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The Interaction between TcpA Pilus and pⅢCTX in Vibrio Cholerae El Tor

Author: XiongLiFeng
Tutor: ZuoZuo
School: Disease Control and Prevention Center
Course: Pathogen Biology
Keywords: TcpA pIIICTX Protein interactions GST-pull down SPR-BIACORE
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 27
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Abstract


Vibrio cholerae is the causative agent of cholera (Vibrio cholerae). According to V. cholerae bacteria (O) antigen serotype different programs established cholera can be divided into more than 200 serogroups. But it can only cholerae pathogenic serogroups O1 and O139 two. Which, O1 serogroup can be divided into classical and El Tor biotype type two, the former has triggered the sixth worldwide cholera epidemic, and now the world is still in the seventh pandemic El Tor cholera by arc caused by bacteria. Severe diarrhea of ??cholera caused by cholera toxin. The cholera toxin is produced by cholera chromosome prophage (lysogenic filamentous phage) CTXΦ ctxAB carried genes encoding. Under certain conditions, could be induced prophage CTXΦ into a phage infection and attB site-specific integration into the chromosome, resulting ctxAB genes and non-producing strains producing strains between the horizontal transfer, and then caused by the emergence of new epidemic strains. And from different serogroups or biotypes CTXΦ between the existence of certain differences. So, study the phage CTXΦ different sources and different hosts Vibrio cholerae interactions for understanding Vibrio cholerae virulence gene horizontal transfer, Vibrio cholerae strains of different periods of transition, as well as predict the emergence of new epidemic strains and so have significance. CTXΦ Vibrio cholerae infection process, first CTXΦ specific receptors on host identification and interactions. Genetic evidence indicates that there is an indirect, CTXΦ cholera vibrio receptor Tcp pilus (TcpA protein), and the phage ligands for the p Ⅲ CTX proteins. But there has been no direct experimental evidence to verify that the establishment of an inference. First select the topic caused seventh world pandemic Vibrio cholerae El Tor as the main object, using FITC fluorescence, GST fusion protein deposition (GST-pull down) and surface plasmon resonance (SPR) technology to Research TcpA pilin and p Ⅲ CTX-protein interaction, the interaction between them to find direct evidence for further study and host lysogenic CTXΦ Vibrio cholerae causes disease outbreak and the relationship between preparation. We first constructed with a His-tagged protein and p Ⅲ CTX TcpA protein, and the method by affinity chromatography purification. By FITC-labeled way to make from the El Tor biotype of V. cholerae N16961 p Ⅲ CTX bring FITC-labeled protein, and then let a good p Ⅲ CTX marker protein expression conditions induced through pili classical and El Tor Vibrio cholerae were role in the confocal microscope p Ⅲ CTX in vitro protein binding of Vibrio cholerae. The results show that, from the El Tor biotype of V. cholerae N16961 p Ⅲ CTX protein and the presence of V. cholerae classical in vitro binding, and we were done by constructing method deficient strains confirmed this in vitro binding by TcpA pilin mediated. Meanwhile, we also constructed and purified with GST tag p Ⅲ CTX proteins. With GST fusion protein deposition technique (GST-pull down), we confirmed: from the El Tor biotype of V. cholerae N16961 p Ⅲ CTX-type protein and from El Tor (N16961) and classical (0395) between proteins of Vibrio cholerae TcpA there were a direct interaction in vitro. Based on the above study, we also used surface plasmon resonance (SPR-BIACORE) technique to quantitatively analyze the interaction between these two proteins. From Vibrio cholerae El Tor N16961 fusion protein GST-p Ⅲ CTX-protein coupled to the CM5 chip, to capture the flow through the chip surface TcpA protein solution, and then analyzes the interaction of these two proteins affinity constant. In this study, the interaction through a variety of in vitro methods, confirmed the Vibrio cholerae El Tor N16961 which TcpA protein and p Ⅲ CTX direct interaction between proteins, also found: El Tor vibrio cholerae N16961 of p Ⅲ CTX protein Vibrio cholerae classical TcpA 0395 of interactions between proteins, while the p Ⅲ CTX classical and classical type TcpA pilin, and between the p Ⅲ CTX classical and El Tor type TcpA pilin not observed directly between interactions, suggesting that we come from different serogroups p Ⅲ CTX and TcpA protein interactions there are differences, this difference is reflected in carrying cholera toxin genes ctxAB phage infection CTXΦ differences between different hosts, it has been possible cholera whether pathogenic Vibrio different serogroups cause epidemic and an important reason and judgment basis. In a subsequent study, we will take this as a starting point, from different serogroups p Ⅲ CTX and TcpA protein interactions and their differences for further research to explore different serogroups of V. cholerae is causing disease outbreaks and Popular real reason.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria
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