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, σ^V is required for lysozyme resistance, and σ^V is activated in response to lysozyme. Once activated, σ^V, encoded by csfV, directs transcription of genes necessary for lysozyme resistance. Here, we analyze the contribution of individual genes in the σ^V regulon to lysozyme resistance. Using CRISPR-Cas9-mediated mutagenesis we constructed in-frame deletions of single genes in the csfV operon. We find 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, encoded by 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 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 C. difficile.

中文翻译：

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艰难梭菌中的溶菌酶抗性依赖于两种肽聚糖脱乙酰酶。


艰难梭菌( Clostridium difficile) 是导致抗生素相关性腹泻的医院获得性感染的主要原因。艰难梭菌对溶菌酶表现出非常高水平的抗性。细菌通常通过改变细胞壁来抵抗溶菌酶。在艰难梭菌中，溶菌酶抗性需要 σ ^V ，并且 σ ^V会响应溶菌酶而被激活。一旦激活，由csfV编码的 σ ^V就会指导溶菌酶抗性所需基因的转录。在这里，我们分析了 σ ^V调节子中各个基因对溶菌酶抗性的贡献。使用 CRISPR-Cas9 介导的诱变，我们构建了csfV操纵子中单个基因的框内删除。我们发现编码肽聚糖脱乙酰酶的pdaV是溶菌酶抗性的部分原因。然后，我们进行了 CRISPR 抑制 (CRISPRi)，以鉴定由pgdA编码的第二种肽聚糖脱乙酰酶，该酶对于溶菌酶抗性非常重要。删除pgdA或pdaV会导致溶菌酶抗性适度下降。然而，同时删除pgdA和pdaV会导致溶菌酶抗性降低 1,000 倍。此外，胞肽分析表明，PgdA 或 PdaV 的缺失对肽聚糖脱乙酰化影响不大，但 PgdA 和 PdaV 的缺失导致肽聚糖脱乙酰化几乎完全缺失。这表明 PgdA 和 PdaV 是多余的肽聚糖脱乙酰酶。 我们还使用CRISPRi比较了其他溶菌酶抗性机制，并得出肽聚糖脱乙酰化是艰难梭菌溶菌酶抗性的主要机制的结论。