Characterization of a β-lactamase that contributes to intrinsic β-lactam resistance inClostridioides difficile

Abstract

ABSTRACTClostrididioides difficilecauses severe antibiotic-associated diarrhea and colitis.C. difficileis an anaerobic, Gram-positive spore former that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance ofC. difficileto β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance inC. difficileare not fully understood. Our data reinforce prior evidence thatC. difficileproduces 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 inC. difficilestrain 630Δermand 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 theblaXDoperon in response to β-lactams. These results provide support for the function of a β-lactamase inC. difficileantibiotic resistance, and reveal the unique roles of a co-regulated lipoprotein and reducing environment in β-lactamase activity.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal human pathogen. One of the highest risk factors for contractingC. difficileinfection is antibiotic treatment, which causes microbiome dysbiosis.C. difficileis resistant to β-lactam antibiotics, the most commonly prescribed class of antibiotics.C. difficileproduces 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 inC. difficile, as well as the transcriptional regulation of the gene,blaD, by a BlaRI system. In addition, our data demonstrate co-transcription ofblaDwithblaX, which encodes a membrane protein of previously unknown function. Furthermore, we provide evidence that BlaX enhances β-lactamase activity in a portion ofC. difficilestrains. 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.