Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases

Gabriela M. Kaus; Lindsey F. Snyder; Ute Müh; Matthew J. Flores; David L. Popham; Craig D. Ellermeier

doi:10.1128/JB.00421-20

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Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases

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Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases

Gabriela M. Kaus, Lindsey F. Snyder, Ute Müh, Matthew J. Flores, David L. Popham and Craig D. Ellermeier

Journal of bacteriology, Vol.202(22), p.1

10/22/2020

DOI: 10.1128/JB.00421-20

PMCID: PMC7585060

PMID: 32868404

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Abstract

Clostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV 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 C. difficile . We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation. 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 , 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 . IMPORTANCE Clostridioides difficile is the leading cause of hospital-acquired diarrhea. C. difficile is highly resistant to lysozyme. We previously showed that the csfV 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 C. difficile . We show that two peptidoglycan deacetylases in C. difficile are partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation, and PdaV, encoded by a part of the csfV operon, provides lysozyme-induced peptidoglycan deacetylation.

Details

Title: Subtitle: Lysozyme Resistance in Clostridioides difficile Is Dependent on Two Peptidoglycan Deacetylases
Creators: Gabriela M. Kaus - University of Iowa
Lindsey F. Snyder - University of Iowa
Ute Müh - University of Iowa
Matthew J. Flores - Virginia Tech
David L. Popham - Virginia Tech
Craig D. Ellermeier - University of Iowa
Contributors: Michael J. Federle (Editor)
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.202(22), p.1
DOI: 10.1128/JB.00421-20
PMID: 32868404
PMCID: PMC7585060
ISSN: 0021-9193
eISSN: 1098-5530
Grant note: DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: R01AI087834; DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: T32AI007511
Language: English
Date published: 10/22/2020
Academic Unit: Microbiology and Immunology
Record Identifier: 9984297319602771

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