RESUMO
Vibrio vulnificus is a causative agent of fatal septicemia and necrotic wound infection and the pathogen infection became an important public health problem in many counties. Vibrio vulnificus causes RtxA1 toxin-induced acute cell death. We tried to identify natural products that inhibit the acute cytotoxicity of V. vulnificus using a lactate hydrogenase assay. A polyphenol pyrogallol protected HeLa cells from V. vulnificus-induced cytotoxicity. Pyrogallol also decreased the growth of V. vulnificus; this inhibitory effect was more significant during log phase than stationary phase. To further elucidate the inhibitory mechanism, pyrogallol-induced toxicity was compared between a V. vulnificus catalase-peroxidase mutant (katG-) and the isogenic wild-type MO6-24/O strains. No growth was observed for the katG- mutant in the presence of pyrogallol (50 µg/mL) even after 24 h, whereas the wild-type strain demonstrated growth recovery following a prolonged lag phase. Pyrogallol-mediated growth inhibition of the katG- mutant strain was partially rescued by exogenous catalase treatment. These results indicate that the mechanism by which pyrogallol inhibits the growth and cytotoxicity of V. vulnificus likely involves polyphenol-induced prooxidant damage. Taken together, these results suggest that pyrogallol has potential for development as a new paradigm drug to treat infectious diseases.
Assuntos
Antibacterianos/farmacologia , Catalase/genética , Pirogalol/farmacologia , Vibrioses/tratamento farmacológico , Vibrioses/enzimologia , Vibrio vulnificus/efeitos dos fármacos , Antioxidantes/farmacologia , Toxinas Bacterianas/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Vibrioses/genética , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
TonB systems actively transport iron-bound substrates across the outer membranes of Gram-negative bacteria. Vibrio vulnificus CMCP6, which causes fatal septicemia and necrotizing wound infections, possesses three active TonB systems. It is not known why V. vulnificus CMCP6 has maintained three TonB systems throughout its evolution. The TonB1 and TonB2 systems are relatively well characterized, while the pathophysiological function of the TonB3 system is still elusive. A reverse transcription-PCR (RT-PCR) study showed that the tonB1 and tonB2 genes are preferentially induced in vivo, whereas tonB3 is persistently transcribed, albeit at low expression levels, under both in vitro and in vivo conditions. The goal of the present study was to elucidate the raison d'être of these three TonB systems. In contrast to previous studies, we constructed in-frame single-, double-, and triple-deletion mutants of the entire structural genes in TonB loci, and the changes in various virulence-related phenotypes were evaluated. Surprisingly, only the tonB123 mutant exhibited a significant delay in killing eukaryotic cells, which was complemented in trans with any TonB operon. Very interestingly, we discovered that flagellum biogenesis was defective in the tonB123 mutant. The loss of flagellation contributed to severe defects in motility and adhesion of the mutant. Because of the difficulty of making contact with host cells, the mutant manifested defective RtxA1 toxin production, which resulted in impaired invasiveness, delayed cytotoxicity, and decreased lethality for mice. Taken together, these results indicate that a series of virulence defects in all three TonB systems of V. vulnificus CMCP6 coordinately complement each other for iron assimilation and full virulence expression by ensuring flagellar biogenesis.
Assuntos
Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Flagelos/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/genética , Vibrio vulnificus/patogenicidade , Animais , Proteínas de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Transporte Biológico/genética , Linhagem Celular Tumoral , Feminino , Flagelos/genética , Células HeLa , Humanos , Ferro/metabolismo , Proteínas de Membrana/biossíntese , Camundongos , Cavidade Peritoneal/microbiologia , Ratos , Ratos Sprague-Dawley , Vibrioses/microbiologia , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
An exoprotease of Vibrio vulnificus, VvpS, exhibits an autolytic function during the stationary phase. To understand how vvpS expression is controlled, the regulators involved in vvpS transcription and their regulatory mechanisms were investigated. LeuO was isolated in a ligand-fishing experiment, and experiments using a leuO-deletion mutant revealed that LeuO represses vvpS transcription. LeuO bound the extended region including LeuO-binding site (LBS)-I and LBS-II. Further screening of additional regulators revealed that SmcR and cyclic adenosine monophosphate-receptor protein (CRP) play activating roles in vvpS transcription. SmcR and CRP bound the regions overlapping LBS-I and -II, respectively. In addition, the LeuO occupancy of LBS-I and LBS-II was competitively exchanged by SmcR and CRP, respectively. To examine the mechanism of stationary-phase induction of vvpS expression, in vivo levels of three transcription factors were monitored. Cellular level of LeuO was maximal at exponential phase, while those of SmcR and CRP were maximal at stationary phase and relatively constant after the early-exponential phase, respectively. Thus, vvpS transcription was not induced during the exponential phase by high cellular content of LeuO. When entering the stationary phase, however, LeuO content was significantly reduced and repression by LeuO was relieved through simultaneous binding of SmcR and CRP to LBS-I and -II, respectively.
