Identification of a novel two-component system involved in peptidoglycan synthesis in Clostridioides difficile

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ABSTRACT Clostridioides difficile is a spore-forming, anaerobic bacterium that can cause severe disease, including antibiotic-associated diarrhea and pseudomembranous colitis, in humans. Vancomycin is a first-line drug for treating C. difficile infection; it targets peptidoglycan biosynthesis, a pathway specific for prokaryotic cells and essential for the formation of the bacterial cell wall and growth. Vancomycin binds to the D-Ala-D-Ala residues of the peptidoglycan intermediates and prevents their incorporation into mature peptidoglycan. The C. difficile vanG operon, analogs of which confer vancomycin resistance in other bacterial species due to the replacement of the D-Ala-D-Ala moiety of peptidoglycan with D-Ala-D-Ser, is positively regulated by a two-component system, VanRS. Uniquely, neither vancomycin-induced nor high, constitutive expression of the vanG operon confers by itself resistance to vancomycin in C. difficile. Nevertheless, many clinical and laboratory-generated vancomycin-resistant C. difficile strains contain vanRS mutations that increase vanG expression, strongly suggesting that high expression of the operon contributes, together with other mutations, to the development of the resistance. We have found that in the absence of the C. difficile vancomycin-sensing histidine kinase, VanS, another histidine kinase, not yet genetically identified and provisionally named as KinX, also responds to vancomycin and is able to replace VanS and induce the vanG operon. A regulated histidine kinase crosstalk in response to the same environmental signal, in this case vancomycin, is unusual. In contrast to VanS, KinX also responds to at least one more antibiotic that interferes with peptidoglycan synthesis. Therefore, it is critically important to understand in detail the function of KinX, which is activated in response to a clinically used antibiotic, may contribute to the emerging resistance of C. difficile to vancomycin via the regulation of the vanG operon, and is very likely to regulate additional genes that are involved in peptidoglycan metabolism. Using several independent unbiased or targeted approaches, including RNA-Seq and CRISPRi, we propose to identify the novel histidine kinase, KinX, and, likely, its cognate response regulator that control expression of genes of peptidoglycan biosynthesis. Using gene-specific and global expression analyses, we will determine the contribution of KinX to the regulation of the vanG operon and define the KinX regulon. Our results will shed new light on peptidoglycan biosynthesis and mechanisms of vancomycin sensitivity and resistance in C. difficile. Vancomycin-resistant strains are commonly detected in the clinic, and the spread of the resistance may become a serious issue in treating C. difficile infection. Detailed knowledge on the regulation of the vanG operon and other genes of peptidoglycan metabolism is critical for understanding the development of vancomycin resistance and designing new antimicrobials that target peptidoglycan.