Lysozyme resistance inC. difficileis dependent on two peptidoglycan deacetylases

Abstract

Clostridioides (Clostridium) difficile is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In C. difficile σV is required for lysozyme resistance and σV is activated in response to lysozyme. Once activated σV, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in the csfV regulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find pdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, pgdA, that is important for lysozyme resistance. Deletion of either pgdA or pdaV resulted in modest decreases in lysozyme resistance. However, deletion of both pgdA and pdaV resulted in a 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile.

HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

“…subtilis strain BS49 (Christie et al, 1987). CRISPR-Cas9 plasmids were built on the backbone of pJK02 (McAllister et al, 2017) with some modification (Kaus et al, 2020).…”

Pathogenicity and virulence of Clostridioides difficile

“…σV is activated upon lysozyme detection, resulting in expression of peptidoglycan deacetylases PgdA and PdaV [ 148 ]. Classical microbiology has shown the synergistic effects of these two proteins in their contribution to lysozyme resistance – deletion of a single protein resulted in small reductions in resistance, whereas deletion of both proteins simultaneously resulted in 1000× reduction in resistance, through almost complete loss of peptidoglycan deacetylation [ 149 ]. Deacetylation is an effective method of lysozyme resistance, since interaction between the activate site of lysozyme, and acetyl groups on peptidoglycan enables efficient hydrolysis [ 150 ].…”