Assuntos
Exopeptidases/biossíntese , Fatores de Transcrição/metabolismo , Vibrio vulnificus/metabolismo , Proteínas de Bactérias/metabolismo , Indução Enzimática , Exopeptidases/genética , Exopeptidases/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Ligação Proteica , Serina Proteases/biossíntese , Serina Proteases/genética , Serina Proteases/metabolismo , Vibrio vulnificus/enzimologia , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O2-dependent HemF and O2-independent HemN. The growth of the hemF mutant HF1 was similar to wild-type cells at pH 7.5 under 2% O2 conditions where HemN was active and had a half-life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S-adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM-limiting conditions, a mutation arose in hemN, encoding HemNY74F . Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemNY74F for the cofactor SAM1, which coordinates the 4Fe-4S cluster, was approximately sixfold higher than that of HemN. The Km of HemNY74F for the co-substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.
Assuntos
Coenzimas/metabolismo , Coproporfirinogênio Oxidase/metabolismo , Heme/biossíntese , S-Adenosilmetionina/metabolismo , Vibrio vulnificus/enzimologia , Vibrio vulnificus/metabolismo , Coproporfirinogênio Oxidase/genética , Meios de Cultura/química , Deleção de Genes , Concentração de Íons de Hidrogênio , Cinética , Mutação de Sentido Incorreto , Oxigênio/metabolismo , Ligação Proteica , Supressão Genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
This study was undertaken to determine whether cyclic AMP (cAMP) or cAMP-receptor protein (CRP) modulates the activity of the autoinducer (AI)-2-mediated quorum sensing (QS) system in response to glucose availability in Vibrio vulnificus. A mutation in crp impaired V. vulnificus growth, decreased AI-2 production, and repressed the expression of smcR encoding the master regulator SmcR (a Vibrio harveyi LuxR homolog) of the AI-2-QS system, and these changes were prevented by in trans complementation of wild-type crp. Furthermore, glucose repressed smcR expression in the presence of CRP but not in its absence. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP synthesis, also impaired V. vulnificus growth and repressed smcR expression, and these changes were recovered by in trans complementation of wild-type cyaA. These results indicate that cAMP or CRP modulates the AI-2-QS system in response to glucose availability in V. vulnificus, demonstrating the presence of a connection between catabolite repression and quorum sensing in V. vulnificus.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Homosserina/análogos & derivados , Lactonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Vibrio vulnificus/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/genética , Glucose/metabolismo , Homosserina/metabolismo , Homosserina/farmacologia , Humanos , Lactonas/metabolismo , Mutação , Transativadores/genética , Transativadores/metabolismo , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismoRESUMO
Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., has many beneficial biological activities. However, there are relatively few reports of the effects of curcumin on pathogen infections. This study examined the effect of curcumin on a Vibrio vulnificus infection. The cytotoxicity of V. vulnificus to HeLa cells was significantly inhibited by curcumin (at 10 or 30 µM). To further examine the inhibitory mechanism of curcumin against V. vulnificus-mediated cytotoxicity, the level of bacterial growth, bacterial motility, cell adhesion, RTX toxin expression and host cell reactions were evaluated. Curcumin inhibited V. vulnificus growth in HI broth. Curcumin inhibited both bacterial adhesion and RTX toxin binding to the host cells, which can be considered the major protective mechanisms for the decrease in V. vulnificus cytotoxicity. Curcumin also inhibited host cell rounding and actin aggregation, which are the early features of cell death caused by V. vulnificus. In addition, curcumin decreased the V. vulnificus-induced NF-κB translocation in HeLa cells. Finally, curcumin protected mice from V. vulnificus-induced septicemia. In conclusion, curcumin may be an alternative antimicrobial agent against fatal bacterial infections.
