Culture Conditions for Stimulating Cholera Toxin Production by <i>Vibrio cholerae</i> O1 El Tor

Iwanaga, Masaaki; Yamamoto, Koichiro; Higa, Naomi; Ichinose, Yuki; Nakasone, Noboru; Tanabe, Masao

doi:10.1111/j.1348-0421.1986.tb03037.x

Cited by 262 publications

(280 citation statements)

References 13 publications

(1 reference statement)

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“…In vitro, virulence genes can be induced using a set of artificial conditions, such as liquid AKI medium for El Tor strains or 30°C and pH 6.5 for classical strains (6,7). Culturing under AKI conditions requires an initial phase in the absence of shaking, suggesting that oxygen levels may have an effect on virulence gene induction in El Tor strains.…”

Section: Resultsmentioning

confidence: 99%

“…Further studies are required The reversibility of the effect of oxygen on AphB. aphB deletion mutants containing pP tcpP -luxCDABE and either pBAD-aphB wt (wells 1, 3, 5, and 7) or pBAD-aphB C235S (wells 2, 4, 6, and 8) were grown aerobically (wells 1-4) and anaerobically (wells [5][6][7][8] in the presence of 0.01% arabinose for 4 h. Arabinose was then removed, and cultures were shifted to anaerobic (wells 3 and 4) or aerobic (wells 7 and 8) conditions for an additional 4 h. Cultures were then transferred to 96-well plates, and photographs were taken in the dark using a LAS4010 Imager (GE Healthcare).…”

Section: Discussionmentioning

confidence: 99%

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Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB

Li¹,

Yang²,

Peterfreund³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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Bacterial pathogens have evolved sophisticated signal transduction systems to coordinately control the expression of virulence determinants. For example, the human pathogen Vibrio cholerae is able to respond to host environmental signals by activating transcriptional regulatory cascades. The host signals that stimulate V. cholerae virulence gene expression, however, are still poorly understood. Previous proteomic studies indicated that the ambient oxygen concentration plays a role in V. cholerae virulence gene expression. In this study, we found that under oxygen-limiting conditions, an environment similar to the intestines, V. cholerae virulence genes are highly expressed. We show that anaerobiosis enhances dimerization and activity of AphB, a transcriptional activator that is required for the expression of the key virulence regulator TcpP, which leads to the activation of virulence factor production. We further show that one of the three cysteine residues in AphB, C 235 , is critical for oxygen responsiveness, as the AphB C235S mutant can activate virulence genes under aerobic conditions in vivo and can bind to tcpP promoters in the absence of reducing agents in vitro. Mass spectrometry analysis suggests that under aerobic conditions, AphB is modified at the C 235 residue. This modification is reversible between oxygen-rich aquatic environments and oxygen-limited human hosts, suggesting that V. cholerae may use a thiol-based switch mechanism to sense intestinal signals and activate virulence. thiol-modification | virulence activatorsT he Gram-negative bacterium Vibrio cholerae, the causative agent of the acute, dehydrating diarrheal disease cholera, has figured prominently in the history of infectious diseases as a cause of periodic, deadly pandemics. V. cholerae resides in aquatic environments between epidemics, and human infection normally starts with the ingestion of contaminated food or water. Vibrio cells surviving passage through the acidic gastric environment enter the small intestine, where they must produce an array of virulence factors including cholera toxin (CT) and the toxin co-regulated pilus (TCP) that are transcriptionally regulated by multiple systems (1). The primary, direct transcriptional activator of virulence genes is ToxT, whose transcription is regulated by the ToxRS and TcpPH proteins. Two additional activators encoded by unlinked genes, AphA and AphB, regulate the transcription of tcpPH.The environmental cues within the host and their effect on the expression of virulence genes in V. cholerae in vivo remain poorly characterized. It has been shown that anaerobiosis serves as one of the host environmental factors that modulate virulence factor production (2). This is not surprising because it is generally presumed that the oxygen concentration in the intestine is low (3). A recent report showed that under anaerobic conditions, tcpP expression is higher and this effect depends on AphB (4). However, whether and how this AphB-mediated tcpP expression contributes to anaerobic virulence indu...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB

Li¹,

Yang²,

Peterfreund³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

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“…O395 and E7946 are wild-type classical and El Tor (respectively) V. cholerae strains. Classical strains were grown overnight in LB at 30ЊC and El Tor strains were grown in AKI medium at 37ЊC (Iwanaga et al, 1986).…”

Section: Resultsmentioning

confidence: 99%

“…V. cholerae strains were grown at 25-30ЊC in LB medium or in AKI conditions at ᮊ Blackwell Science Ltd, Molecular Microbiology, 23, 323-331 37ЊC (Iwanaga et al, 1986). Antibiotics were used at the following concentrations: ampillicin, 100 g ml ¹1 ; tetracycline, 12.5 g ml ¹1 ; kanamycin, 30 g ml ¹1 ; and streptomycin, 100 g ml ¹1 .…”

