UVM Theses and Dissertations
Format:
Online
Author:
Fimlaid, Kelly
Title:
Dept./Program:
Microbiology and Molecular Genetics
Year:
2016
Degree:
PhD
Abstract:
Clostridium difficile is a Gram-positive spore-forming strict anaerobe that can cause severe colitis in humans. C. difficile is best known as the leading cause of nosocomial-acquired diarrhea, particularly in people undergoing antibiotic therapies, since it is naturally resistant to most antibiotics. A clinical feature that makes C. difficile infection, or CDI, particularly difficult to treat is the organism's inherent ability to resist antibiotic therapies while in its spore form. Since oxygen is toxic to C. difficile, spores are the major transmissive form; they are also resilient to most disinfectants, which makes them extremely difficult to eliminate to prevent additional infections.
While over fifty years of studies on the spore-forming model organism Bacillus subtilis laid the foundation of how sporulation and germination occurs, little was known about how C. difficile regulates spore formation and/or what proteins are necessary for sporulation and germination processes. The work presented in this dissertation addresses how C. difficile regulates sporulation, identifies genes that are regulated during sporulation, and characterizes some key proteins that are required for either sporulation or germination.
Guided by our RNA-Sequencing results, we constructed targeted gene mutations in spoIIQ and spoIIIA-H, which are important for forming a channel known as the "feeding tube" in B. subtilis. We demonstrated that these proteins are necessary for maintaining forespore integrity, tethering the coat to the forespore, and engulfment. Using metabolic labeling, we show that while spoIIQ and spoIIIA mutants cannot finish the phagocytic-like process of engulfment, they are capable of transforming peptidoglycan, which is a necessary step for engulfment to occur.
We also constructed a targeted gene mutation in a gene that is highly transcribed during sporulation, now known as gerS. We show that a gerS mutant cannot degrade cortex during germination and is required for SleC-mediated cortex hydrolysis, making GerS a novel regulator of C. difficile spore germination. Altogether, this research provides a framework for understanding how the pathogen C. difficile undergoes sporulation and is therefore capable of infecting humans. Further, our studies reveal important factors that mediate the essential process of engulfment during sporulation and an important factor that mediates cortex hydrolysis during germination. This work has demonstrated that C. difficile regulates sporulation and germination differently than what has previously been described in other Firmicutes.
While over fifty years of studies on the spore-forming model organism Bacillus subtilis laid the foundation of how sporulation and germination occurs, little was known about how C. difficile regulates spore formation and/or what proteins are necessary for sporulation and germination processes. The work presented in this dissertation addresses how C. difficile regulates sporulation, identifies genes that are regulated during sporulation, and characterizes some key proteins that are required for either sporulation or germination.
Guided by our RNA-Sequencing results, we constructed targeted gene mutations in spoIIQ and spoIIIA-H, which are important for forming a channel known as the "feeding tube" in B. subtilis. We demonstrated that these proteins are necessary for maintaining forespore integrity, tethering the coat to the forespore, and engulfment. Using metabolic labeling, we show that while spoIIQ and spoIIIA mutants cannot finish the phagocytic-like process of engulfment, they are capable of transforming peptidoglycan, which is a necessary step for engulfment to occur.
We also constructed a targeted gene mutation in a gene that is highly transcribed during sporulation, now known as gerS. We show that a gerS mutant cannot degrade cortex during germination and is required for SleC-mediated cortex hydrolysis, making GerS a novel regulator of C. difficile spore germination. Altogether, this research provides a framework for understanding how the pathogen C. difficile undergoes sporulation and is therefore capable of infecting humans. Further, our studies reveal important factors that mediate the essential process of engulfment during sporulation and an important factor that mediates cortex hydrolysis during germination. This work has demonstrated that C. difficile regulates sporulation and germination differently than what has previously been described in other Firmicutes.