Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases

( ) is a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. exhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. In , σ is required for lysozyme resistance, and σ is activated in response to lysozyme. Once activated, σ , encoded by , directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σ regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the operon. We find that , which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase, encoded by , that is important for lysozyme resistance. Deletion of either or resulted in modest decreases in lysozyme resistance. However, deletion of both and resulted in a 1,000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed that loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but that 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 used CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in is the leading cause of hospital-acquired diarrhea. is highly resistant to lysozyme. We previously showed that the operon is required for lysozyme resistance. Here, we used CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism in We show that two peptidoglycan deacetylases in are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the operon, provides lysozyme-induced peptidoglycan deacetylation.