Essential role of an adenylate cyclase in regulating Vibrio vulnificus virulence

Vibrio vulnificus, a halophilic estuarine bacterium, causes a fatal septicemia and necrotizing wound infection. To investigate the role of cAMP in V. vulnificus virulence regulation, an in-frame deletion mutant of the cya gene encoding adenylate cyclase was constructed. The cya null mutation resulted in a pleiotropic change of virulence phenotypes. The production of hemolysin and protease, the motility, and the cytotoxicity were decreased by the cya mutation. The defects in the cya mutant were functionally complemented in trans by a plasmid carrying the wild type cya allele. The V. vulnificus cya mutant exhibited a 100-fold increase in LD50 to mice. The result indicates that cAMP plays an essential role in the global regulation of V. vulnificus virulence.     

Regulation of fatty acid metabolism by FadR is essential for Vibrio vulnificus to cause infection of mice

The opportunistic bacterial pathogen Vibrio vulnificus causes severe wound infection and fatal septicemia. We used alkaline phosphatase insertion mutagenesis in a clinical isolate of V. vulnificus to find genes necessary for virulence, and we identified fadR, which encodes a regulator of fatty acid metabolism. The fadR::mini-Tn5Km2phoA mutant was highly attenuated in a subcutaneously inoculated iron dextran-treated mouse model of V. vulnificus disease, was hypersensitive to the fatty acid synthase inhibitor cerulenin, showed aberrant expression of fatty acid biosynthetic (fab) genes and fatty acid oxidative (fad) genes, produced smaller colonies on agar media, and grew slower in rich broth than did the wild-type parent. Deletion of fadR essentially recapitulated the phenotypes of the insertion mutant, and the DeltafadR mutation was complemented in trans with the wild-type gene. Further characterization of the DeltafadR mutant showed that it was not generally hypersensitive to envelope stresses but had decreased motility and showed an altered membrane lipid profile compared to that of the wild type. Supplementation of broth with the unsaturated fatty acid oleate restored wild-type growth in vitro, and infection with oleate in the inoculum increased the ability of the DeltafadR mutant to infect mice. We conclude that fadR and regulation of fatty acid metabolism are essential for V. vulnificus to be able to cause disease in mammalian hosts.     

Isolation and characterization of a cold-induced nonculturable suppression mutant of Vibrio vulnificus

The viable but nonculturable (VBNC) suppression mutant formed platable cells at low temperature stress after inoculation in artificial seawater (ASW). Suppression subtractive hybridization was used to identify differentially expressed genes among cDNAs of the VBNC suppression mutant and the wild-type Vibrio vulnificus strain. Glutathione S-transferase was identified as a responsive gene of the VBNC suppression mutant in our assay, and was highly expressed from the VBNC suppression mutant at low temperature stress. Culturability tests revealed that the wild-type cells were sensitive to oxidative stress in the hydrogen peroxide (H(2)O(2)) and to 1-chloro-2,4-dinitrobenzene (CDNB) compared with the VBNC suppression mutant cells. Adding glutathione showed that many wild-type V. vulnificus cells maintained culturability in cold ASW. These results suggest that non-nutritional growth inhibitors, such as peroxide that accumulates at low temperatures, influence VBNC in V. vulnificus cells.     

霍乱弧菌中调控aphB 的基因筛选及其功能

【目的】筛选霍乱弧菌C6706-中调控LysR家族蛋白AphB表达的基因。【方法】将霍乱弧菌埃尔托型菌株C6706-aphB启动子区克隆到2个报告质粒pBBRLux和pKP302上,并将其导入霍乱弧菌C6706-中,以此作为出发菌株。利用出发菌株与转座子pSC123接合构建LZV630-302转座子随机突变文库,通过测定化学发光强度检测aphB启动子的表达水平,筛选aphB表达受影响的突变株。利用随机PCR方法检测转座子插入位点,并测序比对分析基因。【结果】从7个转座子库中(共约4万个突变株)得到能影响aphB表达(均导致下降)的2株突变株T1和T2。测序比对发现T1中转座子插入在vc1585读码框内,T2中转座子插入在距vc1602基因末端7 bp处。【结论】获得aphB表达改变的突变株,基因vc1585和vc1602可能直接或间接影响aphB表达,为进一步研究aphB表达调控影响因素奠定了基础。     

Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.     

