ABSTRACT
Clostrididioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive spore former that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics 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. Our data reinforce prior evidence that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. We identified an 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 this β-lactamase, herein named 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 via insertional disruption 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 β-lactamase activity.
IMPORTANCE Clostridioides difficile is an anaerobic, gastrointestinal human pathogen. One of the highest risk factors for contracting C. difficile infection is antibiotic treatment, which causes microbiome dysbiosis. C. difficile is resistant to β-lactam antibiotics, the most commonly prescribed class of antibiotics. C. difficile produces a recently discovered β-lactamase, which cleaves and inactivates numerous β-lactams. In this study, we report the contribution of this anaerobic β-lactamase to ampicillin resistance in C. difficile, as well as the transcriptional regulation of the gene, blaD, by a BlaRI system. In addition, our data demonstrate co-transcription of blaD with blaX, which encodes a membrane protein of previously unknown function. Furthermore, we provide evidence that BlaX enhances β-lactamase activity in a portion of C. difficile strains. This study demonstrates a novel interaction between a β-lactamase and a membrane protein in a Gram-positive pathogen, and due to the anaerobic nature of the β-lactamase activity, suggests that more β-lactamases are yet to be identified in other anaerobes.
