Evidence that fish death after Vibrio vulnificus infection is due to an acute inflammatory response triggered by a toxin of the MARTX family.

Vibrio vulnificus is an emerging zoonotic pathogen associated with fish farms that is capable of causing a hemorrhagic septicemia known as warm-water vibriosis. According to a recent transcriptomic and functional study, the death of fish due to vibriosis is more related to the inflammatory response of the host than to the tissue lesions caused by the pathogen. In this work, we hypothesize that the RtxA1 toxin (a V. vulnificus toxin of the MARTX (Multifunctional Autoprocessing Repeats in Toxin) family) is the key virulence factor that would directly or indirectly trigger this fatal inflammatory response. Our hypothesis was based on previous studies that showed that rtxA1-deficient mutants maintained their ability to colonize and invade, but were unable to kill fish. To demonstrate this hypothesis, we infected eels (model of fish vibriosis) by immersion with a mutant deficient in RtxA1 production and analyzed their transcriptome in blood, red blood cells and white blood cells during early vibriosis (0, 3 and 12 h post-infection). The transcriptomic results were compared with those obtained in the previous study in which eels were infected with the V. vulnificus parental strain, and were functionally validated. Overall, our results confirm that fish death after V. vulnificus infection is due to an acute, early and atypical inflammatory response triggered by RtxA1 in which red blood cells seem to play a central role. These results could be relevant to other vibriosis as the toxins of this family are widespread in the Vibrio genus.

TolCV1 Has Multifaceted Roles During Vibrio vulnificus Infection.

RtxA1 is a major cytotoxin of Vibrio vulnificus (V. vulnificus) causing fatal septicemia and necrotic wound infections. Our previous work has shown that RpoS regulates the expression and secretion of V. vulnificus RtxA1 toxin. This study was conducted to further investigate the potential mechanisms of RpoS on RtxA1 secretion. First, V. vulnificus TolCV1 and TolCV2 proteins, two Escherichia coli TolC homologs, were measured at various time points by Western blotting. The expression of TolCV1 was increased time-dependently, whereas that of TolCV2 was decreased. Expression of both TolCV1 and TolCV2 was significantly downregulated in an rpoS deletion mutation. Subsequently, we explored the roles of TolCV1 and TolCV2 in V. vulnificus pathogenesis. Western blot analysis showed that RtxA1 toxin was exported by TolCV1, not TolCV2, which was consistent with the cytotoxicity results. Furthermore, the expression of TolCV1 and TolCV2 was increased after treatment of the host signal bile salt and the growth of tolCV1 mutant was totally abolished in the presence of bile salt. A tolCV1 mutation resulted in significant reduction of V. vulnificus induced-virulence in mice. Taken together, TolCV1 plays key roles in RtxA1 secretion, bile salt resistance, and mice lethality of V. vulnificus, suggesting that TolCV1 could be an attractive target for the design of new medicines to treat V. vulnificus infections.

DIDS inhibits Vibrio vulnificus cytotoxicity by interfering with TolC-mediated RtxA1 toxin secretion.

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.

Vibrio vulnificus RtxA1 cytotoxin targets filamin A to regulate PAK1- and MAPK-dependent cytoskeleton reorganization and cell death.

Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491-1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491-1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.

Identification and Validation of an Antivirulence Agent Targeting HlyU-Regulated Virulence in Vibrio vulnificus.

Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality. Here, we developed an inhibitor-screening reporter platform to target HlyU, a master transcriptional regulator of virulence factors in V. vulnificus by assessing rtxA1 transcription under its control. The inhibitor-screening platform includes wild type and ΔhlyU mutant strains of V. vulnificus harboring the reporter construct P rtxA1::luxCDABE for desired luminescence signal detection and control background luminescence, respectively. Using the inhibitor-screening platform, we identified a small molecule, fursultiamine hydrochloride (FTH), that inhibits the transcription of the highly invasive repeat-in-toxin (rtxA1) and hemolysin (vvhA) along with other HlyU regulated virulence genes. FTH has no cytotoxic effects on either host cells or pathogen at the tested concentrations. FTH rescues host cells from the necrotic cell-death induced by RtxA1 and decreases the hemolytic activity under in vitro conditions. The most important point is that FTH treatment does not induce the antivirulence resistance. Current study validated the antivirulence strategy targeting the HlyU virulence transcription factor and toxin-network of V. vulnificus and demonstrated that FTH, exhibits a potential to inhibit the pathogenesis of deadly, opportunistic human pathogen, V. vulnificus without inducing AMR.

Vibrio vulnificus RtxA1 Toxin Expression Upon Contact With Host Cells Is RpoS-Dependent.

