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For bacteria to thrive in an environment with competitors, phages and environmental cues, they use different strategies, including Type VI Secretion Systems (T6SSs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) to compete for space. Bacteria often use quorum sensing (QS), to coordinate their behavior as the cell density increases. Like other aliivibrios, Aliivibrio wodanis 06/09/139 harbors two QS systems, the main LuxS/LuxPQ system and an N-acyl homoserine lactone (AHL)-mediated AinS/AinR system and a master QS regulator, LitR. To explore the QS and survival strategies, we performed genome analysis and gene expression profiling on A. wodanis and two QS mutants (ΔainS and ΔlitR) at two cell densities (OD600 2.0 and 6.0) and temperatures (6 and 12°C). Genome analysis of A. wodanis revealed two CRISPR systems, one without a cas loci (CRISPR system 1) and a type I-F CRISPR system (CRISPR system 2). Our analysis also identified three main T6SS clusters (T6SS1, T6SS2, and T6SS3) and four auxiliary clusters, as well about 80 potential Type VI secretion effectors (T6SEs). When comparing the wildtype transcriptome data at different cell densities and temperatures, 13–18% of the genes were differentially expressed. The CRISPR system 2 was cell density and temperature-independent, whereas the CRISPR system 1 was temperature-dependent and cell density-independent. The primary and auxiliary clusters of T6SSs were both cell density and temperature-dependent. In the ΔlitR and ΔainS mutants, several CRISPR and T6SS related genes were differentially expressed. Deletion of litR resulted in decreased expression of CRISPR system 1 and increased expression of CRISPR system 2. The T6SS1 and T6SS2 gene clusters were less expressed while the T6SS3 cluster was highly expressed in ΔlitR. Moreover, in ΔlitR, the hcp1 gene was strongly activated at 6°C compared to 12°C. AinS positively affected the csy genes in the CRISPR system 2 but did not affect the CRISPR arrays. Although AinS did not significantly affect the expression of T6SSs, the hallmark genes of T6SS (hcp and vgrG) were AinS-dependent. The work demonstrates that T6SSs and CRISPR systems in A. wodanis are QS dependent and may play an essential role in survival in its natural environment.

The Type VI Secretion System (T6SS) is a multiprotein device that has emerged as an important fitness and virulence factor for many Gram-negative bacteria through the injection of effector proteins into prokaryotic or eukaryotic cells via a contractile mechanism. While some effector proteins specifically target bacterial or eukaryotic cells, others can target both types of cells (trans-kingdom effectors). In Salmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, which are differentially distributed among serotypes. Salmonella enterica serotype Dublin (S. Dublin) is a cattle-adapted pathogen that harbors both T6SSSPI-6 and T6SSSPI-19. Interestingly, while both systems have been linked to virulence and host colonization in S. Dublin, an antibacterial activity has not been detected for T6SSSPI-6 in this serotype. In addition, there is limited information regarding the repertoire of effector proteins encoded within T6SSSPI-6 and T6SSSPI-19 gene clusters in S. Dublin. In the present study, we demonstrate that T6SSSPI-6 and T6SSSPI-19 of S. Dublin CT_02021853 contribute to interbacterial competition. Bioinformatic and comparative genomic analyses allowed us to identify genes encoding three candidate antibacterial effectors located within SPI-6 and two candidate effectors located within SPI-19. Each antibacterial effector gene is located upstream of a gene encoding a hypothetic immunity protein, thus conforming an effector/immunity (E/I) module. Of note, the genes encoding these effectors and immunity proteins are widely distributed in Salmonella genomes, suggesting a relevant role in interbacterial competition and virulence. Finally, we demonstrate that E/I modules SED_RS01930/SED_RS01935 (encoded in SPI-6), SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both encoded in SPI-19) contribute to interbacterial competition in S. Dublin CT_02021853.

The increase in antibiotic resistance in bacteria has prompted the efforts in developing new alternative strategies for pathogenic bacteria. We explored the feasibility of targeting Vibrio cholerae by neutralizing bacterial cellular processes rather than outright killing the pathogen. We investigated the efficacy of delivering engineered regulatory small RNAs (sRNAs) to modulate gene expression through DNA conjugation. As a proof of concept, we engineered several sRNAs targeting the type VI secretion system (T6SS), several of which were able to successfully knockdown the T6SS activity at different degrees. Using the same strategy, we modulated exopolysaccharide production and motility. Lastly, we delivered an sRNA targeting T6SS into V. cholerae via conjugation and observed a rapid knockdown of the T6SS activity. Coupling conjugation with engineered sRNAs represents a novel way of modulating gene expression in V. cholerae opening the door for the development of novel prophylactic and therapeutic applications., Importance: Given the prevalence of antibiotic resistance, there is an increasing need to develop alternative approaches to managing pathogenic bacteria. In this work, we explore the feasibility of modulating the expression of various cellular systems in Vibrio cholerae using engineered regulatory sRNAs delivered into cells via DNA conjugation. These sRNAs are based on regulatory sRNAs found in V. cholerae and exploit its native regulatory machinery. By delivering these sRNAs conjugatively along with a real-time marker for DNA transfer, we found that complete knockdown of a targeted cellular system could be achieved within one cell division cycle after sRNA gene delivery. These results indicate that conjugative delivery of engineered regulatory sRNAs is a rapid and robust way of precisely targeting V. cholerae ., Competing Interests: The authors declare no conflict of interest.

Type VI secretion systems (T6SSs) are complex molecular machines that allow bacteria to deliver toxic effector proteins to neighboring bacterial and eukaryotic cells. Although initial work focused on the T6SS as a virulence mechanism of human pathogens, the field shifted to examine the use of T6SSs for interbacterial competition in various environments, including in the plant rhizosphere. Genes encoding the T6SS are estimated to be found in a quarter of all Gram-negative bacteria and are especially highly represented in Proteobacteria , a group which includes the most important bacterial phytopathogens. Many of these pathogens encode multiple distinct T6SS gene clusters which can include the core components of the apparatus as well as effector proteins. The T6SS is deployed by pathogens at multiple points as they colonize their hosts and establish an infection. In this review, we describe what is known about the use of T6SS by phytopathogens against plant hosts and non-plant organisms, keeping in mind that the structure of plants requires unique mechanisms of attack that are distinct from the mechanisms used for interbacterial interactions and against animal hosts. While the interactions of specific effectors (such as phospholipases, endonucleases, peptidases, and amidases) with targets have been well described in the context of interbacterial competition and in some eukaryotic interactions, this review highlights the need for future studies to assess the activity of phytobacterial T6SS effectors against plant cells., Competing Interests: The authors declare no conflict of interest.

