Dissertation
Lysozyme resistance in Clostridioides difficile
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2020
DOI: 10.17077/etd.005665
Abstract
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.
Details
- Title: Subtitle
- Lysozyme resistance in Clostridioides difficile
- Creators
- Gabriela Maria Kaus
- Contributors
- Craig Ellermeier (Advisor)David Weiss (Committee Member)Dominique Limoli (Committee Member)Mary Weber (Committee Member)Scott Moye-Rowley (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Autumn 2020
- DOI
- 10.17077/etd.005665
- Publisher
- University of Iowa
- Number of pages
- xii, 131 pages
- Copyright
- Copyright 2020 Gabriela Maria Kaus
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 111-131).
- Public Abstract (ETD)
Clostridioides difficile, colloquially known as “C. diff,” is an anaerobic bacteria and the most common cause of healthcare-associated diarrhea. It causes an opportunistic infection, generally occurring following treatment with antibiotics when natural intestinal flora has been disrupted. Infection is primarily mediated by two toxins, Toxin A (TcdA) and Toxin B (TcdB). We show that the toxin genes are expressed in a bimodal manner, meaning some cells are Toxin-ON and some cells are Toxin-OFF. We observe a subset of cells that are Toxin-ON and a separate subset that are Toxin-OFF in an otherwise identical population. Our work reveals that the master regulator of toxin, TcdR, is critical for bimodal expression. We further showed two additional regulators, SigD and CodY, greatly influence toxin expression. SigD biases cells toward Toxin-ON state, while CodY biases cells towards Toxin-OFF. Additionally, we show that cells that produce toxin can further differentiate into metabolically-inert and aerotolerant spores.
During infection the host immune system produces multiple factors in an attempt to control and eliminate C. difficile bacterial cells. One of these factors is lysozyme, a hydrolytic enzyme that damages bacterial peptidoglycan, part of the bacterial cell wall, resulting in lysis and cell death. In order to survive in the presence of lysozyme bacteria make multiple modifications to their cell wall. Here, we define the contribution of each cell wall modification mechanism to lysozyme resistance. We demonstrate that deacetylation of peptidoglycan is the major mechanism of resistance in C. difficile. We also show that C. difficile produces two proteins that are able to directly bind and inhibit lysozyme activity.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984035795202771
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