Molecular characterization of a putative adhesin Csp1 in Clostridium difficile

• Main Authors: Zih-CianSu, 蘇資茜
• Other Authors: I-Hsiu Huang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/25453058743918092331

碩士 === 國立成功大學 === 微生物及免疫學研究所 === 102 === Clostridium difficile is a Gram-positive, endospore forming human pathogen and the most common cause of antibiotic-associated diarrhea within hospital settings worldwide. Disruption of the host’s indigenous microflora by broad-spectrum antibiotics is one of the major triggers for C. difficile infections (CDI). The emergence of hypervirulent C. difficile strains with high mortality rates have resulted in major epidemics in many parts of the world. C. difficile can express two exotoxins TcdA and TcdB, and in some strains a binary toxin CDT. Both TcdA and TcdB are potent toxins and are responsible for the extensive gastrointestinal inflammation and epithelial tissue damages found in an infected host. In addition, C. difficile is known to express a wide-variety of surface proteins such as S-layer proteins, fibronectin and cell wall proteins (Cwps). Surface proteins of Gram-positive bacteria are known to play important roles during infections. Sortase, a membrane anchored transpeptidase found ubiquitously in Gram-positive bacteria, promotes the covalent anchoring of surface proteins to the cell wall envelope. To determine the role of sortase-dependent surface proteins in C. difficile pathogenesis, I identified at least seven putative sortase substrates in C. difficile strain 630 by bioinformatics analysis. In this study, I focused on one of the putative cell surface proteins, Csp1. Csp1, encoded by CD2831, is a predicted protein of 972 amino acids and is annotated as a collagen-binding protein. The protein has a PPKTG motif suggesting that Csp1 could be a putative substrate of C. difficile sortase B. The aim of this study is to characterize molecular properties of Csp1. I constructed csp1 insertion mutant, KYC01, and defined the localization of Csp1 by immunoblotting using polyclonal anti-Csp1. Due to the low expression level of Csp1, I also overexpressed Csp1 in both CD630 and KYC01. Cellular fractionation and immunoblotting analysis demonstrated that Csp1 can be found anchored to the cell wall. Interestingly, the majority of Csp1 appear to be secreted into the medium. In-vitro binding experiments demonstrated the ability of Csp1 recombinant protein to adhere to Collagen I. In summary, the results generated from this project revealed a novel adhesin in C. difficile.