The Type VI secretion system (T6SS) is a multicomponent apparatus, present in many Gram-negative bacteria, which can inhibit bacterial prey in various ecological niches. Pseudomonas aeruginosa assembles one of its three T6SS (H1-T6SS) to respond to attacks from adjacent competing bacteria. Surprisingly, repeated assemblies of the H1-T6SS, termed dueling, were described in a monoculture in the absence of an attacker strain; however, the underlying mechanism was unknown. Here, we explored the role of H2-T6SS of P. aeruginosa in triggering H1-T6SS assembly. We show that H2-T6SS inactivation in P. aeruginosa causes a significant reduction in H1-T6SS dueling and that H2-T6SS activity directly triggers retaliation by the H1-T6SS. Intraspecific competition experiments revealed that elimination of H2-T6SS in non-immune prey cells conferred protection from H1-T6SS. Moreover, we show that the H1-T6SS response is triggered independently of the characterized lipase effectors of the H2-T6SS, as well as those of Acinetobacter baylyi and Vibrio cholerae . Our results suggest that H1-T6SS response to H2-T6SS in P. aeruginosa can impact intraspecific competition, particularly when the H1-T6SS effector-immunity pairs differ between strains, and could determine the outcome of multistrain colonization.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa harbors three different Type VI secretion systems (H1, H2, and H3-T6SS), which can translocate toxins that can inhibit bacterial competitors or inflict damage to eukaryotic host cells. Unlike the unregulated T6SS assembly in other Gram-negative bacteria, the H1-T6SS in P. aeruginosa is precisely assembled as a response to various cell damaging attacks from neighboring bacterial cells. Surprisingly, it was observed that neighboring P. aeruginosa cells repeatedly assemble their H1-T6SS toward each other. Mechanisms triggering this "dueling" behavior between sister cells were unknown. In this report, we used a combination of microscopy, genetic and intraspecific competition experiments to show that H2-T6SS initiates H1-T6SS dueling. Our study highlights the interplay between different T6SS clusters in P. aeruginosa , which may influence the outcomes of multistrain competition in various ecological settings such as biofilm formation and colonization of cystic fibrosis lungs., Competing Interests: The authors declare no conflict of interest.

Vibrio cholerae ( V. cholerae ), the etiological agent of cholera, employs various virulence factors to adapt and thrive within both aquatic and human host environments. Among these factors, the type VI secretion system (T6SS) stands out as one of the crucial determinants of its pathogenicity. Valine glycine repeat protein G1 (VgrG1) and hemolysin coregulated protein (HCP) are considered major effector molecules of T6SS. Previous studies have highlighted that VgrG1 interacts with HCP proteins. Additionally, it has been shown that VgrG1 possesses an actin cross-linking domain (ACD) with actin-binding activity. Interestingly, it was reported that purified HCP protein treatment increased the stress fibers within cells. Therefore, we hypothesize that HCP may interact with host cell actin, potentially playing a role in the cytoskeletal rearrangement during V. cholerae infection. To test this hypothesis, we characterized HCP from the V. cholerae O139 serotype and demonstrated its interaction with actin monomers. In silico analysis and experimental validation revealed the presence of an actin-binding site within HCP. Furthermore, overexpression of HCP resulted in its colocalization with actin stress fibers in host cells. Our findings establish HCP as an effector molecule for potent host cell actin cytoskeleton remodeling during V. cholerae infection, providing new insights into bacterial pathogenicity mechanisms. Understanding the interplay between bacterial effectors and host cell components is crucial for developing targeted therapeutic interventions against cholera and related infectious diseases.

Previous studies revealed that the type VI secretion system (T6SS) has an essential role in bacterial competition and virulence in many gram-negative bacteria. However, the role of T6SS in virulence in Pectobacterium atrosepticum remains controversial. We examined a closely related strain, PccS1, and discovered that its T6SS comprises a single-copy cluster of 17 core genes with a higher identity to homologs from P. atrosepticum. Through extensive phenotypic and functional analyses of over 220 derivatives of PccS1, we found that three of the five VgrGs could be classified into group I VgrGs. These VgrGs interacted with corresponding DUF4123 domain proteins, which were secreted outside of the membrane and were dependent on either the T6SS or type IV secretion system (T4SS). This interaction directly governed virulence and competition. Meanwhile, supernatant proteomic analyses with strains defective in the T6SS and/or T4SS confirmed that effectors, such as FhaB, were secreted redundantly to control the virulence and suppress host callose deposition in the course of infection. Notably, this redundant secretion mechanism between the T6SS and T4SS is believed to be the first of its kind in bacteria., Competing Interests: The author(s) declare no conflict of interest.

Burkholderia thailandensis is a clinically underestimated conditional pathogen in the genus Burkholderia, the pathogenicity of the infection caused by B. thailandensis remains poorly understood. According to previous studies, Type-VI secretion system (T6SS) is a protein secreting device widely existing in Gram-negative bacilli. Valine-glycine repeat protein G (VgrG) is not only an important component of T6SS, but also a virulence factor of many Gram-negative bacilli. In one of our previous studies, a unique T6SS vgrG gene (vgrG2 gene) was present in a virulent B. thailandensis strain BPM (BPM), but not in the relatively avirulent B. thailandensis strain E264 (E264). Meanwhile, transcriptome analysis of BPM and E264 showed that the vgrG2 gene was strongly expressed in BPM, but not in E264. Therefore, we identified the function of the vgrG2 gene by constructing the mutant and complemented strains in this study. In vitro, the vgrG2 gene was observed to be involved in the interactions with host cells. The animal model experiment showed that the deletion of vgrG2 gene significantly led to the decrease in the lethality of BPM and impaired its ability to trigger host immune response. In conclusion, our study provides a new perspective for studying the pathogenicity of B. thailandensis and lays the foundation for discovering the potential T6SS effectors.

The type VI secretion system (T6SS) is an important translocation apparatus that is widely employed by Gram-negative bacteria to deliver toxic effectors into eukaryotic and prokaryotic target cells, causing host damage and providing competitive advantages in polymicrobial environments. The genome of Pseudomonas aeruginosa harbors three T6SS clusters (H1-T6SS, H2-T6SS, H3-T6SS). Activities of these systems are tightly regulated by a complicated signaling network which remains largely elusive. In this study, we focused on a previously characterized two-component system FleS/FleR, and performed comparative transcriptome analysis between the PAO1 wild-type strain and its isogenic DfleR mutant, which revealed the important role of FleS/FleR in regulating multiple physiological pathways including T6SS. Gene expression and bacterial killing assays showed that the expression and activity of H1-T6SS are repressed in the wildtype strain owing to the high intracellular c-di-GMP content. Further explorations demonstrated that c-di-GMP relies on the transcription factor FleQ to repress H1-T6SS and its synthesis is controlled by a global regulator AmrZ which is induced by the active FleS/FleR. Interestingly, repression of H1-T6SS by FleS/FleR in PAO1 is independent of RetS which is known to regulate H1-T6SS by controlling the central post-transcriptional factor RsmA. Together, our results identified a novel regulator of H1-T6SS and provided detailed mechanisms of this signaling pathway in PAO1. [ABSTRACT FROM AUTHOR]