Assuntos
Antibacterianos/farmacologia , Curcumina/farmacologia , Vibrioses/prevenção & controle , Vibrio vulnificus/efeitos dos fármacos , Animais , Toxinas Bacterianas/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Células HeLa , Humanos , Locomoção/efeitos dos fármacos , Camundongos , NF-kappa B/biossíntese , Vibrio vulnificus/crescimento & desenvolvimento , Vibrio vulnificus/patogenicidadeRESUMO
Pathogens have evolved sophisticated mechanisms to survive oxidative stresses imposed by host defense systems, and the mechanisms are closely linked to their virulence. In the present study, ahpCl, a homologue of Escherichia coli ahpC encoding a peroxiredoxin, was identified among the Vibrio vulnificus genes specifically induced by exposure to H(2)O(2). In order to analyze the role of AhpCl in the pathogenesis of V. vulnificus, a mutant, in which the ahpCl gene was disrupted, was constructed by allelic exchanges. The ahpCl mutant was hypersusceptable to killing by reactive oxygen species (ROS) such as H(2)O(2) and t-BOOH, which is one of the most commonly used hydroperoxides in vitro. The purified AhpCl reduced H(2)O(2) in the presence of AhpF and NADH as a hydrogen donor, indicating that V. vulnificus AhpCl is a NADH-dependent peroxiredoxin and constitutes a peroxide reductase system with AhpF. Compared to wild type, the ahpCl mutant exhibited less cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. In addition, the ahpCl mutant was significantly diminished in growth with INT-407 epithelial cells, reflecting that the ability of the mutant to grow, survive, and persist during infection is also impaired. Consequently, the combined results suggest that AhpCl and the capability of resistance to oxidative stresses contribute to the virulence of V. vulnificus by assuring growth and survival during infection.
Assuntos
Proteínas de Bactérias/fisiologia , Viabilidade Microbiana , Estresse Oxidativo , Peroxirredoxinas/fisiologia , Estresse Fisiológico , Vibrio vulnificus/fisiologia , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Contagem de Colônia Microbiana , Células Epiteliais/microbiologia , Técnicas de Inativação de Genes , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , NAD/metabolismo , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/isolamento & purificação , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Alinhamento de Sequência , Vibrioses/microbiologia , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimento , Vibrio vulnificus/patogenicidade , VirulênciaRESUMO
Proteomic analysis led to identification of the proteins of Vibrio vulnificus that were induced upon exposure to INT-407 cells, and 7 of which belong to the functional categories such as amino acid transport/metabolism, nucleotide transport/metabolism, posttranslational modification/protein turnover/chaperones, and translation. Among the genes encoding the host-induced proteins, disruption of purH, trpD, tsaA, and groEL2 resulted in reduced cytotoxicity. The purH, trpD, and tsaA mutants showed impaired growth in the INT-407 lysate; however, the growth rate of the groEL2 mutant was not significantly changed, indicating that the possible roles of the host-induced proteins in the virulence of V. vulnificus are rather versatile.
