Genetic Characterization of the Anaerobic β-lactamase of Clostridioides difficile Open Access
Sandhu, Brindar (Summer 2019)
Abstract
Clostridioides difficile is an intestinal bacterial pathogen that causes severe antibiotic-associated diarrhea and colitis. C. difficile infections are responsible for billions of dollars in healthcare expenses in the United States every year. C. difficile is an anaerobic bacterium that is highly resistant to β-lactams, the most commonly prescribed antibiotics. Treatment with β-lactam antibiotics causes microbiome dysbiosis, and the resistance of C. difficile to β-lactams allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. We have shown that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. We have characterized the C. difficile operon encoding a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of the β-lactamase, BlaD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a co-transcribed lipoprotein, BlaX, aids in BlaD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated that BlaRI controls transcription of the blaXD operon in response to β-lactams. These results provide support for the function of a β-lactamase in C. difficile antibiotic resistance, and reveal the unique roles of a co-regulated lipoprotein and reducing environment in C. difficile β-lactamase activity.
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Tables and Figures
Chapter 1: Introduction……………………………………………………………………………………..…..1
Chapter 2: Regulation and anaerobic function of the Clostridioides difficile β-lactamase…..32
Chapter 3: Discussion…………………………………………………………………………………..………94
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