Gram‐negative bacteria use type VI secretion systems (T6SSs) to deliver toxic effector proteins into neighboring cells. Cargo effectors are secreted by binding noncovalently to the T6SS apparatus. Occasionally, effector secretion is assisted by an adaptor protein, although the adaptor itself is not secreted. Here, we report a new T6SS secretion mechanism, in which an effector and a co‐effector are secreted together. Specifically, we identify a novel periplasm‐targeting effector that is secreted together with its co‐effector, which contains a MIX (marker for type sIX effector) domain previously reported only in polymorphic toxins. The effector and co‐effector directly interact, and they are dependent on each other for secretion. We term this new secretion mechanism "a binary effector module," and we show that it is widely distributed in marine bacteria. Synopsis: Gram‐negative bacteria use type VI secretion systems to inject toxins. Here, a new mode of T6SS delivery is reported, where a MIX domain‐containing co‐effector mediates the secretion of a second protein with toxic activity. Tricistronic operons mediating interbacterial competition and encoding two T6SS‐secreted proteins are widespread in marine bacteria.The two secreted proteins interact with each other and with the T6SS spike, and require each other for secretion.MIX domain‐containing proteins can function as co‐effectors enabling delivery of a toxin, rather than being a toxin.A novel antibacterial T6SS toxin targets the periplasm, leading to cell lysis. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis is a clinically underestimated conditional pathogen in the genus Burkholderia , the pathogenicity of the infection caused by B. thailandensis remains poorly understood. According to previous studies, Type-VI secretion system (T6SS) is a protein secreting device widely existing in Gram-negative bacilli. Valine-glycine repeat protein G (VgrG) is not only an important component of T6SS, but also a virulence factor of many Gram-negative bacilli. In one of our previous studies, a unique T6SS vgrG gene (vgrG2 gene) was present in a virulent B. thailandensis strain BPM (BPM), but not in the relatively avirulent B. thailandensis strain E264 (E264). Meanwhile, transcriptome analysis of BPM and E264 showed that the vgrG2 gene was strongly expressed in BPM, but not in E264. Therefore, we identified the function of the vgrG2 gene by constructing the mutant and complemented strains in this study. In vitro , the vgrG2 gene was observed to be involved in the interactions with host cells. The animal model experiment showed that the deletion of vgrG2 gene significantly led to the decrease in the lethality of BPM and impaired its ability to trigger host immune response. In conclusion, our study provides a new perspective for studying the pathogenicity of B. thailandensis and lays the foundation for discovering the potential T6SS effectors. [ABSTRACT FROM AUTHOR]

Avian pathogenic E. coli is the causative agent of extra-intestinal infections in birds known as colibacillosis, which can manifest as localized or systemic infections. The disease affects all stages of poultry production, resulting in economic losses that occur due to morbidity, carcass condemnation and increased mortality of the birds. APEC strains have a diverse virulence trait repertoire, which includes virulence factors involved in adherence to and invasion of the host cells, serum resistance factors, and toxins. However, the pathogenesis of APEC infections remains to be fully elucidated. The Type 6 secretion (T6SS) system has recently gained attention due to its role in the infection process and protection of bacteria from host defenses in human and animal pathogens. Previous work has shown that T6SS components are involved in the adherence to and invasion of host cells, as well as in the formation of biofilm, and intramacrophage bacterial replication. Here, we analyzed the frequency of T6SS genes hcp, impK, evpB, vasK and icmF in a collection of APEC strains and their potential role in virulence-associated phenotypes of APECO18. The T6SS genes were found to be significantly more prevalent in APEC than in fecal E. coli isolates from healthy birds. Expression of T6SS genes was analyzed in culture media and upon contact with host cells. Mutants were generated for hcp, impK, evpB, and icmF and characterized for their impact on virulence-associated phenotypes, including adherence to and invasion of host model cells, and resistance to predation by Dictyostelium discoideum. Deletion of the aforementioned genes did not significantly affect adherence and invasion capabilities of APECO18. Deletion of hcp reduced resistance of APECO18 to predation by D. discoideum, suggesting that T6SS is involved in the virulence of APECO18. [ABSTRACT FROM AUTHOR]

Aeromonas hydrophila is a Gram-negative bacterium that is commonly distributed in aquatic surroundings and has been considered as a pathogen of fish, amphibians, reptiles, and mammals. In this study, a virulent strain A. hydrophila GD18, isolated from grass carp (Ctenopharyngodon idella), was characterized to belong to a new sequence type ST656. Whole-genome sequencing and phylogenetic analysis showed that GD18 was closer to environmental isolates, however distantly away from the epidemic ST251 clonal group. The type VI secretion system (T6SS) was known to target both eukaryotic and prokaryotic cells by delivering various effector proteins in diverse niches by Gram-negative bacteria. Genome-wide searching and hemolysin co-regulated protein (Hcp) expression test showed that GD18 possessed a functional T6SS and is conditionally regulated. Further analysis revealed that VasH, a σ54-transcriptional activator, was strictly required for the functionality of T6SS in A. hydrophila GD18. Mutation of vasH gene by homologous recombination significantly abolished the bactericidal property. Then the virulence contribution of VasH was characterized in both in vitro and in vivo models. The results supported that VasH not only contributed to the bacterial cytotoxicity and resistance against host immune cleaning, but also was required for virulence and systemic dissemination of A. hydrophila GD18. Taken together, these findings provide a perspective for understanding the VasH-mediated regulation mechanism and T6SS-mediated virulence and bactericidal effect of A. hydrophila.

Avian pathogenic E. coli is the causative agent of extra-intestinal infections in birds known as colibacillosis, which can manifest as localized or systemic infections. The disease affects all stages of poultry production, resulting in economic losses that occur due to morbidity, carcass condemnation and increased mortality of the birds. APEC strains have a diverse virulence trait repertoire, which includes virulence factors involved in adherence to and invasion of the host cells, serum resistance factors, and toxins. However, the pathogenesis of APEC infections remains to be fully elucidated. The Type 6 secretion (T6SS) system has recently gained attention due to its role in the infection process and protection of bacteria from host defenses in human and animal pathogens. Previous work has shown that T6SS components are involved in the adherence to and invasion of host cells, as well as in the formation of biofilm, and intramacrophage bacterial replication. Here, we analyzed the frequency of T6SS genes hcp, impK, evpB, vasK and icmF in a collection of APEC strains and their potential role in virulence-associated phenotypes of APECO18. The T6SS genes were found to be significantly more prevalent in APEC than in fecal E. coli isolates from healthy birds. Expression of T6SS genes was analyzed in culture media and upon contact with host cells. Mutants were generated for hcp, impK, evpB, and icmF and characterized for their impact on virulence-associated phenotypes, including adherence to and invasion of host model cells, and resistance to predation by Dictyostelium discoideum. Deletion of the aforementioned genes did not significantly affect adherence and invasion capabilities of APECO18. Deletion of hcp reduced resistance of APECO18 to predation by D. discoideum, suggesting that T6SS is involved in the virulence of APECO18.

ABSTRACT The type VI secretion system (T6SS) belongs to the evolutionarily related group of contractile injection systems that employ a contractile outer sheath to inject a rigid spear-like inner tube into target bacterial and eukaryotic cells. The tip of the rigid tube is often decorated by a PAAR-repeat protein as a key structural component. Many members of the PAAR protein family can also have additional and diverse functions by serving as toxins for those with extended domains or as carriers for interacting toxins. A plethora of toxin modules or modules of unknown functions have been bioinformatically predicted to be associated with PAAR either as a fused domain or as an interacting partner, and yet only a small number of PAAR proteins have been studied, highlighting the exciting and dire need for future research to better understand the diverse PAAR-mediated functions.

