Pleiotropic effects of the twin-arginine translocation system on biofilm formation, colonization, and virulence in Vibrio cholerae.

State Key Laboratory for Infectious Disease Prevention and Control, Department of Diarrheal Diseases, Chinese Center for Disease Control and Prevention, Beijing, PR China.
BMC Microbiology (Impact Factor: 2.98). 02/2009; 9:114. DOI: 10.1186/1471-2180-9-114
Source: PubMed

ABSTRACT The Twin-arginine translocation (Tat) system serves to translocate folded proteins, including periplasmic enzymes that bind redox cofactors in bacteria. The Tat system is also a determinant of virulence in some pathogenic bacteria, related to pleiotropic effects including growth, motility, and the secretion of some virulent factors. The contribution of the Tat pathway to Vibrio cholerae has not been explored. Here we investigated the functionality of the Tat system in V. cholerae, the etiologic agent of cholera.
In V. cholerae, the tatABC genes function in the translocation of TMAO reductase. Deletion of the tatABC genes led to a significant decrease in biofilm formation, the ability to attach to HT-29 cells, and the ability to colonize suckling mouse intestines. In addition, we observed a reduction in the output of cholera toxin, which may be due to the decreased transcription level of the toxin gene in tatABC mutants, suggesting an indirect effect of the mutation on toxin production. No obvious differences in flagellum biosynthesis and motility were found between the tatABC mutant and the parental strain, showing a variable effect of Tat in different bacteria.
The Tat system contributes to the survival of V. cholerae in the environment and in vivo, and it may be associated with its virulence.

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    ABSTRACT: Bacterial proteins destined for the Tat pathway are folded before crossing the inner membrane and are typically identified by an N-terminal signal peptide containing a twin arginine motif. Translocation by the Tat pathway is dependent on the products of genes which encode proteins possessing the binding site of the signal peptide and mediating the actual translocation event. In the fully virulent CO92 strain of Yersinia pestis, the tatA gene was deleted. The mutant was assayed for loss of virulence through various in vitro and in vivo assays. Deletion of the tatA gene resulted in several consequences for the mutant as compared to wild-type. Cell morphology of the mutant bacteria was altered and demonstrated a more elongated form. In addition, while cultures of the mutant strain were able to produce a biofilm, we observed a loss of adhesion of the mutant biofilm structure compared to the biofilm produced by the wild-type strain. Immuno-electron microscopy revealed a partial disruption of the F1 antigen on the surface of the mutant. The virulence of the ΔtatA mutant was assessed in various murine models of plague. The mutant was severely attenuated in the bubonic model with full virulence restored by complementation with the native gene. After small-particle aerosol challenge in a pneumonic model of infection, the mutant was also shown to be attenuated. In contrast, when mice were challenged intranasally with the mutant, very little difference in the LD50 was observed between wild-type and mutant strains. However, an increased time-to-death and delay in bacterial dissemination was observed in mice infected with the ΔtatA mutant as compared to the parent strain. Collectively, these findings demonstrate an essential role for the Tat pathway in the virulence of Y. pestis in bubonic and small-aerosol pneumonic infection but less important role for intranasal challenge.
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    ABSTRACT: ABSTRACT The twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects. IMPORTANCE This work reports the first characterization of the twin-arginine translocation (Tat) system in the gastric pathogen Helicobacter pylori. While tatB mutants were easily obtained, only single-crossover partial tatC mutants or conditional tatC mutants could be generated, indicating that tatC is essential in H. pylori, a surprising finding given the fact that only four proteins are predicted to be translocated by the Tat system in this bacterium. The levels of activity of hydrogenase and catalase, two of the predicted Tat-dependent enzymes, were affected in these mutants. In addition, all tat mutants displayed cell envelope defects, and tatC mutants were deficient in mouse colonization.
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    ABSTRACT: Edwardsiella tarda is a Gram-negative, facultative aerobic pathogen which infects multifarious hosts including fish, amphibians, and human beings. A twin-arginine translocation (Tat) gene cluster important for high-salt tolerance in E. tarda was previously identified. Here the genetic structure and pleiotropic roles of the Tat system in physiological adaptation of the bacterium were further characterized. Functional analysis indicated that tatD was not required for Tat export process and tatE might be an allelic gene of tatA in the bacterium. The results showed that disruption in Tat system did not affect morphology and biofilm formation in E. tarda, but affected motility, hemagglutination, cell aggregation and infection of eukaryotic cells (e.g., macrophage J774a). Comparative proteomics analysis of subcellular proteins using two-dimensional gel electrophoresis and a qualitative shotgun protein sequencing method were implemented to identify proteins differentially expressed in E. tarda EIB202 vs. ∆tatABCD. The results revealed a large repertoire of differentially expressed proteins (n=61), sheding light on Tat system associated with the virulence and stress-associated processes in E. tarda. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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