Date of Award

Summer 7-31-2024

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Andrew Gewirtz

Second Advisor

Didier Merlin

Third Advisor

Jun Zou

Fourth Advisor

Kuk Jeong Chin

Abstract

ABSTRACT

The goal of this thesis project was to understand how fecal microbiota transplant treats Clostridioides difficile (C. difficile) infection, potentially inspiring more effective noninvasive therapies to restore the microbiome as a means of mitigating the disease burden caused by this pathogen. Accordingly, I sought to identify taxa that provide colonization resistance against C. difficile. I devised a gnotobiotic ASF (Altered Schaedler Flora) mouse model of CDI that provided a tractable and defined platform for studying microbiota’s role in CDI. Challenge of ASF mice with 105 spores of hypervirulent C. difficile strain VPI10463 (acute challenge) led to complete mortality within three days; challenge with 102 spores resulted in low mortality and mice that remained chronically infected by C. difficile (chronic CDI model) and, concomitantly, chronic CDI symptoms. Furthermore, I found a previously described Clostridia preparation (Kim et al., 2017) protected ASF mice from acute challenge, and led to cessation of VPI10563 shedding and resolution of CDI symptoms in chronically infected ASF mice. Sequencing-based microbiome analysis revealed an association between Lachnospiraceae microbes and recovery. Bulk culture of the Clostridia preparation was used to generate a Lachnospiraceae consortium that protected mice against acute and chronic CDI. Deep sequencing found that recovery of chronic CDI mice strongly corelated with the appearance of a Lachnospiraceae species, namely Uncultured Bacteria and Archaea (UBA) 3401 a bacterium, which, as its name implies, was only known to exist as from databases of shotgun sequencing. While iterative removal of non-UBA3401 microbes from the Lachnospiraceae consortium enabled in vitro isolation of PCR positive-UBA3401 colonies, subculture of such colonies failed to propagate UBA3401. Thus, I performed iterative limiting dilution fecal transplants of the Lachnospiraceae consortium resulting in generation of a consortium predominated by UBA3401, albeit accompanied by a few other microbes (UBA3401 consortium). Inoculation of mice with the UBA3401 consortium was tracked longitudinally. Sequencing and PCR testing showed UBA3401 became detectable 3 days post inoculation and peaked in absolute and relative abundance at 18 days post inoculation. The UBA3401 consortium-colonized mice and age matched ASF controls underwent acute challenge with VPI10463. Severe CDI followed resulting in complete mortality of the control group, however UBA3401-consortium mice suffered no mortality and demonstrated minimal illness, showing that despite the simple, defined composition of the UBA3401 consortium, significant protection was retained. A concentrated chloroform extract of UBA3401 mouse feces was shown to result in reduced in-vitro growth of VPI10463 compared to ASF feces chloroform extract. Genomic sequencing of UBA3401 uncovered a probable biosynthetic gene cluster (BSC) responsible for production of a Thiopeptide bacteriocin. Canonic thiopeptide BSC genes, including the critical YCAO enzyme, were predicted along with adjacent genes for transport and localization (Vinogradov & Suga, 2020). The UBA3401 genome was predicted to encode many genes involved with quorum sensing, potentially regulating bacteriocin production (M. Kleerebezem et al., 1997), suggesting that a threshold of UBA3401 is required to induce thiopeptide production. Collectively, my results provide a more tractable model to study CDI and yield UBA3401 as potential means of providing microbiota-mediated CDI resistance.

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