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...

We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2␤ (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host "addiction" genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.Vibrio anguillarum is a marine pathogen that causes vibriosis in close to 50 species of fish, including cultured and wild fish, mollusks, and crustaceans, in marine, brackish, and fresh water (1). Vibriosis is a hemorrhagic septicemia with dire consequences for fish rearing, especially in countries that depend heavily on fish for their food consumption. Despite the fact that V. anguillarum is a dramatic cause of vibriosis in fish, little is known about the genomic composition of this important pathogen. Although 23 serotypes have been reported in V. anguillarum, the O1 and O2 serotypes are the major causative agent of fish vibriosis (32,66,75). Many O1 serotype strains harbor 65-kb pJM1-type plasmids, which carry the siderophore anguibactin biosynthesis and transport genes, a main virulence factor of V. anguillarum, while one of the O1 serotype strains and other serotypes, such as all of the O2 strains, are plasmidless (1, 13, 33, 76). Many virulence factors have been characterized, but we are still far from getting the whole picture of the virulence mechanisms of this pathogen (1, 50). The fact that the pJM1 plasmid is an important component of virulence for the 775 strain but that the other three strains examined do not harbor this plasmid and are still virulent indicates that they must have different mechanisms to cause disease. Moreover, O1 serotype strains cause disease in salmonid fish, whereas O2␤ strains are usually isolated from cod and other nonsalmonids (1, 32, 43). We have previously performed random genome sequencing of V. anguillarum 775 genomic DNA and identified potent...

Vibrio vulnificus multiplies rapidly in host tissues under iron-overloaded conditions. To understand the effects of iron in the physiology of this pathogen, we performed a genome-wide transcriptional analysis of V. vulnificus growing at three different iron concentrations, i.e., iron-limiting [Trypticase soy broth with 1.5% NaCl (TSBS) plus ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA)], low-iron (1 g Fe/ml; TSBS), and iron-rich (38 g Fe/ml; TSBS plus ferric ammonium citrate) concentrations. A few genes were upregulated under the last two conditions, while several genes were expressed differentially under only one of them. A gene upregulated under both conditions encodes the outer membrane porin, OmpH, while others are related to the biosynthesis of amino sugars. An ompH mutant showed sensitivity to sodium dodecyl sulfate (SDS) and polymyxin B and also had a reduced competitive index compared with the wild type in the iron-overloaded mice. Under iron-limiting conditions, two of the TonB systems involved in vulnibactin transport were induced. These genes were essential for virulence in the iron-overloaded mice inoculated subcutaneously, underscoring the importance of active iron transport in infection, even under the high-iron conditions of this animal model. Furthermore, we demonstrated that a RyhB homologue is also essential for virulence in the iron-overloaded mouse. This novel information on the role of genes induced under iron limitation in the iron-overloaded mouse model and the finding of new genes with putative roles in virulence that are expressed only under iron-rich conditions shed light on the many strategies used by this pathogen to multiply rapidly in the susceptible host.Vibrio vulnificus is an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels (17,43). This estuarine bacterium preferentially affects individuals with underlying hepatic diseases and other compromised conditions, such as hemochromatosis and beta thalassemia. In humans, this pathogen frequently causes fatal primary septicemia with a very rapid progress, resulting in a mortality rate of Ͼ50% within a few days (17). The common theme in susceptible individuals appears to be high serum iron levels; however, changes in the innate immune response in the host cannot be discarded (17,19).Iron is an important element in almost all known organisms. In many bacterial pathogens, the ability to acquire this metal from the host is an important virulence factor (7). Seminal work in Oliver's laboratory showed the importance of iron in V. vulnificus infections (74), as the laboratory identified two siderophores, vulnibactin and a hydroxamate-type siderophore, both involved in iron acquisition (59). 70) studied the role of the siderophore vulnibactin in the uptake of iron from transferrin and hemoglobin and identified genes involved in vulnibactin biosynthesis and transport as well as the heme receptor and Fur, a regulator that represses several genes under iron-limiting conditions (13). More recently, K...

SummaryIn Vibrio vulnificus, HlyU upregulates the expression of the large RTX toxin gene. In this work we identified the binding site of HlyU to -417 to -376 bp of the rtxA1 operon transcription start site. lacZ fusions for a series of progressive deletions from the rtxA1 operon promoter showed that transcriptional activity increased independently of HlyU when its binding site was absent. Thus HlyU must regulate the rtxA1 operon expression by antagonizing a negative regulator. Concomitantly we found that an hns mutant resulted in an increase in the expression of the rtxA1 operon genes. Multiple copies of HlyU can increase the promoter activity only in the presence of H-NS underscoring the hypothesis that HlyU must alleviate the repression by this protein. H-NS binds to a region that extends upstream and downstream of the rtxA1 operon promoter. In the upstream region it binds to five AT-rich sites of which two overlap the HlyU binding site. Competitive footprinting and gel shift data demonstrate HlyU's higher affinity as compared with H-NS resulting in the de-repression and a corresponding increased expression of the rtxA1 operon.

SummaryA chromosomal gene cluster encoding vanchrobactin biosynthesis and transport genes was identified in the Vibrio anguillarum serotype O1 strain, 775(pJM1), harbouring the anguibactin biosynthetic genes in the pJM1 plasmid. In this strain only anguibactin is produced as the vanchrobactin chromosome cluster has a RS1 transposition insertion into vabF, one of the vanchrobactin biosynthesis genes. Removal of this RS1 generating 775(pJM1)Δtnp, still resulted in the detection of only anguibactin in specific bioassays. Surprisingly, when the pJM1 plasmid was not present as in the plasmidless strain H775-3, removal of the RS1 resulted in the detection of only vanchrobactin. These results thus can be interpreted as if presence of the pJM1 plasmid or of anguibactin itself is associated with the lack of detection of the vanchrobactin siderophore in bioassays. As high-performance liquid chromatography (HPLC) and mass spectrometry analysis demonstrated that both vanchrobactin and anguibactin were indeed produced in 775(pJM1)Δtnp, it is clear that the pJM1-encoded anguibactin siderophore has higher affinity for iron than the vanchrobactin system in strains in which both systems are expressed at the same time. Our results underscore the importance of the anguibactin system in the survival of V. anguillarum 775 under conditions of iron limitation.