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Bollinger Thesis Revised3.63 MB
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Dissertation
Discovery and characterization of a novel L,D-transpeptidase domain that makes essential 3-3 crosslinks in Clostridioides difficile
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2025
DOI: 10.25820/etd.008245
Abstract
Clostridioides difficile is the most common cause of nosocomial infectious diarrhea in the US and is considered an urgent threat by the CDC due to the high-cost burden on the US healthcare system and poor quality of life for patients with C. difficile infections (CDI). A large issue with CDI is the high rate of recurrent infections, with about 1 in 5 people treated for C. difficile developing a recurrent CDI. Novel antibiotics for CDI are needed, and the cell wall is a common target for antibiotics due to its essentiality in most bacteria. Peptidoglycan (PG) is the major component of the cell wall, and it consists of a repeating N-acetylglucosamine and N-acetylmuramic acid disaccharide chain with a peptide sidechain attached to the N-acetylmuramic acid. The repeating disaccharide subunits form a strand that must be crosslinked to another strand of PG to fortify the cell wall. Transpeptidases use the peptide sidechains to crosslink the strands (of PG) together. There are two types of transpeptidases. Penicillin-binding proteins (PBPs) are essential in most bacteria and utilize a pentapeptide sidechain as their substrate to make 4-3 crosslinks. L,D-transpeptidases (LDTs) are not known to be essential and utilize a tetrapeptide substrate to make 3-3 crosslinks. In most bacteria, 4-3 crosslinks make up the vast majority (~90%) of crosslinks in the cell wall. However, in C. difficile 3-3 crosslinks make up about 70% of the total crosslinks.
In this thesis, we investigated the LDTs and 3-3 crosslinking in C. difficile. We discovered that VanW domains are a novel class of LDTs. Additionally, we found that 3-3 crosslinks and the LDTs are essential for viability in C. difficile, making it the first organism in which LDTs were shown to be essential. Loss of the LDTs in C. difficile eliminated 3-3 crosslinks with no compensatory increase in the 4-3 crosslinks, resulting in cell lysis. PG precursors are synthesized with a pentapeptide sidechain rather than a tetrapeptide sidechain, so we investigated how C. difficile generates the tetrapeptides utilized by LDTs for crosslinking. We discovered that two D-alanyl-D-alanine carboxypeptidases (DD-CPases), DacA and DacC, are responsible for converting the pentapeptides to tetrapeptides during vegetative growth. Deletion of dacA and dacC prevented this conversion and provided C. difficile with enough pentapeptide substrate for the PBPs to efficiently crosslink the cell wall and bypass the normal essentiality of LDTs. This engineered C. difficile strain lacking dacA, dacC, and all the LDTs, called ∆dacAC∆ldt, was remarkably healthy. Its only major phenotypic defect was an inability to sporulate. To understand why, we conducted a suppressor screen that revealed loss of dacB, a sporulation specific DD-CPase, restored sporulation to WT levels in the ∆dacAC∆ldt background. Thus, during both sporulation and vegetative growth C. difficile has high levels of carboxypeptidase activity that deprive PBPs of substrates and make the LDTs essential for PG crosslinking. We hypothesize that the LDTs may be good targets for novel narrow-spectrum antibiotics against C. difficile.
Details
- Title: Subtitle
- Discovery and characterization of a novel L,D-transpeptidase domain that makes essential 3-3 crosslinks in Clostridioides difficile
- Creators
- Kevin W. Bollinger
- Contributors
- Craig Ellermeier (Advisor)David Weiss (Advisor)Michael Gebhardt (Committee Member)Richard Roller (Committee Member)Mary Weber (Committee Member)Adrian Elcock (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Autumn 2025
- DOI
- 10.25820/etd.008245
- Publisher
- University of Iowa
- Number of pages
- xv, 204 pages
- Copyright
- Copyright 2025 Kevin W. Bollinger
- Language
- English
- Date submitted
- 12/08/2025
- Description illustrations
- Illustrations, graphs, charts, tables
- Description bibliographic
- Includes bibliographical references (pages 173-204).
- Public Abstract (ETD)
The bacterial cell wall is a structure that is important for protecting and separating the bacteria from the outside environment. I studied the cell wall of Clostridioides difficile, a major pathogen for which new antibiotic treatment options are needed. A good analogy for the cell wall is a chain linked fence; multiple strands, or chains, of PG have to be crosslinked together for the cell wall to have sufficient strength and structure. My research focused on crosslinks called 3-3 crosslinks in the cell wall that are made by proteins called LDTs. Unlike in most bacteria, 3-3 crosslinks are the most common type of crosslink in the C. difficile cell wall. I therefore hypothesized that the 3-3 crosslinks are essential for survival in C. difficile. Indeed, my results show that LDTs are essential and that loss of them results in a drastic reduction in cell wall integrity that C. difficile cannot survive. My discovery that LDTs are essential in C. difficile is the first time LDTs have been shown to be essential in an organism. This makes the LDTs good potential drug targets for new antibiotics to treat C. difficile. For C. difficile infections, narrow-spectrum antibiotics that specifically kill C. difficile are desired because they will reduce the frequency of relapse. Thus, my work elucidating the essentiality of LDTs in C. difficile may be important for future antibiotic development.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9985134948702771
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