Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae.

Vibrio cholerae controls the pathogenicity of interactions with arthropod hosts via the activity of the CrbS/R two-component system. This signaling pathway regulates the consumption of acetate, which in turn alters the relative virulence of interactions with arthropods, including Drosophila melanogaster CrbS is a histidine kinase that links a transporter-like domain to its signaling apparatus via putative STAC and PAS domains. CrbS and its cognate response regulator are required for the expression of acetyl coenzyme A (acetyl-CoA) synthetase (product of acs), which converts acetate to acetyl-CoA. We demonstrate that the STAC domain of CrbS is required for signaling in culture; without it, acs transcription is reduced in LB medium, and V. cholerae cannot grow on acetate minimal media. However, the strain remains virulent toward Drosophila and expresses acs similarly to the wild type during infection. This suggests that there is a unique signal or environmental variable that modulates CrbS in the gastrointestinal tract of Drosophila Second, we present evidence in support of CrbR, the response regulator that interacts with CrbS, binding directly to the acs promoter, and we identify a region of the promoter that CrbR may target. We further demonstrate that nutrient signals, together with the cAMP receptor protein (CRP)-cAMP system, control acs transcription, but regulation may occur indirectly, as CRP-cAMP activates the expression of the crbS and crbR genes. Finally, we define the role of the Pta-AckA system in V. cholerae and identify redundancy built into acetate excretion pathways in this pathogen.IMPORTANCE CrbS is a member of a unique family of sensor histidine kinases, as its structure suggests that it may link signaling to the transport of a molecule. However, mechanisms through which CrbS senses and communicates information about the outside world are unknown. In the Vibrionaceae, orthologs of CrbS regulate acetate metabolism, which can, in turn, affect interactions with host organisms. Here, we situate CrbS within a larger regulatory framework, demonstrating that crbS is regulated by nutrient-sensing systems. Furthermore, CrbS domains may play various roles in signaling during infection and growth in culture, suggesting a unique mechanism of host recognition. Finally, we define the roles of additional pathways in acetate flux, as a foundation for further studies of this metabolic nexus point.

A Putative Acetylation System in Vibrio cholerae Modulates Virulence in Arthropod Hosts.

Acetylation is a broadly conserved mechanism of covalently modifying the proteome to precisely control protein activity. In bacteria, central metabolic enzymes and regulatory proteins, including those involved in virulence, can be targeted for acetylation. In this study, we directly link a putative acetylation system to metabolite-dependent virulence in the pathogen Vibrio cholerae We demonstrate that the cobB and yfiQ genes, which encode homologs of a deacetylase and an acetyltransferase, respectively, modulate V. cholerae metabolism of acetate, a bacterially derived short-chain fatty acid with important physiological roles in a diversity of host organisms. In Drosophila melanogaster, a model arthropod host for V. cholerae infection, the pathogen consumes acetate within the gastrointestinal tract, which contributes to fly mortality. We show that deletion of cobB impairs growth on acetate minimal medium, delays the consumption of acetate from rich medium, and reduces virulence of V. cholerae toward Drosophila These impacts can be reversed by complementing cobB or by introducing a deletion of yfiQ into the ΔcobB background. We further show that cobB controls the accumulation of triglycerides in the Drosophila midgut, which suggests that cobB directly modulates metabolite levels in vivo In Escherichia coli K-12, yfiQ is upregulated by cAMP-cAMP receptor protein (CRP), and we identified a similar pattern of regulation in V. cholerae, arguing that the system is activated in response to similar environmental cues. In summary, we demonstrate that proteins likely involved in acetylation can modulate the outcome of infection by regulating metabolite exchange between pathogens and their colonized hosts.IMPORTANCE The bacterium Vibrio cholerae causes severe disease in humans, and strains can persist in the environment in association with a wide diversity of host species. By investigating the molecular mechanisms that underlie these interactions, we can better understand constraints affecting the ecology and evolution of this global pathogen. The Drosophila model of Vibrio cholerae infection has revealed that bacterial regulation of acetate and other small metabolites from within the fly gastrointestinal tract is crucial for its virulence. Here, we demonstrate that genes that may modify the proteome of V. cholerae affect virulence toward Drosophila, most likely by modulating central metabolic pathways that control the consumption of acetate as well as other small molecules. These findings further highlight the many layers of regulation that tune bacterial metabolism to alter the trajectory of interactions between bacteria and their hosts.