Importance of opgHXcv of Xanthomonas campestris pv. vesicatoria in host-parasite interactions

Tn5 insertion mutants of Xanthomonas campestris pv. vesicatoria were inoculated into tomato and screened for reduced virulence. One mutant exhibited reduced aggressiveness and attenuated growth in planta. Southern blot analyses indicated that the mutant carried a single Tn5 insertion not associated with previously cloned pathogenicity-related genes of X. campestris pv. vesicatoria. The wild-type phenotype of this mutant was restored by one recombinant plasmid (pOPG361) selected from a genomic library of X. campestris pv. vesicatoria 91-118. Tn3-gus insertion mutagenesis and sequence analyses of a subclone of pOPG361 identified a 1,929-bp open reading frame (ORF) essential for complementation of the mutants. The predicted protein encoded by this ORF was highly homologous to the previously reported pathogenicity-related HrpM protein of Pseudomonas syringae pv. syringae and OpgH of Erwinia chrysanthemi. Based on homology, the new locus was designated opgHXcv. Manipulation of the osmotic potential in the intercellular spaces of tomato leaves by addition of mannitol at low concentrations (25 to 50 mM) compensates for the opgHXcv mutation.     

Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.     

Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice

A mutant exhibiting decreased cytotoxic activity toward INT-407 intestinal epithelial cells and carrying a mutation in the rtx gene cluster that consists of rtxCA and rtxBDE operons was screened from a library of V. vulnificus mutants. The functions of the rtxA gene, assessed by constructing an isogenic mutant and evaluating its phenotypic changes, demonstrated that RtxA is essential for the virulence of V. vulnificus in mice as well as in tissue cultures.     

Nonribosomal peptide synthase is responsible for the biosynthesis of siderophore in Vibrio vulnificus MO6-24/O

Vibrio vulnificus produces siderophores, lowmolecular- weight iron-chelating compounds, to obtain iron under conditions of iron deprivation. To identify genes associated with the biosynthesis of siderophore in V. vulnificus MO6-24/ O, we screened clones with mini-Tn5 random insertions for those showing decreased production of siderophore. Among 6,000 clones screened, nine such clones were selected. These clones contain the transposon inserted in VV2_0830 (GenBank accession number) that is a homolog of a nonribosomal peptide synthetase (NRPS). There is an another NRPS module, VV2_0831, 49-bp upstream to VV2_0830. We named these two genes vvs (Vibrio vulnificus siderophore synthetase) A and B, respectively. Mutation of either vvsA or vvsB showed a decreased production of siderophore. The expression of an NRPS-lux fusion was negatively modulated by the presence of iron, and the regulation was dependent on Fur (ferric uptake regulator). However, the expression of the NRPS genes was still not fully derepressed in the iron-rich condition, even in fur-null mutant cells, suggesting that some other unknown factors are involved in the regulation of the genes. We also demonstrated that the NRPS genes are important for virulence of the pathogen in a mice model.     

Proteomic identification and characterization of Vibrio vulnificus proteins induced upon exposure to INT-407 intestinal epithelial cells

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.     

Construction of a Tn5-Tagged Mutant Library of Xanthomonas oryzae pv. oryzicola as An Invaluable Resource for Functional Genomics

