Strain competition restricts colonization of an enteric pathogen and prevents colitis

The microbiota is a major source of protection against intestinal pathogens; however, the specific bacteria and underlying mechanisms involved are not well understood. As a model of this interaction, we sought to determine whether colonization of the murine host with symbiotic non‐toxigenic Bacteroides fragilis could limit acquisition of pathogenic enterotoxigenic B. fragilis. We observed strain‐specific competition with toxigenic B. fragilis, dependent upon type VI secretion, identifying an effector–immunity pair that confers pathogen exclusion. Resistance against host acquisition of a second non‐toxigenic strain was also uncovered, revealing a broader function of type VI secretion systems in determining microbiota composition. The competitive exclusion of enterotoxigenic B. fragilis by a non‐toxigenic strain limited toxin exposure and protected the host against intestinal inflammatory disease. Our studies demonstrate a novel role of type VI secretion systems in colonization resistance against a pathogen. This understanding of bacterial competition may be utilized to define a molecularly targeted probiotic strategy.     

Use of ELISA and GUS‐transformed strains to study competition between pathogenic and non‐pathogenic Fusarium oxysporum for root colonization

To characterize the ability of different strains of Fusarium oxysporum to colonize roots, and to analyze competition for root colonization between pathogenic and non‐pathogenic strains of F. oxysporum, it was necessary to develop specific labelling techniques for quantification of root colonization. Two methods were selected: the production of polyclonal antibodies, and the use of GUS‐transformed strains of F. oxysporum. The polyclonal antibodies recognized infected plants, and gave a minimum reaction with healthy plants, but were not specific for individual strains of F. oxysporum. These antibodies enabled total density of F. oxysporum to be assessed on roots, by ELISA. Metabolic activity of the root population of GUS‐marked strains was assessed by measuring the glucuronidase activity. Strains showed a diversity in their ability to colonize roots: patterns of root colonization were similar, but the intensity and the speed of colonization differed according to the plant—fungus combination used. Results demonstrated competition between the pathogenic and the non‐pathogenic strains for root colonization. In the presence of the non‐pathogenic strain Fo 47, the competition seems to be reciprocal, affecting both the pathogen and non‐pathogenic strain. Other non‐pathogenic strains reduced root colonization by the pathogenic strain, but some strains did not reduce the metabolic activity of the pathogen, suggesting that different mechanisms are involved in the interaction between pathogenic and non‐pathogenic F. oxysporum.     

Prevalence of type III secretion system genes in cholera vibrios from different serogroups

Prevalence of vcs genes coding the type III secretion system (T3SS) in cholera vibrios of different serogroups isolated in Russia and neighboring countries was studied for the first time. Virulent strains of O1 and O139 serogroups as well as toxigenic Vibrio cholerae strains of other serogroups contained no T3SS genes. Unlike mentioned strains, 29.2% of atoxigenic non O1/non O139 cholera vibrios isolated from patients in Russia and neighboring countries contained the T3SS genes cluster, which might contribute to the pathogenic properties of these strains.     

Proteomic Adaptation of Australian Epidemic Bordetella pertussis

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non‐ptxP3/non‐prn2). Cluster I was mostly responsible for the 2008–2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high‐resolution multiple reaction monitoring (MRM‐hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.     

Characterization of V. cholerae T3SS‐dependent cytotoxicity in cultured intestinal epithelial cells

AM‐19226 is a pathogenic, non‐O1/non‐O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co‐culturing the Caco2‐BBE human intestinal epithelial cell line with AM‐19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2‐BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell‐cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM‐19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner.     

