Sporadic type VI secretion in seventh pandemic Vibrio cholerae.

Vibrio cholerae is a pathogen that causes disease in millions of people every year by colonizing the small intestine and then secreting the potent cholera toxin. How the pathogen overcomes the colonization barrier created by the host's natural microbiota is, however, still not well understood. In this context, the type VI secretion system (T6SS) has gained considerable attention given its ability to mediate interbacterial killing. Interestingly, and in contrast to non-pandemic or environmental V. cholerae isolates, strains that are causing the ongoing cholera pandemic (7PET clade) are considered T6SS-silent under laboratory conditions. Since this idea was recently challenged, we performed a comparative in vitro study on T6SS activity using diverse strains or regulatory mutants. We show that modest T6SS activity is detectable in most of the tested strains under interbacterial competition conditions. The system's activity was also observed through immunodetection of the T6SS tube protein Hcp in culture supernatants, a phenotype that can be masked by the strains' haemagglutinin/protease. We further investigated the low T6SS activity within the bacterial populations by imaging 7PET V. cholerae at the single-cell level. The micrographs showed the production of the machinery in only a small fraction of cells within the population. This sporadic T6SS production was higher at 30 °C than at 37 °C and occurred independently of the known regulators TfoX and TfoY but was dependent on the VxrAB two-component system. Overall, our work provides new insight into the heterogeneity of T6SS production in populations of 7PET V. cholerae strains in vitro and provides a possible explanation of the system's low activity in bulk measurements.

The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains.

The human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent seventh pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole-genome sequences and de novo assemblies, we identify several strain-specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain-specific alterations.

QstR-dependent regulation of natural competence and type VI secretion in Vibrio cholerae.

During growth on chitinous surfaces in its natural aquatic environment Vibrio cholerae develops natural competence for transformation and kills neighboring non-immune bacteria using a type VI secretion system (T6SS). Activation of these two phenotypes requires the chitin-induced regulator TfoX, but also integrates signals from quorum sensing via the intermediate regulator QstR, which belongs to the LuxR-type family of regulators. Here, we define the QstR regulon using RNA sequencing. Moreover, by mapping QstR binding sites using chromatin immunoprecipitation coupled with deep sequencing we demonstrate that QstR is a transcription factor that binds upstream of the up- and down-regulated genes. Like other LuxR-type family transcriptional regulators we show that QstR function is dependent on dimerization. However, in contrast to the well-studied LuxR-type biofilm regulator VpsT of V. cholerae, which requires the second messenger c-di-GMP, we show that QstR dimerization and function is c-di-GMP independent. Surprisingly, although ComEA, which is a periplasmic DNA-binding protein essential for transformation, is produced in a QstR-dependent manner, QstR-binding was not detected upstream of comEA suggesting the existence of a further regulatory pathway. Overall, these results provide detailed insights into the function of a key regulator of natural competence and type VI secretion in V. cholerae.

Molecular mechanism of plasmid elimination by the DdmDE defense system.

Seventh pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. Here we use cryogenic electron microscopy to reveal the mechanistic basis for plasmid defense by DdmDE. A structure of the DdmD helicase-nuclease reveals it adopts an auto-inhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. A structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the non-target DNA strand. In vitro studies reveal that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.

Genome sequences of Vibrio cholerae strains isolated in the DRC between 2009 and 2012.

Vibrio cholerae has caused seven cholera pandemics in the past two centuries. The seventh and ongoing pandemic has been particularly severe on the African continent. Here, we report long read-based genome sequences of six V. cholerae strains isolated in the Democratic Republic of the Congo between 2009 and 2012.

Single nucleotide polymorphism determines constitutive versus inducible type VI secretion in Vibrio cholerae.

Vibrio cholerae is a well-studied human pathogen that is also a common inhabitant of marine habitats. In both environments, the bacterium is subject to interbacterial competition. A molecular nanomachine that is often involved in such competitive behavior is the type VI secretion system (T6SS). Interestingly and in contrast to non-pandemic or environmental isolates, the T6SS of the O1 El Tor clade of V. cholerae, which is responsible for the ongoing 7th cholera pandemic, is largely silent under standard laboratory culture conditions. Instead, these strains induce their full T6SS capacity only under specific conditions such as growth on chitinous surfaces (signaled through TfoX and QstR) or when the cells encounter low intracellular c-di-GMP levels (TfoY-driven). In this study, we identified a single nucleotide polymorphism (SNP) within an intergenic region of the major T6SS gene cluster of V. cholerae that determines the T6SS status of the cell. We show that SNP conversion is sufficient to induce T6SS production in numerous pandemic strains, while the converse approach renders non-pandemic/environmental V. cholerae strains T6SS-silent. We further demonstrate that SNP-dependent T6SS production occurs independently of the known T6SS regulators TfoX, QstR, and TfoY. Finally, we identify a putative promoter region adjacent to the identified SNP that is required for all forms of T6SS regulation in V. cholerae.

Human commensal gut Proteobacteria withstand type VI secretion attacks through immunity protein-independent mechanisms.

