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.
