A ToxR Homolog from Vibrio anguillarum Serotype O1 Regulates Its Own Production, Bile Resistance, and Biofilm Formation

Abstract

ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.Vibrio anguillarum is a highly pathogenic bacterium that causes terminal hemorrhagic septicemia in marine fish, resulting in great economic losses within aquaculture (1, 3). The disease vibriosis is associated with high rates of mortality and shares many features with invasive septicemic diseases in humans (1). The infection model of V. anguillarum in salmonid fish has been suggested to be a useful model for studying host-pathogen interactions.Although the exact mode of infection for V. anguillarum is still unclear, it most likely involves attachment to and colonization of host surfaces, followed by penetration of the tissues. Chemotactic motility has been suggested to aid the entry of V. anguillarum into the fish host (47). Furthermore, adhesion and invasion studies using a Chinook salmon embryo cell line and a set of isogenic motility mutants showed that invasion of but not adhesion to the cell line was significantly decreased in nonmotile or partially motile mutants (45). A smooth-swimming, chemotactic mutant, however, was hyperinvasive.V. anguillarum utilizes fish skin and intestinal mucus as chemoattractants (48). This mucus should induce smooth swimming, which may aid the entry of V. anguillarum into the fish through the mucus layers. These studies strongly suggest that active motility is required for invasion of the fish host. Once the bacterium has invaded the fish, motility is no longer needed for the progression of vibriosis (37,45,47).Several factors have been suggested to be important in the...

Role for the major outer-membrane protein from Vibrio anguillarum in bile resistance and biofilm formation

“…Furthermore, the major OMP from serotype O1 exhibits weak cation selectivity and possesses a moderate surface charge (Simó n et al, 1996). The N-terminal amino acid sequence suggests that these two major OMPs are possible homologues of OmpU from V. cholerae (Okuda et al, 2001;Wang et al, 2002).…”

“…Previously, the amino-terminal sequence of the major OMP from V. anguillarum serotype O1 was sequenced and shown to be 75 % identical to that of OmpU from V. cholerae (Wang et al, 2002). Using the amino-terminal sequence of V. anguillarum OmpU and an internal amino acid sequence from V. cholerae OmpU, two degenerate inosine-containing oligonucleotides were designed and used to PCR amplify a 560 bp DNA fragment from the V. anguillarum ompU gene.…”

“…PCR was performed as described previously (Wang et al, 2002). Unless otherwise stated, all conditions for the various DNA techniques were as described by Sambrook et al (1989).…”

“…Using the amino-terminal sequence of V. anguillarum OmpU and an internal amino acid sequence from V. cholerae OmpU, two degenerate inosine-containing oligonucleotides were designed and used to PCR amplify a 560 bp DNA fragment from the V. anguillarum ompU gene. Primer OmpU-A [59-GGTGAGCTCTAIAAICAIGA(TC)GG(ACTG)AC-39] is complementary to deduced codons for the amino-terminal sequence YNQDGT of OmpU from V. anguillarum (Wang et al, 2002) and OmpU-C [59-GGCACTAGTTCITTITGITC(AG)TC(TC)TG(AG)TC-39] is complementary to V. cholerae ompU codons for amino acids DQDDQNE (Sperandino et al, 1996). The 560 bp DNA fragment was cloned into pBluescript (Stratagene), creating pBSOmpU-AC, and sequenced.…”

“…A transcriptional gene fusion was constructed between the promoter region of ompU and the reporter gene lacZ from E. coli. This fusion was created using previously described methods (Wang et al, 2002). pDM8-OmpU1 is described in Table 1.…”