Role of GacA in virulence of Vibrio vulnificus

Abstract

The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P,0.0003) in a V. vulnificus CMCP6 DgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wildtype strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P50.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P,0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P,0.0005) or systemic infections (P,0.02) in subcutaneously inoculated, noniron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner. INTRODUCTIONVibrio vulnificus is a moderately halophilic, Gram-negative bacterium that inhabits coastal waters and colonizes fish and filter-feeding shellfish (DePaola et al., 1994;Motes et al., 1998;Tamplin & Capers, 1992; Wright et al., 1996). Individuals with underlying conditions such as liver disease, diabetes mellitus, cancer, haemochromatosis (iron-overload) or immune system dysfunction can incur life-threatening systemic disease from consumption of raw oysters or from wound infections (Blake et al., 1979;Jones & Oliver, 2009). The virulence of this bacterium in animal models has been attributed to multiple factors (Gulig et al., 2005;Jones & Oliver, 2009), including capsular polysaccharide (CPS) expression (Amako et al., 1984;Kreger et al., 1984;Simpson et al., 1987;Yoshida et al., 1985), iron acquisition (Litwin et al., 1996;Simpson & Oliver, 1983;Wright et al., 1986), pili (Gander & LaRocco, 1989;Paranjpye & Strom, 2005;Paranjpye et al., 2007), flagella (Kim & Rhee, 2003;Lee et al., 2004), quorum sensing (McDougald et al., 2001) and cytolytic haemolysin encoded by the rtxA1 gene (Lee et al., 2007; Kim et al., 2008;Liu et al., 2007). Other cytotoxin/haemolysin genes, including rtxA2 and rtxA3 (J. L. Joseph and P. A. Gulig, unpublished results) and vvhA (Wright & Morris, 1991), as well as a metalloprotease encoded by vvpE (Jeong et al., 2000;Shao & Hor, 2000), show no apparent role in the vi...

Transcriptome profiling analysis of Vibrio vulnificus during human infection

“…Additional factors have been proposed as potentially associated with virulence, such as phospholipase A (Koo et al, 2007), haemolysin-cytolysin (Gray & Kreger, 1985;Wright & Morris, 1991) and metalloproteases (Gulig et al, 2005). A considerable body of knowledge concerning the virulence mechanisms of V. vulnificus was obtained from mutational analysis experiments using nonhuman cell lines such as mouse intestinal epithelial cells (Jeong & Satchell, 2012), ovarian hamster cells, bovine pulmonary artery cells and peritoneal macrophages from mice (Lee et al, 2004b), or intraperitoneally injected mice (Alice et al, 2008;Gauthier et al, 2010;Kim et al, 2005Kim et al, , 2008Lee et al, 2008b;Shao & Hor, 2000). Nevertheless, it is unclear what is the exact role of the various virulent factors during human infection.…”

Characterization of thegacA-dependent surface and coral mucus colonization by an opportunistic coral pathogenSerratia marcescensPDL100

“…In addition to indirectly regulating carbon metabolism through CsrA, GacA is known to regulate swarming motility and biofilm formation (Goodier & Ahmer, ; Teplitski et al ., a; Gauthier et al ., ). Swarming assays on AB soft (0.4%) agar plates revealed that S. marcescens PDL100 swarms in a branching dendritic pattern (Fig.…”

“…When the wild‐type and gacA mutant strains were grown on low molecular weight fractions of coral mucus, the competitive fitness of the wild type increased and led to a relatively equal proportion of each strain in the culture (competitive index = 0.083). Protease activities (as measured on skim milk agar plates) were not different in the mutant and the wild type (data not shown), thus the degradation of the proteinaceous backbone of mucus is not the likely explanation of the observed phenotype, even though gacA orthologs are known to control proteases in other bacteria (Ovadis et al ., ; Cui et al ., ; Gauthier et al ., ).…”

“…Virulence is often associated with GacA in Gammaproteobacteria , although the contribution of gacA to the phenotype varies depending on the model (Lapouge et al ., ; Gauthier et al ., ). We did not observe any role for gacA in controlling virulence of S. marcescens in surrogate hosts, A. pallida , and Artemia .…”

A High Throughput Amenable Arabidopsis-P. aeruginosa System Reveals a Rewired Regulatory Module and the Utility to Identify Potent Anti-Infectives

“…This mechanism seems to circumvent the need for GacA, a master regulator that has been shown to act upstream of LasR [9]. The role of GacA in controlling a subset of virulence factors is also known in other microbial pathogens [12], [13]. Indeed, in a C. elegans model of host-microbe interaction involving P. aeruginosa PA14, the role of GacA is very prominent and the pathogenicity of a PA14Δ gacA mutants is significantly abrogated [14].…”