Lysozyme resistance in Clostridioides difficile

Clostridioides (Clostridium) difficile is a Gram-positive, spore forming, anaerobic pathogen. It is the major cause of hospital-acquired infections leading to antibiotic-associated diarrhea. C. difficile causes an inflammatory infection, primarily mediated by two large exotoxins called Toxin A (TcdA) and Toxin B (TcdB). Both toxins function by glucosylating Rho family GTPases, leading to cytoskeletal collapse, cell death and inflammation at the site of infection. Transcription of tcdA and tcdB requires the specialized sigma factor, σTcdR, which also directs RNA Polymerase to transcribe tcdR itself. We fused a gene for a red fluorescent protein to the tcdA promoter to study toxin gene expression at the level of individual C. difficile cells. Surprisingly, only a subset of cells became red fluorescent upon entry into stationary phase. Breaking the positive feedback loop that controls σTcdR production by engineering cells to express tcdR from a tetracycline-inducible promoter resulted in uniform fluorescence across the population. Experiments with two regulators of tcdR expression, σD and CodY, revealed neither is required for bimodal toxin gene expression. However, σD biased cells towards the Toxin-ON state, while CodY biased cells towards the Toxin-OFF state. Finally, toxin gene expression was observed in sporulating cells.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 found that 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 evaluate the contribution of other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance in C. difficile.