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Identification of Vibrio cholerae type III secretion system effector proteins.

ABSTRACT: AM-19226 is a pathogenic O39 serogroup Vibrio cholerae strain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ?49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a ?-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins were bona fide effectors translocated into HeLa cells in vitro in a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only in V. cholerae and Vibrio parahaemolyticus T3SS-positive strains and has not been previously studied. A vopX deletion reduces the ability of strain AM-19226 to colonize in vivo, and the bile-induced expression of a vopX-lacZ transcriptional fusion in vitro is regulated by the T3SS-encoded transcriptional regulators VttR(A) and VttR(B). An RLM1 yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that the V. cholerae T3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.

SUBMITTER: Alam A

PROVIDER: S-EPMC3067535 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Identification of Vibrio cholerae type III secretion system effector proteins.

Alam Ashfaqul A Miller Kelly A KA Chaand Mudit M Butler J Scott JS Dziejman Michelle M

Infection and immunity 20110131 4

AM-19226 is a pathogenic O39 serogroup Vibrio cholerae strain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified ...[more]

PMID: 21282418

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Project description:UNLABELLED:The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. IMPORTANCE:The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents.

Project description:Edwardsiella ictaluri, a major pathogen in channel catfish aquaculture, encodes a type III secretion system (T3SS) that is essential for intracellular replication and virulence. Previous work identified three putative T3SS effectors in E. ictaluri, and in silico analysis of the E. ictaluri genome identified six additional putative effectors, all located on the chromosome outside the T3SS pathogenicity island. To establish active translocation by the T3SS, we constructed translational fusions of each effector to the amino-terminal adenylate cyclase (AC) domain of the Bordetella pertussis adenylate cyclase toxin CyaA. When translocated through the membrane of the Edwardsiella-containing vacuole (ECV), the cyclic AMP produced by the AC domain in the presence of calmodulin in the host cell cytoplasm can be measured. Results showed that all nine effectors were translocated from E. ictaluri in the ECV to the cytoplasm of the host cells in the wild-type strain but not in a T3SS mutant, indicating that translocation is dependent on the T3SS machinery. This confirms that the E. ictaluri T3SS is similar to the Salmonella pathogenicity island 2 T3SS in that it translocates effectors through the membrane of the bacterial vacuole directly into the host cell cytoplasm. Additional work demonstrated that both initial acidification and subsequent neutralization of the ECV were necessary for effector translocation, except for two of them that did not require neutralization. Single-gene mutants constructed for seven of the individual effectors were all attenuated for replication in CCO cells, but only three were replication deficient in head kidney-derived macrophages (HKDM). IMPORTANCE The bacterial pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), an economically significant disease of farm-raised channel catfish. Commercial catfish production accounts for the majority of the total fin fish aquaculture in the United States, with almost 300,000 tons produced annually, and ESC is the leading cause of disease loss in the industry. We have demonstrated the survival and replication of E. ictaluri within channel catfish cells and identified a secretion system that is essential for E. ictaluri intracellular replication and virulence. We have also identified nine proteins encoded in the E. ictaluri genome that we believe are actively transferred from the bacterium to the cytoplasm of the host cell and act to manipulate host cell physiology to the advantage of the bacterium. The data presented here confirm that the proteins are actually transferred during an infection, which will lead to further work on approaches to preventing or controlling ESC.

Dataset Information

Identification of Vibrio cholerae type III secretion system effector proteins.

Publications

Identification of Vibrio cholerae type III secretion system effector proteins.

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