Cloning and sequencing of a novel hemolysis gene of Vibrio cholerae

Nagamune, Kazuya

doi:10.1016/0378-1097(95)00104-d

Cited by 15 publications

(24 citation statements)

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“…Within the CTX prophage, ctxA and ctxB (VC1456 and VC1457) were also upregulated in all flagellar mutants. Among the additional putative virulence genes that were upregulated in the flagellar mutants were vieA and vieB (VC1651 and VC1652), response regulators involved in modulation of cyclic di-GMP (c-di-GMP) levels and regulation of CT expression (64,65); epsL (VC2725), required for CT secretion (1); hlyA and hlyB (VCA0219 and VCA0220), encoding the hemolysins that cause vacuolation and pore formation in many cell types (2); a gene encoding a thermolabile hemolysin (tlh; VCA0218) that has phospholipase activity (14); an additional hemolysin gene (hlx; VCA0594) (45); the gene encoding the chitin-binding protein GbpA (VCA0811), which mediates binding to epithelial cells as well as chitinous surfaces (28); a putative lipase gene (VC1418); and three genes within the type VI secretion system (T6SS) (VCA0107, VCA0108, and VCA0124) involved in translocating proteins associated with virulence across the gram-negative membranes (52).…”

Section: Resultsmentioning

confidence: 99%

“…VCA0219 encodes the "El Tor" hemolysin HlyA, and the O395 strain used in these studies contains a deletion in hlyA that renders this strain HlyA Ϫ (19); thus, HlyA cannot be the flagellum-regulated hemolysin. VCA0594 encodes a hemolysin, Hlx, which was determined to lyse sheep but not human erythrocytes (45), suggesting that this was also not the flagellum-regulated hemolysin. VCA0218 encodes a thermolabile hemolysin, Tlh, which has phospholipase and lecithinase activity (14).…”

Section: Resultsmentioning

confidence: 99%

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The Vibrio cholerae Flagellar Regulatory Hierarchy Controls Expression of Virulence Factors

et al. 2009

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Vibrio cholerae is a motile bacterium responsible for the disease cholera, and motility has been hypothesized to be inversely regulated with virulence. We examined the transcription profiles of V. cholerae strains containing mutations in flagellar regulatory genes (rpoN, flrA, flrC, and fliA) by utilizing whole-genome microarrays. Results revealed that flagellar transcription is organized into a four-tiered hierarchy. Additionally, genes with proven or putative roles in virulence (e.g., ctx, tcp, hemolysin, and type VI secretion genes) were upregulated in flagellar regulatory mutants, which was confirmed by quantitative reverse transcription-PCR. Flagellar regulatory mutants exhibit increased hemolysis of human erythrocytes, which was due to increased transcription of the thermolabile hemolysin (tlh). The flagellar regulatory system positively regulates transcription of a diguanylate cyclase, CdgD, which in turn regulates transcription of a novel hemagglutinin (frhA) that mediates adherence to chitin and epithelial cells and enhances biofilm formation and intestinal colonization in infant mice. Our results demonstrate that the flagellar regulatory system modulates the expression of nonflagellar genes, with induction of an adhesin that facilitates colonization within the intestine and repression of virulence factors maximally induced following colonization. These results suggest that the flagellar regulatory hierarchy facilitates correct spatiotemporal expression patterns for optimal V. cholerae colonization and disease progression.Vibrio cholerae causes the human diarrheal disease cholera. The bacteria are natural inhabitants of aquatic environments and are introduced into the human population through the ingestion of contaminated food or water. Within the human host, V. cholerae expresses virulence factors that facilitate colonization of the intestine (e.g., toxin-coregulated pilus [TCP]) and that stimulate dramatic fluid loss from host tissues (cholera toxin [CT]) (5, 61). A regulatory cascade consisting of a number of different proteins, including ToxR, TcpP, and ToxT, induces the coordinated expression of CT and TCP maximally within the intestine and under specific in vitro growth conditions (for a review, see reference 7).V. cholerae is a highly motile organism by virtue of its single polar flagellum. Flagellar genes are transcribed in a four-tiered transcriptional hierarchy (51). The single class I gene product FlrA activates 54 -dependent transcription of class II genes, which encode components of the MS ring-switch-export apparatus as well as the two-component system FlrBC (31). Phosphorylated FlrC activates 54 -dependent transcription of class III genes, which encode the basal body-hook and the flagellin FlaA (10, 11). Finally, the antisigma factor FlgM is secreted through the basal body-hook to allow 28 -dependent transcription of class IV genes, which encode four additional flagellins and some of the motor components (9, 30). Motility has been linked to the virulence of V. cholerae. Spontaneous nonmot...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