ABSTRACT Commensal microbes in animal guts often help to exclude bacterial pathogens. In honey bees, perturbing or depleting the gut microbiota increases host mortality rates upon challenge with the opportunistic pathogen Serratia marcescens, suggesting antagonism between S. marcescens and one or more members of the bee gut microbiota. In laboratory culture, S. marcescens uses a type VI secretion system (T6SS) to kill bacterial competitors, but the role of this T6SS within hosts is unknown. Using infection assays, we determined how the microbiota impacts the abundance and persistence of S. marcescens in the gut and visualized colocalization of S. marcescens with specific community members in situ. Using T6SS-deficient S. marcescens strains, we measured T6SS-dependent killing of gut isolates in vitro and compared the persistence of mutant and wild-type strains in the gut. We found that S. marcescens is rapidly eliminated in the presence of the microbiota but persists in microbiota-free guts. Protection is reduced in monocolonized and antibiotic-treated bees, possibly because different symbionts occupy distinct niches. Serratia marcescens uses a T6SS to antagonize Escherichia coli and other S. marcescens strains but shows limited ability to kill bee symbionts. Furthermore, wild-type and T6SS-deficient S. marcescens strains achieved similar abundance and persistence in bee guts. Thus, an intact gut microbiota offers robust protection against this common pathogen, whose T6SSs do not confer the ability to compete with commensal species. IMPORTANCE Bacteria living within guts of animals can provide protection against infection by pathogens. Some pathogens have been shown to use a molecular weapon known as a T6SS to kill beneficial bacteria during invasion of the mouse gut. In this study, we examined how bacteria native to the honey bee gut work together to exclude the opportunistic pathogen Serratia marcescens. Although S. marcescens has a T6SS that can kill bacteria, bee gut bacteria seem resistant to its effects. This limitation may partially explain why ingestion of S. marcescens is rarely lethal to insects with healthy gut communities.

ABSTRACT Human health is threatened by bacterial infections that are increasingly resistant to multiple drugs. A recently emerged strategy consists of disarming pathogenic bacteria by targeting and blocking their virulence factors. The type VI secretion system (T6SS) is a widespread secretion nanomachine encoded and employed by pathogenic strains to establish their virulence process during host invasion. Given the conservation of T6SS in several human bacterial pathogens, the discovery of an effective broad-spectrum T6SS virulence blocker represents an attractive target for development of antivirulence therapies. Here, we identified and validated a protein-protein interaction interface, TssK-TssG, as a key factor in the assembly of the T6SS baseplate (BP) complex in the pathogen enteroaggregative Escherichia coli (EAEC). In silico and biochemical studies revealed that the determinants of the interface are broadly conserved among pathogenic species, suggesting a role for this interface as a target for T6SS inhibition. Based on the high-resolution structure of the TssKFGE wedge complex, we rationally designed a biomimetic cyclic peptide (BCP) that blocks the assembly of the EAEC BP complex and inhibits the function of T6SS in bacterial cultures. Our BCP is the first compound completely designed from prior structural knowledge with anti-T6SS activity that can be used as a model to target human pathogens. IMPORTANCE New therapeutic options are urgently needed to fight drug-resistant and life-threatening infections. In contrast to antibiotics that inhibit the growth pathways of bacteria, the antivirulence strategy is a promising approach to disarm pathogens by interfering with bacterial virulence factors without exerting evolutionary pressure. The type VI secretion system (T6SS) is used by many pathogens, including members of the antibiotic-resistant ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), to establish their virulence during the invasion of the human host. Although the T6SS is undoubtedly involved in pathogenesis, strategies targeting this virulence factor are crucially lacking. Here, we used a combination of genetics, microbiology, biochemical, biophysics, and bioinformatics approaches to rationally design a biomimetic peptide that interferes with T6SS assembly and functioning. This study represents a novel proof of concept for an antivirulence strategy which aims to interfere with the assembly of the T6SS.

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 outcompete 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 peptidoglycandegrading effectors with an unprecedented impact in antagonistic bacterial interactions. [ABSTRACT FROM AUTHOR]

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development. [ABSTRACT FROM AUTHOR]

Abstract Background Acinetobacter baumannii is one of the most important opportunistic pathogens responsible for hospital acquired infections. It displays multi-drug resistance profile and has the ability to colonize surfaces and persist under harsh conditions. A. baumannii two-component signal transduction system BfmRS, consisting of response regulator BfmR and sensor kinase BfmS, has been implicated in the control of various virulence-related traits and has been suggested to act as a global modulator of A. baumannii physiology. Results Here, we assessed the role of BfmR regulator in pellicle formation and bacterial competition, features important for the establishment of A. baumannii in clinical environment. We show that BfmR is required for the pellicle formation of A. baumannii, as ΔbfmRS mutant lacked this phenotype. The loss of bfmRS also greatly reduced the secretion of A. baumannii Hcp protein, which is a component of T6SS secretion system. However, T6SS-mediated killing phenotype was not impaired in ΔbfmRS mutant. On the contrary, the same mutation resulted in the transcriptional activation of contact-dependent inhibition (CDI) system, which A. baumannii used to inhibit the growth of another clinical A. baumannii strain and a closely related species Acinetobacter baylyi. Conclusions The obtained results indicate that BfmR is not only required for the pellicle phenotype induction in A. baumannii, but also, due to the down-regulation of a CDI system, could allow the incorporation of other A. baumannii strains or related species, possibly increasing the likelihood of the pathogens’ survival.

Abstract Background Klebsiella pneumoniae (KP) is an opportunistic pathogen that mainly causes respiratory and urinary tract infections. The frequent occurrence of simultaneously virulent and multiple drug-resistant isolates led WHO to include this species in the list of top priorities for research and development of therapeutic alternatives. The comprehensive knowledge of the molecular mechanisms underlying KP virulence may lead to the proposal of more efficient and specific drugs. One of its virulence factors is the Type VI Secretion System (T6SS), which contributes to bacterial competition, cell invasion and in vivo colonisation. Despite the few studies showing the involvement of T6SS in KP pathogenesis, little is known concerning the regulation of its expression. The understanding of regulatory mechanisms may give more clues about the function of the system and the possibilities of future interference in this process. This work aimed to standardise the annotation of T6SS genes in KP strains and identify mechanisms of their transcriptional regulation through computational predictions. Results We analyzed the genomes of Kp52.145, HS11286 and NTUH-K2044 strains to perform a broad prediction and re-annotation of T6SS genes through similarity searches, comparative and linear discriminant analysis. 38 genes were found in Kp52.145, while 29 in HS11286 and 30 in NTUH-K2044. Genes coding for iron uptake systems are encoded in adjacencies of T6SS, suggesting that KP T6SS might also play a role in ion import. Some of the T6SS genes are comprised in syntenic regions. 17 sigma 70-dependent promoter regions were identified in Kp52.145, 12 in HS11286 and 12 in NTUH-K2044. Using VirtualFootprint algorithm, binding sites for 13 transcriptional regulators were found in Kp52.145 and 9 in HS11286 and 17 in NTUH-K2044. Six of them are common to the 3 strains: OxyR, H-NS, RcsAB, GcvA, Fis, and OmpR. Conclusions The data presented herein are derived from computational analysis. Although future experimental studies are required to confirm those predictions, they suggest that KP T6SS might be regulated in response to environmental signals that are indeed sensed by the bacteria inside the human host: temperature (H-NS), nutrition-limitation (GcvA and Fis), oxidative stress (OxyR) and osmolarity (RscAB and OmpR).

