Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system

Abstract

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...

Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium

“…We generated mutants of both sciG (the clpV ortholog), and sciS (the icmF ortholog), genes which have been established as essential for T6SS assembly and functional secretion in other systems (6,42). When mice were infected with wild-type and mutant strains of S. Typhimurium, mice infected with the mutant strains reached an endpoint approximately 1 to 2 days later than mice infected with wild-type S. Typhimurium (Fig.…”

Salmonella Typhimurium utilizes a T6SS-mediated antibacterial weapon to establish in the host gut

“…Many Gram-negative bacteria encode a molecular machine called the T6SS that is dedicated to target and kill other bacteria and sometimes to inject effector proteins into eukaryotic cells (10). For example, the human pathogen V. cholerae employs the T6SS to kill E. coli or to disable the amoebae Dictyostelium discoideum and macrophage cell lines (14,48,49). Recent studies demonstrated that T6SS organelles in V. cholerae, P. aeruginosa, and entero-aggregative E. coli cells are very dynamic and likely expel their T6SS spike/tube VgrG/Hcp complex toward prokaryotic cells and cause target cell lysis by delivering antibacterial toxins (50,51).…”

Direct and Indirect Impairment of Human Dendritic Cell Function by VirulentFrancisella tularensisSchu S4

“…In other bacteria, type VI secretion systems are responsible for secretion of molecules that are capable of disabling the host immune response. For example, proteins secreted via the type VI secretion system described in Vibrio cholera are responsible for cytotoxicity in mammalian macrophages (38). Porphyromonas gingivalis utilizes type VI secretion to expel gingipain proteases.…”