The HlyU Protein Is a Positive Regulator of rtxA1 , a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen Vibrio vulnificus

Abstract

Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised ironoverloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.Vibrio vulnificus is an opportunistic human pathogen that preferentially affects patients that have underlying hepatic diseases and other compromised conditions, such as hemochromatosis and beta-thalassemia, and heavy alcohol drinkers (6,19,32). This bacterium frequently causes fatal primary septicemia with very rapid progress, resulting in a mortality rate of more than 50% within a few days (6,12,20,33). The common theme in most of these patients is that iron is present at higher than physiological levels. Some of the confirmed or putative virulence factors required for in vivo survival and growth of V. vulnificus include capsule (30, 38), protease (8), flagella (9, 24), pili (22), and siderophore vulnibactin (14). More recently it was reported that antibodies against the V. vulnificus HlyU protein were present in serum of convalescent patients who survived V. vulnificus septicemia and that an hlyU mutation resulted in a 53-fold increase of the 50% lethal dose (LD 50 ) of V. vulnificus in the iron-normal mouse model (7). It was already known that the Vibrio cholerae HlyU predicted protein, containing a putative helix-turn-helix motif (34), activated the expression of the hemolysin gene hlyA as well as a hemolysincoregulated protein gene hcp (35,36). The HlyU homologue in V. vulnificus was reported to up-regulate the cytolysin/hemolysin gene vvhA and a gene encoding an elastolytic protease (7), while the hlyU mutant showed a significant decrease in cytotoxic activity to HeLa cells. It is worth mentioning that the purified V. vulnificus cytolysin (VvhA) exhibited cytolytic activity against Chinese hamster ovary cells (5) and could kill mice at low dosages by intravenous administration (10). However, when the vvhA gene was mutated, the VvhA cytolysinnegative strain still showed the same LD 50 in both iron-normal and iron-overloaded mouse models (37). Furthermore, the cytotoxicity for Hep-2 cells in the cytolysin-negative strain was comparable to that of the wild type (4). There...

A Common Virulence Plasmid in Biotype 2 Vibrio vulnificus and Its Dissemination Aided by a Conjugal Plasmid

“…An rtx gene cluster has also been found in the small chromosome of BT1 V. vulnificus (12) and has been shown to be associated with virulence in the mouse (29,31). The rtxB, rtxC, rtxD, and rtxE genes and the promoter region of this gene cluster in pC4602-2 or pR99 were highly homologous to those of V. cholerae and BT1 V. vulnificus (80% and 94% identical, respectively).…”

Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection

“…In this study, using a quantitative infection strategy, we show that MARTX Vv toxin is a significant virulence factor by the intragastric route of infection. Previous studies had linked the rtxA1 gene to wound-induced infection (7,8). We sought to understand better the distribution of this important factor across V. vulnificus strains by undertaking a gene-sequencing approach.…”

“…In fact, V. vulnificus accounts for 1% of all food-related deaths in the United States (4). Numerous studies recently have described a V. vulnificus multifunctional-autoprocessing RTX toxin (MARTX Vv ) as having a major impact on virulence in mice (5)(6)(7)(8). Toxins of the MARTX family are very large composite toxins.…”

Regulation of Fatty Acid Metabolism by FadR Is Essential for Vibrio vulnificus To Cause Infection of Mice

“…The known virulence factors include the antiphagocytic capsule (43, 50, 51); iron sequestration via siderophores (28); flagella and motility (24,26); type IV pili (36); the fibronectin-binding outer membrane protein OmpU (15); the HlyU protein, which upregulates expression of some toxin genes (22); and the RtxA1 toxin (23,25,29). Although some of the virulence factors discovered to date aid in growth and tissue damage, the levels of attenuation seen in these mutants vary, with some showing only marginal decreases in virulence.…”