The bacterium Vibrio cholerae, like other human pathogens that reside in environmental reservoirs, survives predation by unicellular eukaryotes. Strains of the O1 and O139 serogroups cause cholera, whereas non-O1͞non-O139 strains cause human infections through poorly defined mechanisms. Using Dictyostelium discoideum as a model host, we have identified a virulence mechanism in a non-O1͞non-O139 V. cholerae strain that involves extracellular translocation of proteins that lack N-terminal hydrophobic leader sequences. Accordingly, we have named these genes ''VAS'' genes for virulence-associated secretion, and we propose that these genes encode a prototypic ''type VI'' secretion system. We show that vas genes are required for cytotoxicity of V. cholerae cells toward Dictyostelium amoebae and mammalian J774 macrophages by a contact-dependent mechanism. A large number of Gram-negative bacterial pathogens carry genes homologous to vas genes and potential effector proteins secreted by this pathway (i.e., hemolysin-coregulated protein and VgrG). Mutations in vas homologs in other bacterial species have been reported to attenuate virulence in animals and cultured macrophages. Thus, the genes encoding the VAS-related, type VI secretion system likely play an important conserved function in microbial pathogenesis and represent an additional class of targets for vaccine and antimicrobial drug-based therapies.Dictyostelium discoideum ͉ type VI secretion ͉ virulence-associated secretion C holera is a severe, life-threatening diarrheal disease caused by Vibrio cholerae strains of the O1 and O139 serogroups. In contrast, non-O1, non-O139 strains of V. cholerae are primarily associated with isolated cases of extra-intestinal infection or gastroenteritis. An exception to this pattern was a large outbreak of a cholera-like illness that occurred in 1968 in Sudan, where an O37 strain of V. cholerae caused 460 cases and 125 deaths (1). The virulence mechanisms of O1 and O139 strains involve the elaboration of extracellular factors such as cholera enterotoxin and toxin coregulated pili. In contrast, the virulence mechanisms used by non-O1, non-O139 strains remain poorly defined (2). Using the social amoeba Dictyostelium discoideum as a model host, we have developed an experimental system designed to identify novel virulence mechanisms from pathogenic non-O1, non-O139 strains.D. discoideum is a eukaryotic organism that seeks out and preys on bacteria through its phagocytic feeding behavior. As such, it has been used as a model eukaryotic cell that mimics a mammalian macrophage in aspects of its cell biology and interaction with microbes. Several environmental pathogenic bacteria, including Legionella pneumophila, Mycobacterium marinum, and Pseudomonas aeruginosa (3), resist Dictyostelium predation by producing factors that either kill amoebae or allow successful intracellular survival and multiplication. In these cases, the same virulence mechanisms operative against mammalian cells have also been implicated in resistance to Dictyosteli...

Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae, T6SS have been found to secrete three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro, and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate ''tail spike'' of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS apparatus may assemble a ''cellpuncturing device'' analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells.bacteriophage ͉ cytotoxicity ͉ Vibrio cholerae ͉ virulence

Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth.

SUMMARY The type VI secretion system (T6SS) is a virulence mechanism common to several Gram-negative pathogens. In Vibrio cholerae, VgrG-1 is required for T6SS-dependent secretion. VgrG-1 is also secreted by T6SS and displays a C-terminal actin cross-linking domain (ACD). Using a heterologous reporter enzyme in place of the ACD, we show that the effector and secretion functions of VgrG-1 are genetically dissociable with the ACD being dispensable for secretion, but required for T6SS-dependent phenotypes. Furthermore, internalization of bacteria is required for ACD translocation into phagocytic target cells. Inhibiting bacterial uptake abolishes actin cross-linking while improving intracellular survival enhances it. Otherwise resistant nonphagocytic cells become susceptible to T6SS-mediated actin cross-linking when engineered to take up bacteria. Our results support a model for translocation of VgrG C-terminal effector domains into target cell cytosol by a process that requires trafficking of bacterial cells into an endocytic compartment where translocation is triggered by an unknown signal.