Assuntos
Proteínas de Bactérias/química , Células Epiteliais/microbiologia , Intestinos/microbiologia , Proteômica , Vibrioses/microbiologia , Vibrio vulnificus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Linhagem Celular , Eletroforese em Gel Bidimensional , Interações Hospedeiro-Patógeno , Humanos , Mutação , Vibrio vulnificus/química , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
We recently reported a simple PCR procedure that targets a sequence variation of the virulence-correlated gene locus vcg. It was found that 90% of all clinical isolates possessed the vcgC sequence variant, while 93% of all environmental isolates possessed the vcgE sequence variant. Here we report that the clinical genotype of Vibrio vulnificus is significantly better able to survive in human serum than is the environmental genotype. The presence of a siderophore-encoding gene, viuB, influenced serum survivability among all isolates of V. vulnificus tested. Those strains positive for viuB (all C-type strains but very few E-type strains) showed greater serum survivability than those lacking viuB (most E-type strains). The addition of iron (in the form of ferric ammonium citrate) to human serum restored the survival of E-type strains lacking viuB to levels not significantly different from those of C-type and E-type strains that possess viuB. These findings suggest that viuB may dictate serum survival in both C- and E-type strains of V. vulnificus and may explain why some strains (C- and E-type strains) are pathogenic and others (predominately E-type strains) are not. Additionally, C-type strains exhibited a cross-protective response against human serum, not exhibited by E-type strains, after incubation under nutrient and osmotic downshift conditions that mimicked estuarine waters. This suggests that the nutrient/osmotic environment may influence the survival of V. vulnificus following entry into the human body, leading to selection of the C genotype over the E genotype.
Assuntos
Ferro/farmacologia , Soro/química , Vibrio vulnificus/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Compostos Férricos/farmacologia , Genótipo , Humanos , Compostos de Amônio Quaternário/farmacologia , Vibrioses/microbiologia , Vibrio vulnificus/genética , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
Vibrio vulnificus is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. Generally, clinical isolates have a high lethal effect compared with environmental isolates. However, little is known about the mechanisms by which V. vulnificus causes disease. In this study, we compared the pathogenicity of a clinical isolate, strain M2799, with that of an environmental isolate, strain JCM3731. The clinical isolate showed 100 times higher lethality in mice than the environmental isolate. In strain M2799-inoculated mice, the number of macrophages decreased significantly, whereas there was no appreciable change in the number of macrophages in strain JCM3731-inoculated mice. The clinical isolate showed high cytotoxic activity, especially to macrophages, compared with the environmental isolate in vitro. The growth of the clinical isolate was almost completely inhibited in the presence of macrophages. Moreover, the survival rate of the clinical isolate-inoculated mice increased by recruitment of macrophages. These results indicate that V. vulnificus infection progresses by damage to macrophages during the early phase of infection.
Assuntos
Macrófagos/microbiologia , Macrófagos/patologia , Vibrioses/microbiologia , Vibrioses/patologia , Vibrio vulnificus/patogenicidade , Animais , Modelos Animais de Doenças , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vibrioses/imunologia , Vibrioses/mortalidade , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
We determined the ferrophilic characteristics of Vibrio vulnificus to evaluate the potential usefulness of iron chelation therapy for the prevention of V. vulnificus infection. Readily available non-transferrin-bound iron (NTBI) is required for the initiation of V. vulnificus growth under in vitro iron-limited conditions and human ex vivo conditions. NTBI aided efficient transferrin-bound iron (TBI) use by V. vulnificus, and the vulnibactin-mediated iron-uptake system was expressed after bacterial growth had been started by NTBI. V. vulnificus required higher NTBI levels for the initiation of growth, produced siderophores at lower levels, and used TBI less efficiently than other bacteria. In addition, the growth of V. vulnificus was inhibited by deferiprone, a clinically available iron chelator. These results show that V. vulnificus is a ferrophilic bacterium that requires higher NTBI levels than other pathogens and that iron chelation therapy might be an effective means of preventing the in vivo growth of V. vulnificus in susceptible patients.
