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Screen and Functional Analysis of the Genes Regulate Aphb in Vibrio Cholerae and Inhibitory Activity of Pseudomonas Aeruginosa on Vibrio Cholerae

Author: ChenHaiLi
Tutor: ZhuJun
School: Nanjing Agricultural College
Course: Microbiology
Keywords: Vibrio cholerae aphB quorum-sensing pyocyanin
CLC: R378
Type: Master's thesis
Year: 2011
Downloads: 6
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Abstract

Cholera is a life-threatening diarrheal disease caused by the gram-negative bacterium Vibrio cholerae,usually of serogroup 01. In its severe form,cholera gravis, the clinical disease is characterized by the passage of voluminous stools of rice water character that rapidly lead to dehydration. Hypovolemic shock, acidosis, and death can ensue in adults, as well as in children, if prompt and appropriate treatment is not initiated.The organism colonizes the upper intestine, and the toxin-coregulated pilus (TCP) is the primary factor involved in the infection. The severe diarrhea associated with the disease results from the action of the secreted cholera toxin (CT) on intestinal epithelial cells. The genes required for the biogenesis of TCP are located in an operon on a large pathogenicity island termed the TCP-ACF element, or vibrio pathogenicity island(VPI). The subunits of CT are encoded by the ctxA and ctxB genes on a separate genetic element which comprises the genome of the lysogenic filamentous bacteria phage CTXΦ. ToxT is an AraC-type regulator encoded on the VPI that directly activates the transcription of the tcp, ctx, and accessory colonization factor genes. Expression of toxT is dependent upon cooperation between two homologous pairs of trans-membrane regulatory proteins encoded by the toxRS operon on chromosome I and tcpPH encoded on the VPI. AphB functions synergistically with AphA to activate the expression of tcpPH, and it also appears to contribute to the differences in virulence gene expression between the two major disease-causing biotypes, classical and El Tor.Three parallel quorum-sensing circuits appear to function on together in V. cholerae to control virulence. In addition, it has been shown that quorum sensing negatively influences virulence gene expression in strains of V. cholerae through the repressive action of HapR, the V. cholerae LuxR homologue. HapR influences the virulence cascade by binding to a recognition site in the aphA promoter and decreasing its expression, but did not influence the expression of aphB. In an effort to identify the genes involved in aphB express In this study, we used transposon mutagenesis to screen for C6706 (lacZ’) strains cotaining two plasmids pBBRLux-aphB, pkP302-aphB which detect aphB promoter express level.we screened two candidate mutants which positive regulate aphB. Sequencing of the region flanking the transposon insertion by arbitrary PCR showed that T1 inserts vc1585 and T2 inserts vc1602. Then I constructed in-frame deletion strains to certificate these genes positive regulate aphB.In the other project, we find 10 unknown strains has inhibitory activity on V.cholerae by screening. After API20E identification these strains are Pseudomonas aeruginosa.Finally we find pyocyanin (PCN, a blue pigment) is produced by P. aeruginosa which possibly work in the process. Pyocyanin can inhibit bacteria and fungi and has a strong toxicity for a wide range of organisms. Pyocyanin is regulated by quorum-sensing system.

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