Abstract Objectives In this study we have investigated the in vitro and in vivo virulence characteristics of a new T6SS positive Campylobacter jejuni chicken isolate (SV12) originating from a poultry population in North Romania. A detailed phenotypic characterization was performed and compared to the T6SS negative C. jejuni 81–176 wild strain. Results Our results indicate that the significantly higher capacity to attach and invade HCT-8 cells of C. jejuni SV12 isolate is associated with increased motility, increased resistance to bile salts and serum resistance, when compared to C. jejuni strain 81–76. Mice infected with the SV12 isolate showed statistically higher levels of colonization at both 7- and 14-days post-inoculation and in the stomach, caecum, duodenum and large intestine. Infection with the SV12 strain induced a stronger immune response as the gene transcript levels of IL-17, TNFα and IFNγ were more pronouncedly up-regulated compared to the C. jejuni strain 81–176. The present study showed that the new isolate SV12 had an enhanced virulence capacity compared to the wild strain which was evident in vivo as well. This work also provides an insight on the colonization pattern and host immune response differences between T6SS positive and T6SS negative C. jejuni.

Paraburkholderia phymatum STM815, a rhizobial strain of the Burkholderiaceae family, is able to nodulate a broad range of legumes including the agriculturally important Phaseolus vulgaris (common bean). P. phymatum harbors two type VI Secretion Systems (T6SS-b and T6SS-3) in its genome that contribute to its high interbacterial competitiveness in vitro and in infecting the roots of several legumes. In this study, we show that P. phymatum T6SS-b is found in the genomes of several soil-dwelling plant symbionts and that its expression is induced by the presence of citrate and is higher at 20/28°C compared to 37°C. Conversely, T6SS-3 shows homologies to T6SS clusters found in several pathogenic Burkholderia strains, is more prominently expressed with succinate during stationary phase and at 37°C. In addition, T6SS-b expression was activated in the presence of germinated seeds as well as in P. vulgaris and Mimosa pudica root nodules. Phenotypic analysis of selected deletion mutant strains suggested a role of T6SS-b in motility but not at later stages of the interaction with legumes. In contrast, the T6SS-3 mutant was not affected in any of the free-living and symbiotic phenotypes examined. Thus, P. phymatum T6SS-b is potentially important for the early infection step in the symbiosis with legumes.

Type VI Secretion Systems (T6SSs) are widespread in bacteria and can dictate the development and organisation of polymicrobial ecosystems by mediating contact dependent killing. In Neisseria species, including Neisseria cinerea a commensal of the human respiratory tract, interbacterial contacts are mediated by Type four pili (Tfp) which promote formation of aggregates and govern the spatial dynamics of growing Neisseria microcolonies. Here, we show that N. cinerea expresses a plasmid-encoded T6SS that is active and can limit growth of related pathogens. We explored the impact of Tfp on N. cinerea T6SS-dependent killing within a colony and show that pilus expression by a prey strain enhances susceptibility to T6SS compared to a non-piliated prey, by preventing segregation from a T6SS-wielding attacker. Our findings have important implications for understanding how spatial constraints during contact-dependent antagonism can shape the evolution of microbial communities.

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of catfish (ESC), a devastating disease resulting in significant economic losses in the U.S. catfish industry. Bacterial secretion systems are involved in many bacteria's virulence, and Type VI Secretion System (T6SS) is a critical apparatus utilized by several pathogenic Gram-negative bacteria. E. ictaluri strain 93–146 genome has a complete T6SS operon with 16 genes, but the roles of these genes are still not explored. In this research, we aimed to understand the roles of two hemolysin co-regulated family proteins, Hcp1 (EvpC) and Hcp2. To achieve this goal, single and double E. ictaluri mutants (EiΔevpC, EiΔhcp2, and EiΔevpCΔhcp2) were generated and characterized. Catfish peritoneal macrophages were able to kill EiΔhcp2 better than EiΔevpC, EiΔevpCΔhcp2, and E. ictaluri wild-type (EiWT). The attachment of EiΔhcp2 and EiΔevpCΔhcp2 to ovary cells significantly decreased compared to EiWT whereas the cell invasion rates of these mutants were the same as that of EiWT. Mutants exposed to normal catfish serum in vitro showed serum resistance. The fish challenges demonstrated that EiΔevpC and EiΔevpCΔhcp2 were attenuated completely and provided excellent protection against EiWT infection in catfish fingerlings. Interestingly, EiΔhcp2 caused higher mortality than that of EiWT in catfish fingerlings, and severe clinical signs were observed. Although fry were more susceptible to vaccination with EiΔevpC and EiΔevpCΔhcp2, their attenuation and protection were significantly higher compared to EiWT and sham groups, respectively. Taken together, our data indicated that evpC (hcp1) is involved in E. ictaluri virulence in catfish while hcp2 is involved in adhesion to epithelial cells and survival inside catfish macrophages.

Paraburkholderia phymatum STM815, a rhizobial strain of the Burkholderiaceae family, is able to nodulate a broad range of legumes including the agriculturally important Phaseolus vulgaris (common bean). P. phymatum harbors two type VI Secretion Systems (T6SS-b and T6SS-3) in its genome that contribute to its high interbacterial competitiveness in vitro and in infecting the roots of several legumes. In this study, we show that P. phymatum T6SS-b is found in the genomes of several soil-dwelling plant symbionts and that its expression is induced by the presence of citrate and is higher at 20/28°C compared to 37°C. Conversely, T6SS-3 shows homologies to T6SS clusters found in several pathogenic Burkholderia strains, is more prominently expressed with succinate during stationary phase and at 37°C. In addition, T6SS-b expression was activated in the presence of germinated seeds as well as in P. vulgaris and Mimosa pudica root nodules. Phenotypic analysis of selected deletion mutant strains suggested a role of T6SS-b in motility but not at later stages of the interaction with legumes. In contrast, the T6SS-3 mutant was not affected in any of the free-living and symbiotic phenotypes examined. Thus, P. phymatum T6SS-b is potentially important for the early infection step in the symbiosis with legumes. [ABSTRACT FROM AUTHOR]

