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Molecular Investigation of the Clostridium difficile Binary Toxin

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Title: Molecular Investigation of the Clostridium difficile Binary Toxin
Author: Metcalf, Devon
Department: Department of Pathobiology
Program: Pathobiology
Advisor: Weese, J. Scott
Abstract: This thesis is an investigation of the binary toxin of Clostridium difficile. The aim was to improve the understanding of the role of the binary toxin in pathogenesis. Quantitative real-time PCR (qPCR) was used to study expression of cdtA encoding the binary toxin enzymatic domain, cdtR encoding the binary toxin regulator and tcdB encoding toxin B, in response to growth phase and antimicrobial treatments in 2 C. difficile strains. Validation of a set of stable reference genes was required prior to qPCR analysis of gene expression. A universal set of genes could not be identified and reference genes should be validated on a strain-specific basis. Significant increases or decreases in expression were observed in response to levofloxacin and enrofloxacin exposure. The 2 strains selected were from different ribotypes and did not always share expression patterns. Binary toxin loci were sequenced in and compared between 10 C. difficile strains. A non-sense mutation in the cdtR gene of a ribotype 078 strain was identified and found to be restricted to toxinotype V isolates. This mutation is predicted to result in a truncated, non-functional protein. Despite the mutation, cdtA expression was still detected by qPCR. Finally, an evaluation of commercial nucleic acid extraction kits was performed. All kits produced RNA of adequate quality and yield, however, RNA isolated using the the Roche MagNA Pure LC RNA Isolation Kit could not be analyzed using the Agilent Bioanalyzer. It could not properly assign RNA integrity numbers due to a failure to remove small RNAs which were interpreted as degradation. All kits were suitable for DNA extraction with the exception of the MagNA Pure LC DNA Isolation Kit III which produced sheared DNA. In conclusion, this study demonstrated that the binary toxin regulator isn’t necessary for toxin expression and suggests other regulators of expression exist. Binary toxin gene expression did not necessarily correlate with expression of tcdB and expression levels vary between strains. This study also highlighted how the heterogeneity of C. difficile complicates gene expression experiments and the need for assessment of nucleic acid extraction methods due to critical variations between established commercial systems.
URI: http://hdl.handle.net/10214/4927
Date: 2012-11


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