The expression of virulence genes in bacteria is known to be regulated by various environmental and host factors. Vibrio vulnificus, an estuarine bacterium, experiences a dramatic environmental change during its infection process. We reported that V. vulnificus RtxA1 toxin caused acute cell death only when close contact to host cells was allowed. A sigma factor RpoS is a very important regulator for the maximal survival of pathogens under stress conditions. Here, we studied the role of RpoS in V. vulnificus cytotoxicity and mouse lethality. The growth of rpoS mutant strain was comparable to that of wild-type in heart infusion (HI) media and DMEM with HeLa cell lysate. An rpoS mutation resulted in decreased cytotoxicity, which was restored by in trans complementation. Interestingly, host contact increased the expression and secretion of V. vulnificus RtxA1 toxin, which was decreased and delayed by the rpoS mutation. Transcription of the cytotoxic gene rtxA1 and its transporter rtxB1 was significantly increased after host factor contact, whereas the activity was decreased by the rpoS mutation. In contrast, the rpoS mutation showed no effect on the transcriptional activity of a cytolytic heamolysin gene (vvhA). Additionally, the LD50 of the rpoS mutant was 15-fold higher than that of the wild-type in specific pathogen-free CD-1 female mice. Taken together, these results show that RpoS regulates the expression of V. vulnificus RtxA1 toxin and its transporter upon host contact.

Characterization of Prohibitin 1 as a Host Partner of Vibrio vulnificus RtxA1 Toxin.

RtxA1 toxin, which results in cytoskeletal rearrangement, contact cytotoxicity, hemolysis, tissue invasion, and lethality in mice, is the most potent cytotoxic virulence factor of Vibrio vulnificus. Bioinformatics analysis of rtxA1 predicted 4 functional domains that presumably performed discrete functions during host cell killing. V. vulnificus RtxA1 has a unique domain designated as RtxA1-D2, corresponding to amino acids 1951-2574, which is absent in Vibrio cholerae multifunctional-autoprocessing repeats-in-toxin, suggesting that this domain confers specific biological functions to V. vulnificus RtxA1. HeLa cells expressing green fluorescent protein-RtxA1-D2 became round and lost their viability. A yeast 2-hybrid system identified prohibitin (PHB) 1 as the host partner of RtxA1-D2. The specific interaction of RtxA1-D2 with PHB1 was confirmed by performing immunoprecipitation. Interestingly, V. vulnificus RtxA1 up-regulated PHB1 expression on the cytoplasmic membrane of host cells. Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways were confirmed as being important in the up-regulation of PHB1 by using inhibitors. Down-regulation of PHB1 by small interfering RNAs decreased the cytotoxicity of RtxA1-D2 against HeLa cells. The pretreatment of an anti-PHB1 antibody impaired the cytotoxicity of V. vulnificus RtxA1. These results suggest that the involvement PHB1 in the RtxA1 cytotoxicity has significant implications for the pathogenesis of V. vulnificus infections.

Safety and vaccine efficacy of an attenuated Vibrio vulnificus strain with deletions in major cytotoxin genes.

Vibrio vulnificus is a human pathogen causing a rapidly progressing fatal septicemia. We have previously reported that a V. vulnificus large toxin RtxA1 causes programmed necrotic cell death through calcium-mediated mitochondrial dysfunction. Here we developed a live attenuated vaccine strain (CMM781) having deletions in three genes encoding major virulence factors: RTX cytotoxin (rtxA1), hemolysin/cytolysin (vvhA) and metalloprotease (vvpE) of a clinical isolate strain CMCP6. The CMM781 strain showed significant attenuation in cytotoxicity and mouse lethality. The safety of CMM781 was also confirmed by measuring the transepithelial electric resistance of Caco-2 cell monolayers. Intragastric immunization of mice with the live attenuated V. vulnificus strain resulted in induction of systemic and mucosal antibodies specific to the pathogen. Moreover, the vaccinated mice were protected from challenges with high doses of the virulent strain through various injection routes. These results suggest that CMM781 appears to be a safe and effective vaccine candidate that would provide significant protection against V. vulnificus infection.

Development and characterization of a catalytically inactive cysteine protease domain of RtxA1/MARTXVv as a potential vaccine for Vibrio vulnificus.

Recent studies have defined several virulence factors as vaccine candidates against Vibrio vulnificus. However, most of these factors have the potential to cause pathogenic effects in the vaccinees or induce incomplete protection. To overcome these drawbacks, a catalytically inactive form, CPDVv (C3725S), of the well-conserved cysteine protease domain (CPD) of V. vulnificus multifunctional autoprocessing repeats-in-toxin (MARTXVv /RtxA1) was recombinantly generated and characterized. Notably, active and passive immunization with CPDVv (C3725S) conferred protective immunity against V. vulnificus strains. These results may provide a novel framework for developing safe and efficient subunit vaccines and/or therapeutics against V. vulnificus that target the CPD of MARTX toxins.

Vibrio vulnificus RtxA1 modulated calcium flux contributes reduced internalization in phagocytes.