New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens

Protein secretion systems are critical to bacterial virulence and interactions with other organisms. The Type VI secretion system (T6SS) is found in many bacterial species and is used to target either eukaryotic cells or competitor bacteria. However, T6SS‐secreted proteins have proven surprisingly elusive. Here, we identified two secreted substrates of the antibacterial T6SS from the opportunistic human pathogen, Serratia marcescens. Ssp1 and Ssp2, both encoded within the T6SS gene cluster, were confirmed as antibacterial toxins delivered by the T6SS. Four related proteins encoded around the Ssp proteins (‘Rap’ proteins) included two specifically conferring self‐resistance (‘immunity’) against T6SS‐dependent Ssp1 or Ssp2 toxicity. Biochemical characterization revealed specific, tight binding between cognate Ssp–Rap pairs, forming complexes of 2:2 stoichiometry. The atomic structures of two Rap proteins were solved, revealing a novel helical fold, dependent on a structural disulphide bond, a structural feature consistent with their functional localization. Homologues of the Serratia Ssp and Rap proteins are found encoded together within other T6SS gene clusters, thus they represent founder members of new families of T6SS‐secreted and cognate immunity proteins. We suggest that Ssp proteins are the original substrates of the S. marcescens T6SS, before horizontal acquisition of other T6SS‐secreted toxins. Molecular insight has been provided into how pathogens utilize antibacterial T6SSs to overcome competitors and succeed in polymicrobial niches.     

PAAR‐Rhs proteins harbor various C‐terminal toxins to diversify the antibacterial pathways of type VI secretion systems

The type VI secretion system (T6SS) of bacteria plays a key role in competing for specific niches by the contact‐dependent killing of competitors. Recently, Rhs proteins with polymorphic C‐terminal toxin‐domains that inhibit or kill neighboring cells were identified. In this report, we identified a novel Rhs with an MPTase4 (Metallopeptidase‐4) domain (designated as Rhs‐CT1) that showed an antibacterial effect via T6SS in Escherichia coli. We managed to develop a specific strategy by matching the diagnostic domain‐architecture of Rhs‐CT1 (Rhs with an N‐terminal PAAR‐motif and a C‐terminal toxin domain) for effector retrieval and discovered a series of Rhs‐CTs in E. coli. Indeed, the screened Rhs‐CT3 with a REase‐3 (Restriction endonuclease‐3) domain also mediated interbacterial antagonism. Further analysis revealed that vgrG_O1 and eagR/DUF1795 (upstream of rhs‐ct) were required for the delivery of Rhs‐CTs, suggesting eagR as a potential T6SS chaperone. In addition to chaperoned Rhs‐CTs, neighborless Rhs‐CTs could be classified into a distinct family (Rhs‐Nb) sharing close evolutionary relationship with T6SS2‐Rhs (encoded in the T6SS2 cluster of E. coli). Notably, the Rhs‐Nb‐CT5 was confirmed bioinformatically and experimentally to mediate interbacterial antagonism via Hcp2B‐VgrG2 module. In a further retrieval analysis, we discovered various toxin/immunity pairs in extensive bacterial species that could be systematically classified into eight referential clans, suggesting that Rhs‐CTs greatly diversify the antibacterial pathways of T6SS.     

An AIL family protein promotes type three secretion system‐1‐independent invasion and pathogenesis of Salmonella enterica serovar Typhi

Adhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system‐1 (T3SS‐1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non‐typhoidal Salmonellae. Here, we show that T2942, an AIL‐like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS‐1 independent was proved by ectopic expression of T2942 in the non‐invasive E. coli BL21 and double‐mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host‐binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti‐T2942 antiserum blocked adhesion/invasion of S. Typhi, indicating specificity. A 20‐amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin‐binding T2544 for adhesion and T3SS‐1 for invasion. Finally, T2942 was required and synergistically worked with T3SS‐1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20‐mer peptide may be suitable for vaccine development.     

Role of the acquisition of a type 3 secretion system in the emergence of novel pathogenic strains of Xanthomonas

Cases of emergence of novel plant-pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non-pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non-pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild-type, non-pathogenic strains induced a hypersensitive response (HR)-like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non-pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non-pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter-selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.     

Ancestral acquisitions,gene flow and multiple evolutionary trajectories of the type three secretion system and effectors in Xanthomonas plant pathogens

