Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Program Name/Specialization

Biological and Chemical Sciences

Faculty/School

Faculty of Science

First Advisor

Joel Weadge

Advisor Role

Principle Investigator

Abstract

Most bacterial populations reside within biofilms, which are communities of microorganisms embedded in a self-produced polymer matrix. These biofilms play critical roles in colonization and persistence within targeted niches, often contributing to disease progression. Bacterial species under specific conditions produce and export polymers that undergo post-synthesis chemical modifications, such as acetylation or the addition of phosphoethanolamine, which influence the chemical properties of the polymer, biofilm structure, and organism persistence. While well-studied systems, such as the opportunistic pathogen Pseudomonas aeruginosa, demonstrate the role of acetylation in alginate biofilms and their contribution to colonization and persistence in cystic fibrosis lungs, the mechanisms behind similar modifications in Clostridioides difficile and related pathogenic Clostridia species remained largely uncharacterized. In addition, WssI and WssF, cellulose acetyltransferases from Pseudomonas fluorescens will be functionally characterized in tandem. This research identified and characterized the role of CdCcsI, an acetyltransferase encoded within the C. difficile cellulose synthase operon (ccs), in the acetylation of cellulose biofilms along with WssI and WssF from P. fluorescens. Kinetic and enzymatic assays confirmed that CdCcsI catalyzes acetyl transfer to cellulose substrates via a catalytic triad (Ser344, Asp196, and His198) and an oxyanion hole (Arg366, Tyr367). Chromosomal mutation studies demonstrated that loss of the catalytic residues significantly reduced acetylation activity and biofilm biomass by 71%, as confirmed by iii crystal violet assays and fluorescence microscopy. Inhibitor studies identified lead compounds that effectively reduced CdCcsI activity, highlighting potential targets for antibiofilm strategies. Structural studies, including CD spectroscopy, verified that enzymatic activity loss in the active site variants was due to the specific removal of catalytic residues and not misfolding, reinforcing the functional importance of CdCcsI in acetyl-cellulose biofilm production. This work provided novel insights into the molecular mechanisms of CdCcsI, emphasizing its critical role in biofilm formation and its potential as a target for disrupting pathogenic biofilms in Clostridia. These findings elucidate the mechanisms behind biofilm modification in Clostridia and pave the way for developing strategies to weaken the biofilm barrier, enhancing antimicrobial penetration and effectiveness against infections caused by C. difficile and other Clostridial pathogens.

Convocation Year

2025

Convocation Season

Spring

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