AIMS: Vibrio vulnificusis an opportunistic pathogen that causes primary septicemia and wound infection with high mortality rate. This pathogen produces an RTX toxin (RtxA1) which can cause host cell rounding, cell death and interference with internalization by host phagocytes. However, the mechanism of RtxA1-induced phagocyte paralysis is not clear. MAIN METHODS: Using the murine macrophage cell line RAW264.7, we measured cytotoxicity and phagocytosis of V. vulnificusin normal and calcium-depleted media. To deplete extracellular and cytosolic Ca(2+), cells were exposed to the calcium chelators ethylene glycol tetraacetic acid (EGTA) and 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl esteris (BAPTA-AM), respectively. The cytotoxicity was examined by measuring the activity of lactate dehydrogenase (LDH) released from the damaged cells. The gentamicin protection assay was conducted to determine the number of internalized bacteria, while acridine orange staining was applied to visualize the intracellular bacteria. The fluorescent indicator fura-2-acetoxymethyl ester (fura 2-AM) was used to measure the Ca(2+)signal post-infection. KEY FINDINGS: We revealed that extracellular Ca(2+)was essential for phagocytes to internalize V. vulnificus. Meanwhile, cytosolic Ca(2+)flux in RAW264.7 cells induced by an RtxA1 isogenic mutant was repressed by the parent strain. Furthermore, depletion of extracellular Ca(2+)level by EGTA significantly reduced the cytotoxicity but did not affect the antiphagocytic activity of RtxA1 toxin. SIGNIFICANCE: Our results indicated that RtxA1 may interfere with cytosolic Ca(2+)flux of phagocyte to promote bacteria colonization.

Genetic characterization of Vibrio vulnificus strains isolated from oyster samples in Mexico.

Vibrio vulnificus strains were isolated from oysters that were collected at the main seafood market in Mexico City. Strains were characterized with regard to vvhA, vcg genotype, PFGE, multilocus sequence typing (MLST), and rtxA1. Analyses included a comparison with rtxA1 reference sequences. Environmental (vcgE) and clinical (vcgC) genotypes were isolated at nearly equal percentages. PFGE had high heterogeneity, but the strains clustered by vcgE or vcgC genotype. Select housekeeping genes for MLST and primers that were designed for rtxA1 domains divided the strains into two clusters according to the E or C genotype. Reference rtxA1 sequences and those from this study were also clustered according to genotype. These results confirm that this genetic dimorphism is not limited to vcg genotyping, as other studies have reported. Some environmental C genotype strains had high similarity to reference strains, which have been reported to be virulent, indicating a potential risk for oyster consumers in Mexico City.

Early steps in the European eel (Anguilla anguilla)-Vibrio vulnificus interaction in the gills: role of the RtxA13 toxin.

Vibrio vulnificus is an aquatic gram-negative bacterium that causes a systemic disease in eels called warm-water vibriosis. Natural disease occurs via water born infection; bacteria attach to the gills (the main portal of entry) and spread to the internal organs through the bloodstream, provoking host death by haemorrhagic septicaemia. V. vulnificus produces a toxin called RtxA13 that hypothetically interferes with the eel immune system facilitating bacterial invasion and subsequent death by septic shock. The aim of this work was to study the early steps of warm-water vibriosis by analysing the expression of three marker mRNA transcripts related to pathogen recognition (tlr2 and tlr5) and inflammation (il-8) in the gills of eels infected by immersion with either the pathogen or a mutant deficient in rtxA13. Results indicate a differential response that is linked to the rtx toxin in the expression levels of the three measured mRNA transcripts. The results suggest that eels are able to distinguish innocuous from harmful microorganisms by the local action of their toxins rather than by surface antigens. Finally, the cells that express these transcripts in the gills are migratory cells primarily located in the second lamellae that re-locate during infection suggesting the activation of a specific immune response to pathogen invasion in the gill.

Protection against Vibrio vulnificus infection by active and passive immunization with the C-terminal region of the RtxA1/MARTXVv protein.

Vibrio vulnificus is a foodborne pathogen that is prevalent in coastal waters worldwide. Infection with V. vulnificus causes septicemia with fatality rates exceeding 50% even with aggressive antibiotic therapy. Several vaccine studies to prevent V. vulnificus infection have been performed but have had limited success. In this study, we identified the C-terminal region (amino acids 3491 to 4701) of the V. vulnificus multifunctional autoprocessing RTX (MARTXVv or RtxA1) protein, RtxA1-C, as a promising antigen that induces protective immune responses against V. vulnificus. Vaccination of mice with recombinant RtxA1-C protein with adjuvant elicited a robust antibody response and a dramatic reduction in blood bacterial load in mice infected intraperitoneally. Vaccination resulted in significant protection against lethal challenge with V. vulnificus. Furthermore, intraperitoneal passive immunization with serum raised against the recombinant RtxA1-C protein demonstrated marked efficacy in both prophylaxis and therapy. These results suggest that active and passive immunization against the C-terminal region of the RtxA1 protein may be an effective approach in the prevention and therapy of V. vulnificus infections.