Deciphering the evolutionary history and transmission patterns of virulence determinants is necessary to understand the emergence of novel pathogens. The main virulence determinant of most pathogenic proteobacteria is the type three secretion system (T3SS). The Xanthomonas genus includes bacteria responsible for numerous epidemics in agroecosystems worldwide and represents a major threat to plant health. The main virulence factor of Xanthomonas is the Hrp2 family T3SS; however, this system is not conserved in all strains and it has not been previously determined whether the distribution of T3SS in this bacterial genus has resulted from losses or independent acquisitions. Based on comparative genomics of 82 genome sequences representing the diversity of the genus, we have inferred three ancestral acquisitions of the Hrp2 cluster during Xanthomonas evolution followed by subsequent losses in some commensal strains and re‐acquisition in some species. While mutation was the main force driving polymorphism at the gene level, interspecies homologous recombination of large fragments expanding through several genes shaped Hrp2 cluster polymorphism. Horizontal gene transfer of the entire Hrp2 cluster also occurred. A reduced core effectome composed of xopF1, xopM, avrBs2 and xopR was identified that may allow commensal strains overcoming plant basal immunity. In contrast, stepwise accumulation of numerous type 3 effector genes was shown in successful pathogens responsible for epidemics. Our data suggest that capacity to intimately interact with plants through T3SS would be an ancestral trait of xanthomonads. Since its acquisition, T3SS has experienced a highly dynamic evolutionary history characterized by intense gene flux between species that may reflect its role in host adaptation.     

Pathogenic synergy: mixed intra-abdominal infections

In this article we review our researches into the pathogenesis of mixed infections. These may conveniently be divided into in vitro and in vivo studies. In vitro we confirmed that interference with the killing of aerobes by polymorphonuclear leucocytes (PMN’s) is a property of theBacteroides strains tested and appears to depend on competition for opsonins i.e. complement factors. Further studies are in progress to define which complement factors and which bacterial structures are involved. The influence ofB. fragilis on chemotaxis has also been studied. Our preliminary data suggest thatB. fragilis is itself poorly chemotactic and reduces the chemoattractivity ofProteus mirabilis. This observation is surprising when we consider that abscess formation is the hall-mark ofB. fragilis infections and needs clarification. In vivo we have developed a skin infection model in mice which is economical and gives reproducible and quantitative results. In this model we have demonstrated pathogenic synergy betweenEscherichia coli andB. fragilis. Further studies are planned to assess the role of complement and bacterial factors in this in vivo synergy.     

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3^Pf, is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3^Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3^Pf inner membrane immunity protein.     

The type VI protein secretion system contributes to biofilm formation and seed‐to‐seedling transmission of Acidovorax citrulli on melon

The type VI protein secretion system (T6SS) is essential for the virulence of several Gram‐negative bacteria. In this study, we identified a T6SS gene cluster in Acidovorax citrulli, a plant‐pathogenic bacterium that causes bacterial fruit blotch (BFB) of cucurbits. One T6SS cluster, of approximately 25 kb in length and comprising 17 genes, was found in the A. citrulli AAC00‐1 genome. Seventeen A. citrulli mutants were generated, each with a deletion of a single T6SS core gene. There were significant differences in BFB seed‐to‐seedling transmission between wild‐type A. citrulli strain, xjl12, and ΔvasD, ΔimpK, ΔimpJ and ΔimpF mutants (71.71%, 9.83%, 8.41%, 7.15% and 5.99% BFB disease index, respectively). In addition, we observed that these four mutants were reduced in melon seed colonization and biofilm formation; however, they were not affected in virulence when infiltrated into melon seedling tissues. There were no significant differences in BFB seed‐to‐seedling transmission, melon tissue colonization and biofilm formation between xjl12 and the other 13 T6SS mutants. Overall, our results indicate that T6SS plays a role in seed‐to‐seedling transmission of BFB on melon.     

The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system

In Pseudomonas aeruginosa three type VI secretion systems (T6SSs) coexist, called H1‐ to H3‐T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 (TagJ) is unique to the H1‐T6SS and absent from the H2‐ and H3‐T6SSs. We demonstrate that HsiE1 interacts with a predicted N‐terminal α‐helix in HsiB1 (TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath‐like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ‐like component are often devoid of the predicted N‐terminal helical region, which suggests co‐evolution. We observe that a synthetic peptide corresponding to the N‐terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non‐essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS.     

