model, to elucidate how differential motility and mechanical interactions between bacterial species can create complex spatial structures. In Chapter Four, I introduce the agent-based modeling, which simulates each individual bacterial cell in large populations. Two examples of applications of such agent-based modeling in microfluidic environments are described. All these chapters combine to highlight the effort towards understanding the spatial-temporal dynamics of multi-strain microbial systems quantitatively.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

Bacteria occupy an impressive diversity of environments in which complex networks of interactions shape the microbial communities’ relationships. In this context, an arsenal of bacterial nanoweapons have been selected to increase the relative fitness of each player. Some of these interactions depend on molecules diffused in the media whereas others rely on direct cell-to-cell contact. The type VI secretion system (T6SS) is one of those sophisticated ways to interact with neighboring cells. These supramolecular complexes allow Gram-negative bacteria to puncture the outer membrane of a cell in a contact- dependent manner and subsequently release a cocktail of toxins named effectors. Here we characterized RhsC, an effector of the Rhs family encoded in the Xenorhabdus bovienii SS-2004 genome. The carboxy-terminal domain of this Rhs toxin (RhsC-CT) shows a R-S-ExE catalytic motif characteristic of arginine ADP-ribosyltransferases. We demonstrated that this RhsC-CT uses the NAD+ coenzyme to ADP-ribosylate various protein targets in E. coli. The activity of this RhsC-CT toxin is inhibited via direct interaction with its cognate RhsIc immunity protein for which we obtained a structure., Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Vibrio cholerae is a diverse species that inhabits a wide range of environments from copepods in brackish waterto the intestines of humans. In order to remain competitive, V. cholerae uses the versatile type-VI secretion system (T6SS) tosecrete anti-prokaryotic and anti-eukaryotic effectors. In addition to competing with other bacterial species, V. cholerae strainsalso compete with one another. Some strains are able to coexist, and are referred to as belonging to the same compatibility group.Challenged by diverse competitors in various environments, different V. choleare strains secrete different combination of effectors– presumably to best suit their niche. Interestingly, all pandemic V. cholerae strains encode the same three effectors. In additionto the diversity displayed in the encoded effectors, the regulation of V. cholerae also differs between strains. Two main layersof regulation appear to exist. One strategy connects T6SS activity with behavior that is suited to fighting eukaryotic cells, whilethe other is linked with natural competence – the ability of the bacterium to acquire and incorporate extracellular DNA. Thisrelationship between bacterial killing and natural competence is potentially a source of diversification for V. cholerae as it hasbeen shown to incorporate the DNA of cells recently killed through T6SS activity. It is through this process that we hypothesizethe transfer of virulence factors, including T6SS effector modules, to happen. Switching of T6SS effectors has the potential tochange the range of competitors V. cholerae can kill and to newly define which strains V. cholerae can co-exist with, two importantparameters for survival in diverse environments.

Salmonella enterica serovar Dublin is a zoonotic infection that can be transmitted from cattle to humans through consumption of contaminated milk and milk products. Outbreaks of human infections by S. Dublin have been reported in several countries including high-income countries. A high proportion of S. Dublin cases in humans are associated with invasive disease and systemic illness. The genetic basis of virulence in S. Dublin is not well characterized. Whole genome sequencing was applied to a set of clinical invasive and non-invasive S. Dublin isolates from different countries in order to characterize the putative genetic determinants involved in the virulence and invasiveness of S. Dublin in humans. We identified several virulence factors that form the bacterial invasome and may contribute to increasing bacterial virulence and pathogenicity including mainly Gifsy-2 prophage, two different type 6 secretion systems (T6SSs) harbored by Salmonella pathogenicity islands; SPI-6 and SPI-19 respectively and virulence genes; ggt and PagN. Although Vi antigen and the virulence plasmid have been reported previously to contribute to the virulence of S. Dublin we did not detect them in all invasive isolates indicating that they are not the main virulence determinants in S. Dublin. Several virulence factors within the genome of S. Dublin might contribute to the ability of S. Dublin to invade humans' blood but there were no genomic markers that differentiate invasive from non-invasive isolates suggesting that host immune response play a crucial role in the clinical outcome of S. Dublin infection.

BACKGROUND: Typhoid fever is an infectious disease of global importance that is caused by Salmonella enterica subsp. enterica serovar Typhi (S. Typhi). This disease causes an estimated 200,000 deaths per year and remains a serious global health threat. S. Typhi is strictly a human pathogen, and some recovered individuals become long-term carriers who continue to shed the bacteria in their faeces, thus becoming main reservoirs of infection. RESULTS: A comparative genomics analysis combined with a phylogenomic analysis revealed that the strains from the outbreak and carrier were closely related with microvariations and possibly derived from a common ancestor. Additionally, the comparative genomics analysis with all of the other completely sequenced S. Typhi genomes revealed that strains BL196 and CR0044 exhibit unusual genomic variations despite S. Typhi being generally regarded as highly clonal. The two genomes shared distinct chromosomal architectures and uncommon genome features; notably, the presence of a ~10 kb novel genomic island containing uncharacterised virulence-related genes, and zot in particular. Variations were also detected in the T6SS system and genes that were related to SPI-10, insertion sequences, CRISPRs and nsSNPs among the studied genomes. Interestingly, the carrier strain CR0044 harboured far more genetic polymorphisms (83% mutant nsSNPs) compared with the closely related BL196 outbreak strain. Notably, the two highly related virulence-determinant genes, rpoS and tviE, were mutated in strains BL196 and CR0044, respectively, which revealed that the mutation in rpoS is stabilising, while that in tviE is destabilising. These microvariations provide novel insight into the optimisation of genes by the pathogens. However, the sporadic strain was found to be far more conserved compared with the others. CONCLUSIONS: The uncommon genomic variations in the two closely related BL196 and CR0044 strains suggests that S. Typhi is more