Spatially selective colonization of the arthropod intestine through activation of Vibrio cholerae biofilm formation.

Vibrio cholerae is an estuarine bacterium and the human pathogen responsible for the diarrheal disease cholera. In the environment, arthropods are proposed to be carriers and reservoirs of V. cholerae. However, the molecular basis of the association between V. cholerae and viable arthropods has not been elucidated previously. Here, we show that the V. cholerae Vibrio polysaccharide (VPS)-dependent biofilm is highly activated upon entry into the arthropod intestine and is specifically required for colonization of the arthropod rectum. Although the V. cholerae VPS-dependent biofilm has been studied in the laboratory for many years, the function of this biofilm in the natural habitats of V. cholerae has been elusive. Our results provide evidence that the VPS-dependent biofilm is required for intestinal colonization of an environmental host.

Fly Models of Vibrio cholerae Infection and Colonization.

Studies of Vibrio cholerae pathogenesis in the context of novel eukaryotic model systems have expanded our understanding of genes that underlie V. cholerae interactions with humans, as well as host organisms in the environment. These model systems have also helped uncover new functions for many gene products, revealing previously unknown virulence mechanisms. The Drosophila model for V. cholerae infection is a powerful tool for discovering new genetic pathways that govern bacterial physiology and colonization in the arthropod gastrointestinal tract. Assays to measure both virulence and colonization have been established and are easily adopted in labs unfamiliar with Drosophila work. Experiments to compare survival of flies colonized with different bacterial mutants are simple to perform and can be completed in less than a week, allowing colonization to be quantified and localized easily. The availability of molecular and genetic tools for the fly enables further exploration of host factors that restrict V. cholerae colonization and invasive infection. Based on the Drosophila system, a house fly (Musca domestica) model of V. cholerae colonization has also been developed. The new house fly model may prove a useful tool for examining V. cholerae infection dynamics in the context of a host carrying a complex microbial community, with a fundamentally different ecology that may increase its chances of acting as a vector for cholera disease.

Activation of Vibrio cholerae quorum sensing promotes survival of an arthropod host.

Vibrio cholerae colonizes the human terminal ileum to cause cholera, and the arthropod intestine and exoskeleton to persist in the aquatic environment. Attachment to these surfaces is regulated by the bacterial quorum-sensing signal transduction cascade, which allows bacteria to assess the density of microbial neighbours. Intestinal colonization with V. cholerae results in expenditure of host lipid stores in the model arthropod Drosophila melanogaster. Here we report that activation of quorum sensing in the Drosophila intestine retards this process by repressing V. cholerae succinate uptake. Increased host access to intestinal succinate mitigates infection-induced lipid wasting to extend survival of V. cholerae-infected flies. Therefore, quorum sensing promotes a more favourable interaction between V. cholerae and an arthropod host by reducing the nutritional burden of intestinal colonization.

Pathogenicity of environmental isolates of V. cholerae in mice.

Environmental V. cholerae (Vc) have the potential for virulence in people and they may also be a reservoir of accessory virulence genes. We infected mice with two non-O1, non-O139 Vc (TP and SIO) that were isolated in San Diego County and compared them to Vc O1 El Tor N16961 using a model of pneumonia in adult mice. Live but not heat killed Vc El Tor and TP caused fatal hemorrhagic pneumonia despite a >90% decrease in CFU in 24h suggesting the disease was toxin mediated. SIO did not cause pneumonia in normal mice but neutropenic, gp91phox and complement (C3) mice were more susceptible to all three strains. TP and SIO lack ctx but have rtxA, hlyA, and hapA, genes that encode virulence factors in Vc El Tor. The explanation for the enhanced virulence of TP remains to be determined.

Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens.

