Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A

Robert, A. Budinsky; Silva, Anisia; Benítez, Jorge A.; Rodrı́guez, Boris L.; Fando, Rafael; Campos, Javier; Sengupta, Dilip K.; Boesman-Finkelstein, Mary; Finkelstein, Richard A.

doi:10.1016/s0264-410x(96)00105-3

Cited by 36 publications

(45 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Inactivation of hapA, which encodes HA/protease, has been shown to enhance adherence to mucin-coated polystyrene plates (43), adherence to mucin-secreting differentiated HT29-18N2 cultured cells (4), and colonization of the suckling mouse intestine (41,43). The mucinase activity of HA/protease (13), together with its capacity to cleave the mucin-binding adhesin GbpA at a high cell density, provides a mechanism that supports the "detachase" function attributed to this protein (25).…”

mentioning

confidence: 99%

Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease

Wang

Ayala

et al. 2011

J Bacteriol

View full text Add to dashboard Cite

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ⌬hapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.Cholera is an acute waterborne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. Infecting V. cholerae cells adhere to the intestinal mucosa, where they express major virulence factors such as the toxin coregulated pilus (TCP) (21) and cholera toxin (CT), which is largely responsible for the profuse rice-watery diarrhea typical of this illness (12,26). Cholera patients shed V. cholerae in their stools, which usually contain about 10 8 hyperinfective V. cholerae cells per ml (38). The V. cholerae cells can then survive and persist in fresh water and estuarine aquatic ecosystems to eventually gain entrance to a new host.The Zn-dependent metalloprotease hemagglutinin (HA)/ protease (18) has been proposed to facilitate V. cholerae detachment from the intestinal mucosa when infecting V. cholerae cells reach a high density (4,14,43,44). Inactivation of hapA, which encodes HA/protease, has been shown to enhance adherence to mucin-coated polystyrene plates (43), adherence to mucin-secreting differentiated HT29-18N2 cultured cells (4), and colonization of the suckling mouse intestine (41, 43). The mucinase activity of HA/protease (13), together with its capacity to cleave the mucin-binding adhesin GbpA at a high cell density, provides a mechanism that supports the "detachase" function attributed to this protein (25). In addition, HA/protease has been reported to perturb the paracellular barrier of cultured intestinal epithelial cells (37, 55) by acting on tight junction-associated proteins (56). In agreement with this finding, we have shown that HA/protease enhances enterotoxicity in the rabbit ileal loop model (43).The expression of flagellar motility ...

show abstract

mentioning

confidence: 99%

Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease

Wang

Ayala

et al. 2011

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Inactivation of hapA increased adherence to mucin synthesized by HT29-18N2 cells (4), and expression of hapA was required for V. cholerae to penetrate a mucin-containing gel in vitro (42). Although analysis of hapA mutants in infant rabbits and suckling mice has not provided evidence that HapA is an essential virulence factor (11,18,36), HapA has been shown to contribute to reactogenicity of live attenuated cholera vaccine strains in humans (3, 13).Expression of hapA requires transcriptional activation by HapR, RpoS, and the cyclic AMP receptor protein (22,41). The regulators LuxO and HapR coordinate cell density-dependent expression of CT, TCP, HapA, and biofilm formation (44,49).…”

mentioning

confidence: 99%

Contribution of Hemagglutinin/Protease and Motility to the Pathogenesis of El Tor Biotype Cholera