• VgrG, the core component of T6SS is a potent mediator of A. baumannii pathogenicity. • Two conserved sequences of vgr G 1159-2196 and vgr G 1159-1502 were identified antigenic. • vgrg 1159-1502 was implanted in Loopless C lobe of N. meningitidis for display. • BALB/c mice were immunized with these proteins and challenged with A. baumannii. • The conserved region of VgrG develops protection against A. baumannii infections. Type VI Secretion System (T6SS) contributes to both virulence and antimicrobial resistance in Acinetobacter baumannii. Valine–glycine repeat protein G (VgrG) is the core component of T6SS that exists in many bacterial pathogens that have emerged as a potent mediator of pathogenicity in A. baumannii. Two conserved sequences of vgr G 1263-2295 and vgr G 1263-1608 were identified antigenic in various strains of Acinetobacter baumannii. The vgrg 1263-1608 sequence was implanted in the Loopless C lobe (LCL) from N. meningitidis for surface display and exposure to functional epitopes. The VgrG and LCL-VgrG were expressed and purified. Groups of BALB/c mice were immunized with these proteins and challenged with A. baumannii. Specific IgG titers, whole-cell ELISA, animal survival rates in active and passive immunizations, the bacterial burden in mice tissues, and cytotoxicity of the proteins were determined. The specific IgG suppressed bacterial burdens in the organs, and increased survival rates were noted in the immunized mice. LCL-VgrG immunization provided better protection against A. baumannii infection than the VgrG immunization. The conserved region of VgrG is probably a safe immunogen to effective vaccine development or an antiserum to control A. baumannii infections. [ABSTRACT FROM AUTHOR]

V. cholerae , the causative agent of cholera epidemic, and V. fluvialis , the emerging foodborne pathogen, share highly homologous T6SS consisting of one large cluster and two small orphan or auxiliary clusters, and each of which was generally recognized as one operon. Here, we showed that the genes in each of the small clusters are organized into two transcriptional units. Specifically, the inner tube coding gene hcp/tssD is highly transcribed as one monocistron, while the tip component vgrG/tssI and its downstream effector and immunity genes are in one polycistron with very low transcriptional level. This conclusion is supported by qPCR analysis of mRNA abundance, reporter fusion analysis and transcriptional unit definition with RT-PCR analysis. Taking tssI 2_ a of V. fluvialis as an example, we further demonstrated that quorum sensing (QS) regulator HapR and global regulator IHF activate vgrG/tssI transcription by directly binding to its promoter region. Taken together, current studies deepen our understanding of T6SS system, highlighting its regulatory complexity during functional execution process. • hcp and vgrG in T6SS orphan clusters are in different transcripts in V. cholerae and V. fluvialis. • hcp is highly transcripted as a monocistron while vgrG is lowly expressed as a polycistron. • Quorum sensing regulator HapR and global regulator IHF activate vgrG transcription by directly binding to its promoter. [ABSTRACT FROM AUTHOR]

Citrobacter freundii is a significant cause of human infections, responsible for food poisoning, diarrhea, and urinary tract infections. We previously identified a highly cytotoxic and adhesive C. freundii strain CF74 expressing a type VI secretion system (T6SS). In this study, we showed that in mice-derived macrophages, C. freundii CF74 activated the Nucleotide Oligomerization Domain -Like Receptor Family, Pyrin Domain Containing 3(NLRP3) inflammasomes in a T6SS-dependent manner. The C. freundii T6SS activated the inflammasomes mainly through caspase 1 and mediated pyroptosis of macrophages by releasing the cleaved gasdermin-N domain. The CF74 T6SS was required for flagellin-induced interleukin 1β release by macrophages. We further show that the T6SS tail component and effector, hemolysin co-regulation protein-2 (Hcp-2), was necessary and sufficient to trigger NLRP3 inflammasome activation. In vivo, the T6SS played a key role in mediating interleukin 1β secretion and the survival of mice during C. freundii infection in mice. These findings provide novel insights into the role of T6SS in the pathogenesis of C. freundii. [ABSTRACT FROM AUTHOR]

Summary: Type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria and functions as a versatile protein export machinery that translocates effectors into eukaryotic or prokaryotic target cells. Growing evidence indicates that T6SS can deliver several effectors to promote bacterial survival in harmful environments through metal ion acquisition. Here, we report that the Pseudomonas aeruginosa H2-T6SS mediates molybdate (MoO42−) acquisition by secretion of a molybdate-binding protein, ModA. The expression of H2-T6SS genes is activated by the master regulator Anr and anaerobiosis. We also identified a ModA-binding protein, IcmP, an insulin-cleaving metalloproteinase outer membrane protein. The T6SS-ModA-IcmP system provides P. aeruginosa with a growth advantage in bacterial competition under anaerobic conditions and plays an important role in bacterial virulence. Overall, this study clarifies the role of T6SS in secretion of an anion-binding protein, emphasizing the fundamental importance of this bacterium using T6SS-mediated molybdate uptake to adapt to complex environmental conditions.

Twenty isolates (9.7%) of Acinetobacter baumannii were obtained from (207) clinical specimens, including (wounds -burns -sputum- blood) from both male and female, different ages, diverse local regions. All the isolates were multidrug resistance against 13 antibiotics from different groups and according to the susceptibility test, the isolates differentiation in two categories, fifteen (75%) MDR isolates were resisted (5-9) antibiotics, while five (25%) XDR isolates were resisted (12 - 13) antibiotics. The MIC was identified for one antibiotic (Imipenem), the results showed MIC values were ranged from (8- ≥1024) µg/ml. Conventional PCR technique was used to detect the hcp gene that is a molecular marker of a functional T6SS, the results inducted the presence of gene 95%, it presented in 19 isolates. The hcp gene gave a low expression after treatment by (128)µg/ml (sub MIC) concentration of Imipenem compared with control in different degrees in 3 isolates with average folding 0.88. The results of sequences hcp amplicon on one isolate showed that the amplified hcp locus exhibited one silent mutation (g.279978C>T) compared with the referring region sequences. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa , respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis . Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis . We further identified YPK_0952 as a DNase dependent on Mg 2+ , Ni 2+ , Mn 2+ , and Co 2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis .IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis . YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli ) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation., Competing Interests: The authors declare no conflict of interest.

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae . Single deletions of the genes encoding the HU subunits ( hupA and hupB ) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans -complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria., Importance: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae , affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis., Competing Interests: The authors declare no conflict of interest.

Introduction Acinetobacter baumannii , designated as a priority pathogen by the World Health Organization (WHO), is responsible for recalcitrant infections in immunocompromised patients. The type VI secretion system (T6SS) is a class of macromolecular secretion machines, contributing to its virulence. The aim of this study is thus to predict the immune-dominant epitope peptides from the Acinetobacter T6SS-associated protein of A. baumannii  (AsaA). Methods AsaA protein retrieval from the bacteria was carried out using computational platforms and the evaluation of antigenicity and allergenicity was performed. The T-cell epitopes of major histocompatibility complex class II binders were identified followed by molecular docking of the immune-dominant epitopes with human leukocyte antigen alleles using the ClusterPro server (https://cluspro.org/help.php). Additionally, the B-cell epitopes were predicted. Results Immune-informatic analysis showed immune-dominant peptides in the most favored regions with promising interactions with HLA alleles DP, DQ, DR, and toll-like receptor showing high binding capacity. Conclusion In the present investigation, epitope 1 (LILFLIGNY) was found to be a promising candidate for the synthesis of vaccines. However, it requires further experimentation for its immunological memory and response., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Ranjan et al.)

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.

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.