The Vibrio cholerae Flagellar Regulatory Hierarchy Controls Expression of Virulence Factors

et al. 2009

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“…Expression of hemolysis by clinical strains of both O139 and non-O1, non-O139 was six times higher when compared to environmental strains with a statistical signi¢cant P-value of 0.02 (Table 3). Hemolysin of V. cholerae is suggested to be a virulence factor contributing towards cholera pathogenesis [20]. Recently, we puri¢ed a bifunctional hemolysin-phospholipase C molecule from V. cholerae O139 showing enterotoxic activity, as shown by £uid accumulation in the ligated rabbit ileal loop and in the intestines of suckling mice [21].…”

Section: Resultsmentioning

confidence: 99%

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of Vibrio cholerae O139 and non-O1, non-O139

Guhathakurta

1999

FEMS Microbiology Letters

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The presence of three major virulence genes toxR, tcpA and ctxA as well as expression of several putative virulence factors were compared in 12 Vibrio cholerae O139 and non-O1,non-O139 strains of clinical and environmental origin. All the strains possessed the gene encoding the regulatory protein TOXR. None of the non-O1, non-O139 strains as well as one of the O139 environmental strains carried the genes for ctxA and tcpA. Statistically significant differences in hemagglutinin and hemolysin production were observed amongst the strains depending on the source of their isolation. Expression of extracellular enzymes such as protease, elastase, neuraminidase, phospholipase A and phospholipase C, however, did not vary significantly from the groups of strains isolated from different sources. ß

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“…Each amplified fragment was cloned into a pT7-Blue vector and then digested with BamHI and PstI. These fragments were cloned into a R6K-ori suicide vector, pKY719 (33), digested with BamHI and PstI. These plasmids were introduced into E. coli SM10pir and then mated with V. parahaemolyticus TH3996.…”

Section: Methodsmentioning

confidence: 99%

Genetic Characterization of DNA Region Containing the trh and ure Genes of Vibrio parahaemolyticus

et al. 2000

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We have demonstrated that possession of the gene for thermostable direct hemolysin-related hemolysin (trh) coincides with the presence of the urease gene among clinical Vibrio parahaemolyticus strains and that the location of the two genes are in close proximity on the chromosome. Here, we cloned and sequenced the 15,754-bp DNA region containing the trh gene and the gene cluster for urease production from the chromosome of clinical V. parahaemolyticus (TH3996). We found 16 open reading frames (ORFs) and a lower G؉C content (41%) compared with the total genome of this bacterium (46 to 47%). The ure cluster consisted of eight genes, namely, ureDABCEFG and ureR. ureR was located 5.2 kb upstream of the other seven genes in the opposite direction. The genetic organization and sequences of the ure genes resembled those found in Proteus mirabilis. Between ureR and the other ure genes, there were five ORFs, which are homologous with the nickel transport operon (nik) of Escherichia coli. We disrupted each of the ureR, ureC, and nikD genes in TH3996 by homologous recombination and analyzed the phenotype of the mutants. In the presence of urea these mutant strains had dramatically less urease activity than the strain they were derived from. Disruption of ureR, nikD, or ureC, however, had no effect on TRH production. The DNA region containing the trh, nik, and ure genes was found in only trh-positive strains and not in Kanagawa phenomenon-positive and environmental V. parahaemolyticus strains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced into V. parahaemolyticus in the past through a mechanism mediated by insertion sequences. This is the first reported case that the genes for an ATP-binding cassette-type nickel transport system, which may play a role in nickel transport through bacterial cytoplasmic membrane, are located adjacent to the ure cluster on the genome of an organism.Vibrio parahaemolyticus is a gram-negative, halophilic marine bacterium which causes gastroenteritis in humans who are infected through consumption of raw or inadequately cooked seafood (15). V. parahaemolyticus strains that are isolated from diarrheal patients produce either the thermostable direct hemolysin (TDH) or the TDH-related hemolysin (TRH), or both, while hardly any isolates from the environment have these properties (15, 41). TDH and TRH, encoded by tdh and trh, respectively, are each composed of 165-amino-acid residues and show approximately 67% identity in their amino acid sequences. TDH and TRH show several common biological activities, such as hemolytic activity, enterotoxicity, cytotoxicity, and cardiotoxicity, and are considered to be the major virulence factors in the pathogenesis of V. parahaemolyticus (15,35,41,47).Urease is an enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide. It is found in a number of bacteria, plants, fungi, and algae (29). Several bacterial urease genes have been isolated and characterized. The types of o...

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Cloning and sequencing of a novel hemolysis gene of Vibrio cholerae

Cited by 15 publications

References 3 publications

The Vibrio cholerae Flagellar Regulatory Hierarchy Controls Expression of Virulence Factors

The Vibrio cholerae Flagellar Regulatory Hierarchy Controls Expression of Virulence Factors

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of Vibrio cholerae O139 and non-O1, non-O139

Genetic Characterization of DNA Region Containing the trh and ure Genes of Vibrio parahaemolyticus

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