et al. 2006

View full text Add to dashboard Cite

Vibrio cholerae is a highly motile organism that secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HapA). HapA has been shown to have mucinase activity and contribute to the reactogenicity of live vaccine candidates, but its role in cholera pathogenesis is not yet clear. The contribution of motility to pathogenesis is not fully understood, since conflicting results have been obtained with different strains, mutants, and animal models. The objective of this work was to determine the contribution of HapA and motility to the pathogenesis of El Tor biotype cholera. To this end we constructed isogenic motility (motY) and mucinase (hapA) single and double mutants of an El Tor biotype V. cholerae strain. Mutants were characterized for the expression of major virulence factors in vitro and in vivo. The motility mutant showed a remarkable increase in cholera toxin (CT), toxin coregulated pilus major subunit (TcpA), and HapA production in vitro. Increased TcpA and CT production could be explained by increased transcription of tcpA, ctxA, and toxT. No effect was detected on the transcription of hapA in the motility mutant. The sodium ionophore monensin diminished production of HapA in the parent but not in the motility mutant. Phenamil, a specific inhibitor of the flagellar motor, diminished CT production in the wild-type and motY strains. The hapA mutant showed increased binding to mucin. In contrast, the motY mutation diminished adherence to biotic and abiotic surfaces including mucin. Lack of HapA did not affect colonization in the suckling mouse model. The motility and mucinase defects did not prevent induction of ctxA and tcpA in the mouse intestine as measured by recombinasebased in vivo expression technology. Analysis of mutants in the rabbit ileal loop model showed that both V. cholerae motility and HapA were necessary for full expression of enterotoxicity.Cholera is an acute diarrheal disease characterized by the passing of voluminous rice water stool. Vibrio cholerae of serogroups O1 and O139 continues to cause seasonal cholera outbreaks that affect highly populated regions in Asia, Africa, and Latin America. V. cholerae is a highly motile organism with a single sheathed polar flagellum. It colonizes the small intestine and expresses a variety of virulence determinants, such as the toxin-coregulated pilus (TCP), cholera toxin (CT), and other factors required to multiply and survive in the host.V. cholerae produces a soluble Zn-metalloprotease, hemagglutinin/protease (HapA), encoded by hapA (18). HapA can proteolytically degrade several physiologically important host proteins including mucin (10). HapA perturbs the paracellular barrier of cultured intestinal epithelial cells (33, 46) by acting on tight junction-associated proteins (47). Inactivation of hapA increased adherence to mucin synthesized by HT29-18N2 cells (4), and expression of hapA was required for V. cholerae to penetrate a mucin-containing gel in vitro (42). Although analysis of hapA mutants in infant rabbits and suckling mice has not ...

show abstract

“…HA/protease perturbs the paracellular barrier of cultured intestinal epithelial cells (31,50) by acting on tight-junction-associated proteins (51) and promotes the detachment of vibrios from monolayers and mucin (4,15). Importantly, HA/protease contributes to the reactogenicity of live attenuated cholera vaccine strains (3,40).…”

mentioning

confidence: 99%

Transcriptional Regulation of Vibrio cholerae Hemagglutinin/Protease by the Cyclic AMP Receptor Protein and RpoS

Silva

Benítez

2004

J Bacteriol

View full text Add to dashboard Cite

Vibrio cholerae secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HA/protease), which is encoded by hapA and displays a broad range of potentially pathogenic activities. Production of HA/protease requires transcriptional activation by the quorum-sensing regulator HapR. In this study we demonstrate that transcription of hapA is growth phase dependent and specifically activated in the deceleration and stationary growth phases. Addition of glucose in these phases repressed hapA transcription by inducing V. cholerae to resume exponential growth, which in turn diminished the expression of a rpoS-lacZ transcriptional fusion. Contrary to a previous observation, we demonstrate that transcription of hapA requires the rpoS-encoded s factor. The cyclic AMP (cAMP) receptor protein (CRP) strongly enhanced hapA transcription in the deceleration phase. Analysis of rpoS and hapR mRNA in isogenic CRP ؉ and CRP ؊ strains suggested that CRP enhances the transcription of rpoS and hapR. Analysis of strains containing hapR-lacZ and hapA-lacZ fusions confirmed that hapA is transcribed in response to concurrent quorum-sensing and nutrient limitation stimuli. Mutations inactivating the stringent response regulator RelA and the HapR-controlled AphA regulator did not affect HA/protease expression. Electrophoretic mobility shift experiments showed that pure cAMP-CRP and HapR alone do not bind the hapA promoter. This result suggests that HapR activation of hapA differs from its interaction with the aphA promoter and could involve additional factors.

show abstract

Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A

Cited by 36 publications

References 26 publications

Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease

Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease

Contribution of Hemagglutinin/Protease and Motility to the Pathogenesis of El Tor Biotype Cholera

Transcriptional Regulation of Vibrio cholerae Hemagglutinin/Protease by the Cyclic AMP Receptor Protein and RpoS

Contact Info

Product

Resources

About