Vibrio cholerae, the causative agent of cholera, could proliferate in aquatic environment and infect humans through contaminated food and water. Enormous microorganisms residing in human gastrointestinal tract establish a special microecological system, which immediately responds to the invasion of V. cholerae, through “colonization resistance” mechanisms, such as antimicrobial peptide production, nutrients competition, and intestinal barrier maintenances. Meanwhile, V. cholerae could quickly sense those signals and modulate the expression of relevant genes to circumvent those stresses during infection, leading to successful colonization on the surface of small intestinal epithelial cells. In this review, we summarized the crosstalks profiles between gut microbiota and V. cholerae in the terms of Type VI Secretion System (T6SS), Quorum Sensing (QS), Reactive Oxygen Species (ROS)/pH stress, and Bioactive metabolites. These mechanisms can also be applied to molecular bacterial pathogenesis of other pathogens in host.

The type VI secretion system (T6SS) is a transmembrane multiprotein nanomachine employed by many Gram-negative bacterial species to translocate, in a contact-dependent manner, effector proteins into adjacent prokaryotic or eukaryotic cells. Typically, the T6SS gene cluster encodes at least 13 conserved core components for the apparatus assembly and other less conserved accessory proteins and effectors. It functions as a contractile tail machine comprising a TssB/C sheath and an expelled puncturing device consisting of an Hcp tube topped by a spike complex of VgrG and PAAR proteins. Contraction of the sheath propels the tube out of the bacterial cell into a target cell and leads to the injection of toxic proteins. Different bacteria use the T6SS for specific roles according to the niche and versatility of the organism. Effectors are present both as cargo (by non-covalent interactions with one of the core components) or specialized domains (fused to structural components). Although several anti-prokaryotic effectors T6SSs have been studied, recent studies have led to a substantial increase in the number of characterized anti-eukaryotic effectors. Against eukaryotic cells, the T6SS is involved in modifying and manipulating diverse cellular processes that allows bacteria to colonize, survive and disseminate, including adhesion modification, stimulating internalization, cytoskeletal rearrangements and evasion of host innate immune responses.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

ABSTRACT Many commensal bacteria antagonize each other or their host by producing syringe-like secretion systems called contractile injection systems (CIS). Members of the Bacteroidales family have been shown to produce only one type of CIS—a contact-dependent type 6 secretion system that mediates bacterium-bacterium interactions. Here, we show that a second distinct cluster of genes from Bacteroidales bacteria from the human microbiome may encode yet-uncharacterized injection systems that we term Bacteroidales injection systems (BIS). We found that BIS genes are present in the gut microbiomes of 99% of individuals from the United States and Europe and that BIS genes are more prevalent in the gut microbiomes of healthy individuals than in those individuals suffering from inflammatory bowel disease. Gene clusters similar to that of the BIS mediate interactions between bacteria and diverse eukaryotes, like amoeba, insects, and tubeworms. Our findings highlight the ubiquity of the BIS gene cluster in the human gut and emphasize the relevance of the gut microbiome to the human host. These results warrant investigations into the structure and function of the BIS and how they might mediate interactions between Bacteroidales bacteria and the human host or microbiome. IMPORTANCE To engage with host cells, diverse pathogenic bacteria produce syringe-like structures called contractile injection systems (CIS). CIS are evolutionarily related to the contractile tails of bacteriophages and are specialized to puncture membranes, often delivering effectors to target cells. Although CIS are key for pathogens to cause disease, paradoxically, similar injection systems have been identified within healthy human microbiome bacteria. Here, we show that gene clusters encoding a predicted CIS, which we term Bacteroidales injection systems (BIS), are present in the microbiomes of nearly all adult humans tested from Western countries. BIS genes are enriched within human gut microbiomes and are expressed both in vitro and in vivo. Further, a greater abundance of BIS genes is present within healthy gut microbiomes than in those humans with with inflammatory bowel disease (IBD). Our discovery provides a potentially distinct means by which our microbiome interacts with the human host or its microbiome.

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.

Summary: The type VI secretion system (T6SS) is a lethal microbial weapon that injects a large needle-like structure carrying toxic effectors into recipient cells through physical penetration. How recipients respond to physical force and effectors remains elusive. Here, we use a series of effector mutants of Vibrio cholerae to determine how T6SS elicits response in Pseudomonas aeruginosa and Escherichia coli. We show that TseL, but no other effectors or physical puncture, triggers the tit-for-tat response of P. aeruginosa H1-T6SS. Although E. coli is sensitive to all periplasmically expressed effectors, P. aeruginosa is most sensitive to TseL alone. We identify a number of stress response pathways that confer protection against TseL. Physical puncture of T6SS has a moderate inhibitory effect only on envelope-impaired tolB and rseA mutants. Our data reveal that recipient cells primarily respond to effector toxicity but not to physical contact, and they rely on the stress response for immunity-independent protection.

Porcine extra-intestinal pathogenic Escherichia coli (ExPEC) causes great economic losses to the pig industry and poses a serious threat to public health worldwide. Some secreted virulence factors have been reported to be involved in the pathogenicity of the infection caused by ExPEC. Type-VI secretion system (T6SS) is discovered in many Gram-negative bacteria and contributes to the virulence of pathogenic bacteria. Valine-glycine repeat protein G (VgrG) has been reported as an important component of the functional T6SS. In our previous studies, a functional T6SS was identified in porcine ExPEC strain PCN033. Further analysis of the PCN033 genome identified two putative vgrGs genes (vgrG1 and 0248) located inside T6SS cluster and another two (vgrG2 and 1588) outside it. This study determined the function of the four putative VgrG proteins by constructing a series of mutants and complemented strains. In vitro, the VgrG1 protein was observed to be involved in the antibacterial ability and the interactions with cells. The animal model experiment showed that the deletion of vgrG1 significantly led to the decrease in the multiplication capacity of PCN033. However, the deletion of 0248 and/or the deletion of vgrG2 and 1588 had no effect on the pathogenicity of PCN033. The study of four putative VgrGs in PCN033 indicated that only VgrG1 plays an important role in the interaction between PCN033 and other bacteria or host cells. This study can provide a novel perspective to the pathogenesis of PCN033 and lay the foundation for discovering potential T6SS effectors.

Abstract Background The Dickeya genus is part of the Pectobacteriaceae family that is included in the newly described enterobacterales order. It comprises a group of aggressive soft rot pathogens with wide geographic distribution and host range. Among them, the new Dickeya fangzhongdai species groups causative agents of maceration-associated diseases that impact a wide variety of crops and ornamentals. It affects mainly monocot plants, but D. fangzhongdai strains have also been isolated from pear trees and water sources. Here, we analysed which genetic novelty exists in this new species, what are the D. fangzhongdai-specific traits and what is the intra-specific diversity. Results The genomes of eight D. fangzhongdai strains isolated from diverse environments were compared to 31 genomes of strains belonging to other Dickeya species. The D. fangzhongdai core genome regroups approximately 3500 common genes, including most genes that encode virulence factors and regulators characterised in the D. dadantii 3937 model strain. Only 38 genes are present in D. fangzhongdai and absent in all other Dickeyas. One of them encodes a pectate lyase of the PL10 family of polysaccharide lyases that is found only in a few bacteria from the plant environment, soil or human gut. Other D. fangzhongdai-specific genes with a known or predicted function are involved in regulation or metabolism. The intra-species diversity analysis revealed that seven of the studied D. fangzhongdai strains were grouped into two distinct clades. Each clade possesses a pool of 100–150 genes that are shared by the clade members, but absent from the other D. fangzhongdai strains and several of these genes are clustered into genomic regions. At the strain level, diversity resides mainly in the arsenal of T5SS- and T6SS-related toxin-antitoxin systems and in secondary metabolite biogenesis pathways. Conclusion This study identified the genome-specific traits of the new D. fangzhongdai species and highlighted the intra-species diversity of this species. This diversity encompasses secondary metabolites biosynthetic pathways and toxins or the repertoire of genes of extrachromosomal origin. We however didn’t find any relationship between gene content and phenotypic differences or sharing of environmental habitats.