To genome-widely mine pathogenesis-related genes of Xanthomonas oryzae pv. oryzicola (Xoc), which is the casual agent of bacterial leaf streak resulting in significant yield loss and poor quality in rice, a Tn5 transposon-mediated mutation library was generated. Twenty-five thousand transformants were produced by using Tn5 transposome, appropriately corresponding to 5 × ORF coverage of the genome, and inoculated into rice and tobacco, individually and respectively, for screening candidate virulence genes. Southern blot and thermal asymmetric interlaced polymerase chain reaction analysis of Tn5 insertion sites of randomly selected mutants suggested a random mode of transposition and a saturation library. Characterization of extracellular polysaccharides, extracellular protease activity, and pigment production of individual mutants in the growth media revealed that 11 mutants enhanced in growth, 12 reduced extracellular polysaccharide production, 12 lost extracellular protease activity completely or partially, and 21 were pigment deficient. In planta pathogenicity assays revealed 253 mutants reduced virulence in rice, but kept triggering hypersensitive response in tobacco; 49 lost the ability to elicit HR in tobacco and pathogenicity in rice; and 3 still induced hypersensitive response in tobacco, but lost pathogenicity in rice. The achieved mutant library of Xoc is of high-quality and nearly saturated and candidate virulence mutants provided a strong basis for functional genomics of Xoc.     

Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or D-galactosamine. Mice with or without iron-overloading died when the bacterial concentration in the blood reached 10(5) cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron-overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor alpha (TNF-alpha), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF-alpha, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron-overloading. These two effects manifest other virulence factors, the host's as well as bacterial. Such factors, other than TNF-alpha stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.     

Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus

During Vibrio vulnificus infection, V. vulnificus reaches the intestine and then invades the bloodstream by crossing the intestinal mucosal barrier of the host, which results in systemic septicemia. Previously, we reported that the RtxA toxin secreted through the RtxE transporter contributes to the cytotoxicity of V. vulnificus against intestinal epithelial cells. Here, we used gene mutants of rtxE and rtxA to determine the role that V. vulnificus RtxA toxin plays in the apoptotic death of human intestinal epithelial cells. The levels of DNA fragmentation were lower in human epithelial cells infected with an rtxE mutant of V. vulnificus than in those that were infected with the wild type. In addition, the rtxE mutant was found to induce lower levels of TUNEL positive cells and cell cycle arrest at the subG(1) than the wild type V. vulnificus. Furthermore, the decreased levels of DNA fragmentation, TUNEL positive cells and subG(1) arrest by the rtxE gene mutation were restored by the complementation of an rtxE gene into the rtxE mutant V. vulnificus. Finally, the rtxA mutant induced significantly lower levels of apoptotic cell death than the wild type. The levels of the PARP, cytochrome c, caspase-3, and mitochondrial membrane depolarization were lower in human epithelial cells infected with the rtxE and rtxA mutants, compared with the wild type and rtxE gene-complemented strains of V. vulnificus. Taken together, these results indicate that V. vulnificus RtxA toxin induces the apoptotic death through a mitochondria-dependent pathway in human intestinal epithelial cells exposed to V. vulnificus.     

Misexpression of acetylcholinesterases in the C. elegans pha-2 mutant accompanies ultrastructural defects in pharyngeal muscle cells

pha-2 is the Caenorhabditis elegans homolog of the vertebrate homeobox gene Hex. Embryonic expression of pha-2 is mostly pharyngeal and the only described mutant allele of pha-2 results in a severe pharyngeal defect in which certain muscle cells (pm5 cells) and neurons are grossly deformed. Here, we performed a detailed characterization of the pha-2 phenotype using cell-type-specific reporters, physical manipulation of the nuclei in pharyngeal muscle cells using "optical tweezers", electron microscopy, staining of the actin cytoskeleton as well as phenotypic rescue and ectopic expression experiments. The main findings of the present study are (i) the pha-2 (ad472) mutation specifically impairs the pharyngeal expression of pha-2; (ii) in the pha-2 mutant, the cytoskeleton of the pm5 cells is measurably weaker than in normal cells and is severely disrupted by large tubular structures and organelles; (iii) the pm5 cells of the pha-2 mutant fail to express the acetylcholinesterase genes ace-1 and ace-2; (iv) ectopic expression of pha-2 can induce ectopic expression of ace-1 and ace-2; and (v) the anc-1 mutant with mislocalized pm5 cell nuclei occasionally shows an isthmus phenotype similar to that of pha-2 worms.