Impact of non‐pathogenic bacteria on insect disease resistance: importance of ecological context

1. The aerial surface of plants is a habitat for large and diverse microbial communities; termed the phyllosphere. These microbes are unavoidably consumed by herbivores, and while the entomopathogens are well studied, the impact of non‐pathogenic bacteria on herbivore life history is less clear. 2. Previous work has suggested that consumption of non‐entomopathogenic bacteria induces a costly immune response that might decrease the risk of infection. However, we hypothesised that insect herbivores should be selective in how they respond to commonly encountered non‐pathogenic bacteria on their host plants to avoid unnecessary and costly immune responses. 3. An ecologically realistic scenario was used in which we fed cabbage looper, Trichoplusia ni Hübner, larvae on cabbage or cucumber leaves treated with the common non‐entomopathogenic phyllosphere bacteria, Pseudomonas fluorescens and P. syringae. Their constitutive immunity and resistance to a pathogenic bacterium (Bacillus thuringiensis; Bt) and a baculovirus (T. ni single nucleopolyhedrovirus) were then examined. 4. While feeding on bacteria‐treated leaves reduced the growth rate and condition of T. ni, there was no effect on immunity (haemolymph antibacterial and phenoloxidase activities and haemocyte numbers). Phyllosphere bacteria weakly affected the resistance of T. ni to Bt but the direction of this effect was concentration dependent; resistance to the virus was unaffected. Host plant had an impact, with cucumber‐fed larvae being more susceptible to Bt. 5. The lack of evidence for a costly immune response to non‐entomopathogenic bacteria suggests that T. ni are probably adapted to consuming common phyllosphere bacteria, and highlights the importance of the evolutionary history of participants in multi‐trophic interactions.     

Genetically distinct pathways guide effector export through the type VI secretion system

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co‐regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone‐like quality of Hcp. Application of this approach to the Hcp secretion island I‐encoded T6SS (H1‐T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (t ype VI s ecretion e xported 4), subsequently shown to act as a potent intra‐specific H1‐T6SS‐delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1‐T6SS effectors, Tse5 and Tse6, which differ from Hcp‐stabilized substrates by the presence of toxin‐associated PAAR‐repeat motifs and genetic linkage to members of the valine‐glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp‐stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1‐T6SS‐exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.     

Hcp and VgrG1 are secreted components of the Helicobacter hepaticus type VI secretion system and VgrG1 increases the bacterial colitogenic potential

The enterohepatic Epsilonproteobacterium Helicobacter hepaticus persistently colonizes the intestine of mice and causes chronic inflammatory symptoms in susceptible mouse strains. The bacterial factors causing intestinal inflammation are poorly characterized. A large genomic pathogenicity island, HHGI1, which encodes components of a type VI secretion system (T6SS), was previously shown to contribute to the colitogenic potential of H. hepaticus. We have now characterized the T6SS components Hcp, VgrG1, VgrG2 and VgrG3, encoded on HHGI1, including the potential impact of the T6SS on intestinal inflammation in a mouse T‐cell transfer model. The H. hepaticus T6SS components were expressed during the infection and secreted in a T6SS‐dependent manner, when the bacteria were cultured either in the presence or in the absence of mouse intestinal epithelial cells. Mutants deficient in VgrG1 displayed a significantly lower colitogenic potential in T‐cell‐transferred C57BL/6 Rag2^?/? mice, despite an unaltered ability to colonize mice persistently. Intestinal microbiota analyses demonstrated only minor changes in mice infected with wild‐typeH. hepaticus as compared with mice infected with VgrG1‐deficient isogenic bacteria. In addition, competitive assays between both wild‐type and T6SS‐deficient H. hepaticus, and between wild‐type H. hepaticus and Campylobacter jejuni or Enterobacteriaceae species did not show an effect of the T6SS on interbacterial competitiveness. Therefore, we suggest that microbiota alterations did not play a major role in the changes of pro‐inflammatory potential mediated by the T6SS. Cellular innate pro‐inflammatory responses were increased by the secreted T6SS proteins VgrG1 and VgrG2. We therefore concluded that the type VI secretion component VgrG1 can modulate and specifically exacerbate the innate pro‐inflammatory effect of the chronic H. hepaticus infection.     

Interbacterial competition and anti-predatory behaviour of environmental Vibrio cholerae strains

Vibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the haemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti-grazing defense relies on the strains' T6SS and its actincross-linking domain (ACD)-containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS-dependent interbacterial killing. We explored the latter phenotype through whole-genome sequencing of 14 isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but non-identical immunity proteins were insufficient to provide cross-immunity among those wild strains.