Assuntos
Compostos Férricos/metabolismo , Ferro/metabolismo , Transferrina/metabolismo , Vibrio vulnificus/metabolismo , Proteínas de Bactérias , Quelantes de Ferro/uso terapêutico , Proteínas Repressoras , Vibrio vulnificus/enzimologia , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
Cyclic AMP-cAMP receptor protein (CRP) complex plays an essential role in the global regulation of Vibrio vulnificus virulence. We found that growth retardation of V. vulnificus caused by mutation of the crp gene encoding CRP was exacerbated under iron-limited conditions. Accordingly, we investigated the effect of crp mutation on the expression of the vulnibactin-mediated iron-uptake system and the ability of V. vulnificus to utilize transferrin-bound iron, and thus to grow in cirrhotic ascites, a human ex vivo system. The production of vulnibactin was suppressed, and the transcription of the vis and vuuA genes, which encode an enzyme required for vulnibactin synthesis and vulnibactin receptor protein, was also suppressed in the crp mutant. Moreover, the crp mutant could not utilize transferrin-bound iron, and its growth was severely suppressed both on transferrin-bound iron and in cirrhotic ascites. All the defects in the crp mutant were recovered by the in trans complementation of the wild-type crp gene. Putative CRP-binding sequences were found in the regulatory regions of the fur, vis and vuuA genes. These results indicate that crp mutation attenuates the ability to grow on transferrin-bound iron and in a human body fluid by down-regulating the vulnibactin-mediated iron-uptake system.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Ferro/metabolismo , Transferrina/metabolismo , Vibrio vulnificus/metabolismo , Amidas/metabolismo , Líquido Ascítico , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Meios de Cultura , Regulação para Baixo , Humanos , Dados de Sequência Molecular , Mutação , Oxazóis/metabolismo , Receptores de AMP Cíclico/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sideróforos/metabolismo , Vibrio vulnificus/crescimento & desenvolvimentoRESUMO
In order to elucidate the role of Vibrio vulnificus metalloprotease VvpE in the uptake of iron from human transferrin, we constructed a VvpE-deficient mutant and a merozygotic vvpE-transcriptional reporter from the wild type strain MO6-24/O. All three strains were able to grow only in deferrated Heart Infusion broth (DF-HI) with human holotransferrin (HT), but not in DF-HI containing partially iron-saturated transferrin or apotransferrin, without noticeable differences among the strains. All strains consumed most iron in the early growth phase. Both the transcription and extracellular production of VvpE proceeded at undetectable levels when bacterial growth was severely retarded in the DF-HI. When HT or FeCl(3) was added to the DF-HI, the retarded bacterial growth was restored and vvpE transcription dramatically increased in the late growth phase, but the extracellular VvpE production was negligible as compared to its transcription. All strains were unable to degrade HT even in normal HI broth containing HT, in which extracellular VvpE activity was remarkably high. The uptake of iron from HT in all strains was consistent with the production of catechol-siderophore rather than hydroxamate-siderophore. Similar results were also observed when clinical isolates from septicemic patients were used. In conclusion, we determined that VvpE was not directly involved in the siderophore-mediated iron-uptake from human transferrin. In addition, the discrepancy between the transcription and extracellular production of VvpE suggests that additional posttranscriptional events are involved in the extracellular production of VvpE.
Assuntos
Ferro/metabolismo , Metaloproteases/fisiologia , Transferrina/metabolismo , Vibrio vulnificus/enzimologia , Meios de Cultura/química , Expressão Gênica , Fusão Gênica , Genes Reporter , Humanos , Metaloproteases/biossíntese , Metaloproteases/genética , Mutação , Sideróforos/análise , Transcrição Gênica , Vibrio vulnificus/crescimento & desenvolvimento , beta-Galactosidase/análiseRESUMO
Vibrio vulnificus expresses a multitude of cell-associated and secreted factors that potentially contribute to pathogenicity, although the specific roles of most of these factors have been difficult to define. Previously we have shown that a mutation in pilD (originally designated vvpD), which encodes a type IV prepilin peptidase/N-methyltransferase, abolishes expression of surface pili, suggesting that they belong to the type IV class. In addition, a pilD mutant exhibits reduced adherence to HEp-2 cells, a block in secretion of several exoenzymes that follow the type II secretion pathway, and decreased virulence. In this study, we have cloned and characterized a V. vulnificus type IV pilin (PilA) that shares extensive homology to group A type IV pilins expressed by many pathogens, including Vibrio cholerae (PilA), Pseudomonas aeruginosa (PilA), and Aeromonas hydrophila (TapA). The V. vulnificus pilA gene is part of an operon and is clustered with three other pilus biogenesis genes, pilBCD. Inactivation of pilA reduces the ability of V. vulnificus to form biofilms and significantly decreases adherence to HEp-2 cells and virulence in iron dextran-treated mice. Southern blot analysis demonstrates the widespread presence of both pilA and pilD in clinical as well as environmental strains of V. vulnificus.