While the major virulence factors for Vibrio cholerae, the cause of the devastating diarrheal disease cholera, have been extensively studied, the initial intestinal colonization of the bacterium is not well understood because non-human adult animals are refractory to its colonization. Recent studies suggest the involvement of an interbacterial killing device known as the type VI secretion system (T6SS). Here, we tested the T6SS-dependent interaction of V. cholerae with a selection of human gut commensal isolates. We show that the pathogen efficiently depleted representative genera of the Proteobacteria in vitro, while members of the Enterobacter cloacae complex and several Klebsiella species remained unaffected. We demonstrate that this resistance against T6SS assaults was mediated by the production of superior T6SS machinery or a barrier exerted by group I capsules. Collectively, our data provide new insights into immunity protein-independent T6SS resistance employed by the human microbiota and colonization resistance in general.

DNA-uptake pili of Vibrio cholerae are required for chitin colonization and capable of kin recognition via sequence-specific self-interaction.

How bacteria colonize surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multipurpose class of cell surface polymers. Here we directly visualize the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth on its natural habitat-chitinous surfaces. As predicted, these pili are highly dynamic and retract before DNA uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA (present in environmental isolates) creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces and are required for chitin colonization under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonization. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.

Neighbor predation linked to natural competence fosters the transfer of large genomic regions in Vibrio cholerae.

Natural competence for transformation is a primary mode of horizontal gene transfer. Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. However, the prevalence of non-degraded DNA with sufficient coding capacity is not well understood. In this context, we previously showed that naturally competent Vibrio cholerae use their type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors. Here, we explored the conditions of the DNA released through T6SS-mediated killing versus passive cell lysis and the extent of the transfers that occur due to these conditions. We show that competent V. cholerae acquire DNA fragments with a length exceeding 150 kbp in a T6SS-dependent manner. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters the exchange of genetic material with sufficient coding capacity to significantly accelerate bacterial evolution.

Molecular insights into Vibrio cholerae's intra-amoebal host-pathogen interactions.

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host's osmoregulatory organelle. In this study, we decipher the molecular mechanisms involved in the maintenance of V. cholerae's intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrate that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, have a key role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

Circulation of a Quorum-Sensing-Impaired Variant of Vibrio cholerae Strain C6706 Masks Important Phenotypes.

Vibrio cholerae, the causative agent of cholera, is a model organism for studying virulence regulation, biofilm formation, horizontal gene transfer, and the cell-to-cell communication known as quorum sensing (QS). As in any research field, discrepancies between data from diverse laboratories are sometimes observed for V. cholerae. Such discrepancies are often caused by the use of diverse patient or environmental isolates. In this study, we investigated the inability of a few laboratories to reproduce high levels of natural transformation, a mode of horizontal gene transfer that is specifically induced on chitinous surfaces. This irreproducibility was mostly related to one specific isolate of V. cholerae: the O1 El Tor C6706 strain. C6706 was previously described as QS proficient, an important prerequisite for the induction of natural competence for transformation. To elucidate the underlying problem, we collected seven isolates of the same C6706 strain from different research laboratories in North America and Europe and compared their phenotypes. Importantly, we observed a split response with respect to QS-related gene expression, including chitin-induced natural competence and type VI secretion (T6S). While approximately half of the strains behaved as reported for several other O1 El Tor pandemic isolates that are commonly studied in the laboratory, the other half were significantly impaired in QS-related expression patterns. This impairment was caused by a mutation in a QS-related gene (luxO). We conclude that the circulation of such QS-impaired wild-type strains is responsible for masking several important phenotypes of V. cholerae, including natural competence for transformation and T6S. IMPORTANCE Phenotypic diversity between laboratory-domesticated bacterial strains is a common problem and often results in the failed reproduction of published data. However, researchers rarely compare such strains to elucidate the underlying mutation(s). In this study, we tested one of the best-studied V. cholerae isolates, O1 El Tor strain C6706 (a patient isolate from Peru), with respect to two main phenotypes: natural competence for transformation and type VI secretion. We recently demonstrated that the two phenotypes are coregulated and specifically induced upon the growth of pandemic V. cholerae O1 El Tor strains on chitinous surfaces. We provide evidence that of seven C6706 strains collected from different laboratories, four were impaired in the tested phenotypes due to a mutation in a QS gene. Collectively, our data indicate that the circulation of such a mutated wild-type strain of C6706 might have had important consequences for QS-related data.

Independent Regulation of Type VI Secretion in Vibrio cholerae by TfoX and TfoY.

Type VI secretion systems (T6SSs) are nanomachines used for interbacterial killing and intoxication of eukaryotes. Although Vibrio cholerae is a model organism for structural studies on T6SSs, the underlying regulatory network is less understood. A recent study showed that the T6SS is part of the natural competence regulon in V. cholerae and is activated by the regulator TfoX. Here, we identify the TfoX homolog TfoY as a second activator of the T6SS. Importantly, despite inducing the same T6SS core machinery, the overall regulons differ significantly for TfoX and TfoY. We show that TfoY does not contribute to competence induction. Instead, TfoY drives the production of T6SS-dependent and T6SS-independent toxins, together with an increased motility phenotype. Hence, we conclude that V. cholerae uses its sole T6SS in response to diverse cues and for distinctive outcomes: either to kill for the prey's DNA, leading to horizontal gene transfer, or as part of a defensive escape reaction.