Section: Methodsmentioning

confidence: 99%

**A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains**

Champion¹,

Neely

Brennan

et al. 1997

Molecular Microbiology

153

144

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SummaryCo-ordinate expression of genes associated with pathogenicity in Vibrio cholerae requires two transcription activators, ToxR and ToxT. Work carried out to date suggests that ToxR activates transcription of the toxT gene and that ToxT directly activates transcription of several genes whose products play a role in colonization or CT production by V. cholerae. Previous work also suggests that ToxR can directly activate transcription of the CT operon (ctxAB) independently of ToxT, thereby implying a degree of complexity in control of the ctxAB operon not found with other genes of the ToxR regulon. We tested the regulatory cascade model of virulence gene expression by constructing strains of classical and El Tor V. cholerae deleted for the coding sequence for the putative DNA-binding domain of toxT. Phenotypic analysis of these strains suggests that V. cholerae has ToxTdependent and ToxT-independent branches of its virulence regulon. The results also raise questions about the precise role for ToxR in activation of ctxAB transcription.

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“…Intriguingly, different in vitro conditions are required to activate virulence genes in different biotypes of V. cholerae. In the El Tor biotype responsible for the current cholera pandemic, AKI medium is used (10), in which peptone is substituted for the tryptone found in LB and oxygen concentrations are lowered by incubating cultures without shaking them. For the classical biotype, LB medium at pH 6.5 and 30°C are required to induce virulence genes.…”

mentioning

confidence: 99%

Bile salt–induced intermolecular disulfide bond formation activates Vibrio cholerae virulence

Yang

Liu

Hughes

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

125

173

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To be successful pathogens, bacteria must often restrict the expression of virulence genes to host environments. This requires a physical or chemical marker of the host environment as well as a cognate bacterial system for sensing the presence of a host to appropriately time the activation of virulence. However, there have been remarkably few such signal-sensor pairs identified, and the molecular mechanisms for host-sensing are virtually unknown. By directly applying a reporter strain of Vibrio cholerae, the causative agent of cholera, to a thin layer chromatography (TLC) plate containing mouse intestinal extracts, we found two host signals that activate virulence gene transcription. One of these was revealed to be the bile salt taurocholate. We then show that a set of bile salts cause dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulfide bonds between cysteine (C)-207 residues in its periplasmic domain. Various genetic and biochemical analyses led us to propose a model in which the other cysteine in the periplasmic domain, C218, forms an inhibitory intramolecular disulfide bond with C207 that must be isomerized to form the active C207-C207 intermolecular bond. We then found bile salt-dependent effects of these cysteine mutations on survival in vivo, correlating to our in vitro model. Our results are a demonstration of a mechanism for direct activation of the V. cholerae virulence cascade by a host signal molecule. They further provide a paradigm for recognition of the host environment in pathogenic bacteria through periplasmic cysteine oxidation.T he human pathogen Vibrio cholerae is the causative agent of the diarrheal disease cholera. The Vibrio life cycle begins with a freeswimming phase in aquatic environments. Human infection normally starts with the ingestion of food or water contaminated with V. cholerae. As it colonizes small intestines of a host and only when it colonizes, V. cholerae produces an array of virulence factors, including cholera toxin (CT), which causes the diarrhea characteristic of cholera and toxin-coregulated pili (TCP), type IV pili required for intestinal colonization both in animal models and in human volunteers (1, 2).Bacterial pathogens have evolved highly sophisticated signal transduction systems to coordinately control the expression of virulence determinants to better infect their hosts. Extensive in vitro studies have revealed details of V. cholerae virulence gene regulation (3): AphA and AphB proteins activate transcription of the transmembrane transcription factor TcpP, which in turn activates toxT transcription together with ToxR, which then completes the cascade by activating toxin and TCP production (Fig. 1A). However, all of the experiments to date used artificial in vitro conditions to induce virulence factor production, leaving unanswered which microenvironmental signals in the intestines activate the V. cholerae virulence cascade. It has been reported that certain environmental conditions such as temperature, oxygen concentra...

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Culture Conditions for Stimulating Cholera Toxin Production by Vibrio cholerae O1 El Tor

Abstract: A method that stimulates cholera toxin (CT) production by Vibrio cholerae culture fluid was established.

Cited by 262 publications

References 13 publications

Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB

Vibrio cholerae anaerobic induction of virulence gene expression is controlled by thiol-based switches of virulence regulator AphB

**A branch in the ToxR regulatory cascade of Vibrio cholerae revealed by characterization of toxT mutant strains**

Bile salt–induced intermolecular disulfide bond formation activates Vibrio cholerae virulence

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