Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.     

Distribution of Vibrio cholerae virulence genes among different Vibrio species isolated in Sardinia, Italy

The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty-two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results.     

Proteomic analysis of pathogenic bacterium Vibrio vulnificus

In this study we have constructed a proteome reference map of the pathogenic bacterium Vibrio vulnificus. From the reference map, we identified several virulence-related proteins, such as ToxR and ToxS, as well as numerous proteins involved in diverse cellular functions. To search for additional virulence-related proteins, we compared the whole proteomes from the wild-type and toxR mutant of V. vulnificus and found that several proteins were up- or down-regulated in the toxR mutant. We suggest that these differentially regulated proteins whose expression is coordinately controlled by a virulence regulator ToxR, some of which are already implicated in virulence, play roles in the pathogenesis of V. vulnificus.     

Virulence genes in halophilic Vibrio spp. isolated in common mussels

Twenty-five Vibrio strains belonging to nine different species, isolated in common mussels, were examined for the presence of different virulence genes: ctxA, tcpA, toxR, toxS, ace, zot and vpi previously found in pathogenic Vibrio cholerae strains. Our results suggest that there is a wide dissemination of Vibrio cholerae virulence genes among the various Vibrio species tested. This finding raises the question of whether a different approach should be taken to study "environmental" Vibrio strains.     

The ToxR protein of Vibrio cholerae forms homodimers and heterodimers.

The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera. Previous experiments with ToxR-alkaline phosphatase (ToxR-PhoA) fusion proteins suggested a model for gene regulation in which the inactive form of ToxR was a monomer and the active form of ToxR was a dimer (V. L. Miller, R. K. Taylor, and J. J. Mekalanos, Cell 48:271-279, 1987). In order to examine whether ToxR exists in a dimeric form in vivo, biochemical cross-linking analyses were carried out. Different dimeric cross-linked species were detected depending on the expression level of ToxR: when overexpressed, ToxR+ToxR homodimers and ToxR+ToxS heterodimers were detected, and when ToxR was expressed at normal levels, exclusively ToxR+ToxS heterodimers were detected. The amount of overexpression was quantitated by using ToxR-PhoA fusion proteins and was found to correspond to 2.7-fold the normal level of ToxR. The formation of both homodimeric ToxR species and heterodimeric ToxR+ToxS species is consistent with previously reported genetic data that suggested that both types of ToxR oligomeric interactions occur. However, variation in the amount of either the homodimeric or heterodimeric form detectable by this cross-linking analysis was not observed to correlate with laboratory culture conditions known to modulate ToxR activity. Thus, genetic and biochemical data indicate that ToxR is able to interact with both itself and ToxS but that these interactions may not explain mechanistically the observed changes in ToxR activity that occur in response to environmental conditions.     

霍乱弧菌ToxS蛋白间的互作增强ToxR蛋白诱导毒力基因的表达

【目的】阐明霍乱弧菌ToxR蛋白功能调控的分子机制。【方法】利用巯基捕获(thiol-trapping)的方法分析DsbA蛋白对ToxR周质空间结构域半胱氨酸残基的氧化作用;采用定点突变的方法构建ToxR半胱氨酸突变株(ToxR_(C236/293S));利用荧光素酶基因作为报告基因分析ToxR野生型(ToxR_(wt))和半胱氨酸突变体(ToxR_(C236/293S))诱导下游基因表达的活性;通过细菌双杂交系统分析ToxR_(wt)和ToxR_(C236/293S)蛋白之间、ToxR与ToxS之间以及ToxS之间的相互作用。【结果】ToxR周质空间结构域半胱氨酸残基确实可以被DsbA蛋白氧化,且当ToxR与ToxS共表达时,ToxR诱导ctxAB转录表达的活性显著增强,且在dsbA基因缺失突变株中ToxR诱导ctxAB转录表达的活性更高;成功构建株霍乱弧菌ToxR半胱氨酸突变株(ToxR_(C236/293S)),在没有ToxS存在的条件下,ToxR_(C236/293S)诱导毒力基因表达的活性与ToxRwt相当;细菌双杂交系统分析发现当ToxR与ToxS共转录表达时,ToxS极大增强ToxR蛋白之间的互作;在dsbA基因缺失突变株中,ToxS之间的相互作用显著增强。【结论】ToxR蛋白本身的氧还状态对其诱导毒力基因表达的活性没有影响;ToxS通过增强ToxR形成二聚体的能力从而增强其诱导毒力基因的表达,而DsbA对ToxS蛋白之间的相互作用具有抑制作用,DsbA通过影响ToxS的蛋白互作从而影响ToxR蛋白的功能。本文为进一步阐明霍乱弧菌毒力基因表达调控的分子机制提供重要的理论依据。     

Vibrio fischeri Outer Membrane Protein OmpU Plays a Role in Normal Symbiotic Colonization

The nascent light-emitting organ of newly hatched juveniles of the Hawaiian sepiolid squid Euprymna scolopes is specifically colonized by cells of Vibrio fischeri that are obtained from the ambient seawater. The mechanisms that promote this specific, cooperative colonization are likely to require a number of bacterial and host-derived factors and activities, only some of which have been described to date. A characteristic of many host-pathogen associations is the presence of bacterial mechanisms that allow attachment to specific tissues. These mechanisms have been well characterized and often involve bacterial fimbriae or outer membrane proteins (OMPs) that act as adhesins, the expression of which has been linked to virulence regulators such as ToxR in Vibrio cholerae. Analogous or even homologous mechanisms are probably operative in the initiation and persistence of cooperative bacterial associations, although considerably less is known about them. We report the presence in V. fischeri of ompU, a gene encoding a 32.5-kDa protein homolog of two other OMPs, OmpU of V. cholerae (50.8% amino acid sequence identity) and OmpL of Photobacterium profundum (45.5% identity). A null mutation introduced into the V. fischeri ompU resulted in the loss of an OMP with an estimated molecular mass of about 34 kDa; genetic complementation of the mutant strain with a DNA fragment containing only the ompU gene restored the production of this protein. The expression of the V. fischeri OmpU was not significantly affected by either (i) iron or phosphate limitation or (ii) a mutation that renders V. fischeri defective in the synthesis of a homolog of the OMP-regulatory protein ToxR. The ompU mutant grew normally in complex nutrient media but was more susceptible to growth inhibition in the presence of either anionic detergents or the antimicrobial peptide protamine sulfate. Interestingly, colonization experiments showed that the ompU null mutant initiated a symbiotic association with juvenile light organ tissue with only about 60% of the effectiveness of the parent strain. When colonization did occur, it proceeded more slowly and resulted in an approximately fourfold-smaller bacterial population. Surprisingly, there was no evidence that in a mixed infection with its parent, the ompU-defective strain had a competitive disadvantage, suggesting that the presence of the parent strain provided a shared compensatory activity. Thus, the OmpU protein appears to play a role in the normal process by which V. fischeri initiates its colonization of the nascent light organ of juvenile squids.