ABSTRACT The type VI secretion system (T6SS) is a critical weapon in bacterial warfare between Gram-negative bacteria. Although invaluable for niche establishment, this machine represents an energetic burden to its host bacterium. Acinetobacter baumannii is an opportunistic pathogen that poses a serious threat to public health due to its high rates of multidrug resistance. In some A. baumannii strains, the T6SS is transcriptionally downregulated by large multidrug resistance plasmids. Other strains, such as the clinical isolate AbCAN2, express T6SS-related genes but lack T6SS activity under laboratory conditions, despite not harboring these plasmids. This suggests that alternative mechanisms exist to repress the T6SS. Here, we used a transposon mutagenesis approach in AbCAN2 to identify novel T6SS repressors. Our screen revealed that the T6SS of this strain is inhibited by a homolog of VgrG, an essential structural component of all T6SSs reported to date. We named this protein inhibitory VgrG (VgrGi). Biochemical and in silico analyses demonstrated that the unprecedented inhibitory capability of VgrGi is due to a single amino acid mutation in a widely conserved C-terminal domain of unknown function, DUF2345. We also show that unlike in other bacteria, the C terminus of VgrG is essential for functional T6SS assembly in A. baumannii. Our study provides insight into the architectural requirements underlying functional assembly of the T6SS of A. baumannii. We propose that T6SS-inactivating point mutations are beneficial to the host bacterium, since they eliminate the energy cost associated with maintaining a functional T6SS, which appears to be unnecessary for A. baumannii virulence. IMPORTANCE Despite the clinical relevance of A. baumannii, little is known about its fundamental biology. Here, we show that a single amino acid mutation in VgrG, a critical T6SS structural protein, abrogates T6SS function. Given that this mutation was found in a clinical isolate, we propose that the T6SS of A. baumannii is probably not involved in virulence; this idea is supported by multiple genomic analyses showing that the majority of clinical A. baumannii strains lack proteins essential to the T6SS. We also show that, unlike in other species, the C terminus of VgrG is a unique architectural requirement for functional T6SS assembly in A. baumannii, suggesting that over evolutionary time, bacteria have developed changes to their T6SS architecture, leading to specialized systems.

Type VI secretion system (T6SS) has been discovered in a variety of gram-negative bacteria as a versatile weapon to stimulate the killing of eukaryotic cells or prokaryotic competitors. Type VI secretion effectors (T6SEs) are well known as key virulence factors for important pathogenic bacteria. In many Burkholderia species, T6SS has evolved as the most complicated secretion pathway with distinguished types to translocate diverse T6SEs, suggesting their essential roles in this genus. Here we attempted to detect and characterize T6SSs and potential T6SEs in target genomes of plant-associated and environmental Burkholderia species based on computational analyses. In total, 66 potential functional T6SS clusters were found in 30 target Burkholderia bacterial genomes, of which 33% possess three or four clusters. The core proteins in each cluster were specified and phylogenetic trees of three components (i.e., TssC, TssD, TssL) were constructed to elucidate the relationship among the identified T6SS clusters. Next, we identified 322 potential T6SEs in the target genomes based on homology searches and explored the important domains conserved in effector candidates. In addition, using the screening approach based on the profile hidden Markov model (pHMM) of T6SEs that possess markers for type VI effectors (MIX motif) (MIX T6SEs), 57 revealed proteins that were not included in training datasets were recognized as novel MIX T6SE candidates from the Burkholderia species. This approach could be useful to identify potential T6SEs from other bacterial genomes.

Motility is required for many bacterial pathogens to reach and colonize target sites. Vibrio cholerae traverses a thick mucus barrier coating the small intestine to reach the underlying epithelium. We screened a transposon library in motility medium containing mucin to identify factors that influence mucus transit. Lesions in structural genes of the type VI secretion system (T6SS) were among those recovered. Two-dimensional (2D) and 3D single-cell tracking was used to compare the motility behaviors of wild-type cells and a mutant that collectively lacked three essential T6SS structural genes (T6SS+). In the absence of mucin, wild-type and T6SS+ cells exhibited similar speeds and run-reverse-flick (RRF) swimming patterns, in which forward-moving cells briefly backtrack before stochastically reorienting (flicking) in a new direction upon resuming forward movement. We show that mucin induced T6SS expression and activity in wild-type bacteria but significantly decreased their swimming speed and flicking, yielding curvilinear or near-surface circular traces for many cells. Conversely, mucin slowed T6SS+ cells to a lesser extent, and many continued to flick and produce RRF-like traces. ΔcheY3 cells, which exclusively swim in the forward direction and thus cannot flick, also produced curvilinear traces with or without mucin present and, on occasion, near-surface circular traces in the presence of mucin. The dependence of flicking on swimming speed suggested that mucininduced T6SS activity further decreased V. cholerae motility and thereby reduced flicking probability during reverse-to-forward transitions. We propose that this encourages cells to continue on their current trajectory rather than reorienting, which may benefit those tracking toward the epithelial surface. IMPORTANCE V. cholerae deploys an arsenal of virulence factors as it attempts to traverse a protective mucus layer and reach the epithelial surface of the distal small intestine. The T6SS used to cull bacterial competition during infection is induced by mucus. We show that this activity may serve an additional purpose by further decreasing motility in the presence of mucin, thereby reducing the probability of speed-dependent, near-perpendicular directional changes. We posit that this encourages cells to maintain course rather than change direction, which may aid those attempting to reach and colonize the epithelial surface. [ABSTRACT FROM AUTHOR]

Vibrio cholerae , the causative agent of cholera, could proliferate in aquatic environment and infect humans through contaminated food and water. Enormous microorganisms residing in human gastrointestinal tract establish a special microecological system, which immediately responds to the invasion of V. cholerae , through "colonization resistance" mechanisms, such as antimicrobial peptide production, nutrients competition, and intestinal barrier maintenances. Meanwhile, V. cholerae could quickly sense those signals and modulate the expression of relevant genes to circumvent those stresses during infection, leading to successful colonization on the surface of small intestinal epithelial cells. In this review, we summarized the crosstalks profiles between gut microbiota and V. cholerae in the terms of Type VI Secretion System (T6SS), Quorum Sensing (QS), Reactive Oxygen Species (ROS)/pH stress, and Bioactive metabolites. These mechanisms can also be applied to molecular bacterial pathogenesis of other pathogens in host. [ABSTRACT FROM AUTHOR]