The CrbS/R two-component signal transduction system is a conserved regulatory mechanism through which specific Gram-negative bacteria control acetate flux into primary metabolic pathways. CrbS/R governs expression of acetyl-CoA synthase (acsA), an enzyme that converts acetate to acetyl-CoA, a metabolite at the nexus of the cell's most important energy-harvesting and biosynthetic reactions. During infection, bacteria can utilize this system to hijack host acetate metabolism and alter the course of colonization and pathogenesis. In toxigenic strains of Vibrio cholerae, CrbS/R-dependent expression of acsA is required for virulence in an arthropod model. Here, we investigate the function of the CrbS/R system in Pseudomonas aeruginosa, Pseudomonas entomophila, and non-toxigenic V. cholerae strains. We demonstrate that its role in acetate metabolism is conserved; this system regulates expression of the acsA gene and is required for growth on acetate as a sole carbon source. As a first step towards describing the mechanism of signaling through this pathway, we identify residues and domains that may be critical for phosphotransfer. We further demonstrate that although CrbS, the putative hybrid sensor kinase, carries both a histidine kinase domain and a receiver domain, the latter is not required for acsA transcription. In order to determine whether our findings are relevant to pathogenesis, we tested our strains in a Drosophila model of oral infection previously employed for the study of acetate-dependent virulence by V. cholerae. We show that non-toxigenic V. cholerae strains lacking CrbS or CrbR are significantly less virulent than are wild-type strains, while P. aeruginosa and P. entomophila lacking CrbS or CrbR are fully pathogenic. Together, the data suggest that the CrbS/R system plays a central role in acetate metabolism in V. cholerae, P. aeruginosa, and P. entomophila. However, each microbe's unique environmental adaptations and pathogenesis strategies may dictate conditions under which CrbS/R-mediated acs expression is most critical.

The acetate switch of an intestinal pathogen disrupts host insulin signaling and lipid metabolism.

Vibrio cholerae is lethal to the model host Drosophila melanogaster through mechanisms not solely attributable to cholera toxin. To examine additional virulence determinants, we performed a genetic screen in V. cholerae-infected Drosophila and identified the two-component system CrbRS. CrbRS controls transcriptional activation of acetyl-CoA synthase-1 (ACS-1) and thus regulates the acetate switch, in which bacteria transition from excretion to assimilation of environmental acetate. The resultant loss of intestinal acetate leads to deactivation of host insulin signaling and lipid accumulation in enterocytes, resulting in host lethality. These metabolic effects are not observed upon infection with ΔcrbS or Δacs1 V. cholerae mutants. Additionally, uninfected flies lacking intestinal commensals, which supply short chain fatty acids (SCFAs) such as acetate, also exhibit altered insulin signaling and intestinal steatosis, which is reversed upon acetate supplementation. Thus, acetate consumption by V. cholerae alters host metabolism, and dietary acetate supplementation may ameliorate some sequelae of cholera.

Mutations in the IMD pathway and mustard counter Vibrio cholerae suppression of intestinal stem cell division in Drosophila.

Vibrio cholerae is an estuarine bacterium and an intestinal pathogen of humans that causes severe epidemic diarrhea. In the absence of adequate mammalian models in which to study the interaction of V. cholerae with the host intestinal innate immune system, we have implemented Drosophila melanogaster as a surrogate host. We previously showed that immune deficiency pathway loss-of-function and mustard gain-of-function mutants are less susceptible to V. cholerae infection. We find that although the overall burden of intestinal bacteria is not significantly different from that of control flies, intestinal stem cell (ISC) division is increased in these mutants. This led us to examine the effect of V. cholerae on ISC division. We report that V. cholerae infection and cholera toxin decrease ISC division. Because IMD pathway and Mustard mutants, which are resistant to V. cholerae, maintain higher levels of ISC division during V. cholerae infection, we hypothesize that suppression of ISC division is a virulence strategy of V. cholerae and that accelerated epithelial regeneration protects the host against V. cholerae. Extension of these findings to mammals awaits the development of an adequate experimental model.

Diversity and distribution of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains.

Cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC(50) = 4.6 +/- 0.4 ng/ml) and crustaceans (Artemia nauplii LD(50) = 10 +/- 2 mug/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-A resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.