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 g/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-Å 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.

Summary 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 (SCFA) 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.

Selection of immune escape variants impairs the ability of the immune system to sustain an efficient antiviral response and to control retroviral infections. Like other retroviruses, mouse mammary tumor virus (MMTV) is not efficiently eliminated by the immune system of susceptible mice. In contrast, MMTV-infected I/LnJ mice are capable of producing IgG2a virus-neutralizing antibodies, sustain this response throughout their life, and secrete antibody-coated virions into the milk, thereby preventing infection of their progeny. Antibodies were produced in response to several MMTV variants and were cross-reactive to them. Resistance to MMTV infection was recessive and was dependent on interferon (IFN)-γ production, because I/LnJ mice with targeted deletion of the INF-γ gene failed to produce any virus-neutralizing antibodies. These findings reveal a novel mechanism of resistance to retroviral infection that is based on a robust and sustained IFN-γ–dependent humoral immune response.

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.ach year, thousands of people living without access to adequate sanitation facilities contract cholera by ingesting food or water that carries the pandemic Vibrio cholerae bacillus (1). After passing through the acidic barrier of the stomach, V. cholerae replicates rapidly in the human gastrointestinal tract and ultimately leaves the body at concentrations as high as 10 9 per mL in a fast-developing, voluminous diarrhea characteristic of the disease. Release of trillions of bacteria into the environment from a single host results in rapid epidemic spread. Once the disease reaches a particular locale, it can become endemic to the region for years, reflecting the ability of V. cholerae to persist in the environment.Both pathogenic and nonpathogenic members of the diverse species of V. cholerae, which comprises more than 200 serogroups, are part of the normal aquatic microbial assemblage in the temperate coastal marine and estuarine waters of the world (2). In these environments, culture-dependent and culture-independent analyses have found V. cholerae in association with marine organisms such as fish (3) and copepods (4, 5). That V. cholerae can also be carried by insects such as chironomids (6) hints that V. cholerae interactions with arthropods are extensive. Indeed, V. cholerae has been isolated from houseflies in areas where cholera is endemic (7-12), signifying that both terrestrial and aquatic arthropods may act as disease reservoirs. Although the presence of arthropods has been correlated with cholera epidemics, a specific interaction between V. cholerae and arthropods has not been described.Bacterial biofilm formation mediates colonization of both biotic and abiotic surfaces. The V. cholerae multilayer biofilm, which is dependent on elaboration of a matrix comprised of the Vibrio polysaccharide (VPS) and several matrix-associated proteins (13, 14), has long been hypothesized to play a role in environmental survival. However, because this biofilm has been studied only in laboratory media, concrete evidence for such a role is lacking.Here, we demonstrate that formation of the V. cholerae VPSdependent biofilm is highly activated upon entry into the fly intestine and absolutely...

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 neighbors. 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 favorable interaction between V. cholerae and an arthropod host by reducing the nutritional burden of intestinal colonization.

Vibrio cholerae has multiple survival strategies which are reflected both in its broad distribution in many aquatic environments and its high genotypic diversity. To obtain additional information regarding the content of the V. cholerae genome, suppression subtractive hybridization (SSH) was used to prepare libraries of DNA sequences from two southern California coastal isolates which are divergent or absent in the clinical strain V. cholerae O1 El Tor N16961. More than 1,400 subtracted clones were sequenced. This revealed the presence of novel sequences encoding functions related to cell surface structures, transport, metabolism, signal transduction, luminescence, mobile elements, stress resistance, and virulence. Flanking sequence information was determined for loci of interest, and the distribution of these sequences was assessed for a collection of V. cholerae strains obtained from southern California and Mexican environments. This led to the surprising observation that sequences related to the toxin genes toxA, cnf1, and exoY are widespread and more common in these strains than those of the cholera toxin genes which are a hallmark of the pandemic strains of V. cholerae. Gene transfer among these strains could be facilitated by a 4.9-kbp plasmid discovered in one isolate, which possesses similarity to plasmids from other environmental vibrios. By investigating some of the nucleotide sequence basis for V. cholerae genotypic diversity, DNA fragments have been uncovered which could promote survival in coastal environments. Furthermore, a set of genes has been described which could be involved in as yet undiscovered interactions between V. cholerae and eukaryotic organisms.Although it is best known as the causative agent of the human disease cholera, Vibrio cholerae is also an autochthonous inhabitant of many aquatic environments, including estuarine and coastal waters (13). Indeed, the great majority of its more than 200 serogroups, excluding O1 and O139, are not associated with epidemic disease. V. cholerae has been isolated routinely from many aquatic environments throughout the world, often in association with plankton, plants, invertebrates, and fish, and there are some reports of its presence in water birds, seals, and diseased farm animals (2,26,27,33,43,56,66). The prevalence of V. cholerae in the environment is influenced by temperature and salinity (reviewed in reference 42) as well concentrations of dissolved organic carbon (44). A number of genes have been previously implicated in environmental survival, and this has already led to a better understanding of the genetic basis for V. cholerae adaptations. Genes have been uncovered which are important for biofilm formation (for examples, see references 68 and 73), zooplankton association (9), survival with filamentous blue cyanobacteria (28), and the degradation of nonbiting midge (Chironomos sp.) egg masses (24). These genes clearly provide V. cholerae with mechanisms to avoid environmental stresses and obtain nutrients in aquatic environments.The evol...

Mice of the I/LnJ inbred strain are unique in their ability to mount a robust and sustained humoral immune response capable of neutralizing infection with a betaretrovirus, mouse mammary tumor virus (MMTV). Virus-neutralizing antibodies (Abs) coat MMTV virions secreted by infected cells, preventing virus spread and hence the formation of mammary tumors. To investigate whether I/LnJ mice resist infection with other retroviruses besides MMTV, the animals were infected with murine leukemia virus (MuLV), a gammaretrovirus. MuLV-infected I/LnJ mice produced virus-neutralizing Abs that block virus transmission and virally induced disease. Generation of virus-neutralizing Abs required gamma interferon but was independent of interleukin-12. This unique mechanism of retrovirus resistance is governed by a single recessive gene, virus infectivity controller 1 (vic1), mapped to chromosome 17. In addition to controlling the antivirus humoral immune response, vic1 is also required for an antiviral cytotoxic response. Both types of responses were maintained in mice of the susceptible genetic background but congenic for the I/LnJ vic1 locus. Although the vic1-mediated resistance to MuLV resembles the mechanism of retroviral recovery controlled by the resistance to Friend virus 3 (rfv3) gene, the rfv3 gene has been mapped to chromosome 15 and confers resistance to MuLV but not to MMTV. Thus, we have identified a unique virus resistance mechanism that controls immunity against two distinct retroviruses.

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.