Genetic Analysis of Fitness Determinants in Phocaeicola vulgatus

Abstract

Over the last few decades, there has been an increasingly large body of research focused on the ecology and function of the gut bacteria, collectively known as the gut microbiome. This work has focused on the human gut microbiome as well as other animals including livestock and model organisms that can be genetically and experimentally manipulated. These organisms include laboratory rodents, fruit flies, worms, and pigs, to name a few. The background and future directions of this field are reviewed in Chapter 1 of this thesis. My work in the laboratory of Dr. John Rawls has focused on several aspects of the gut microbiome in different contexts, including how the gut microbiome is affected by nutritional challenges, host diseases, and lifestyle interventions, as well as the factors affecting microbiome composition, which might inform how we can develop strategies to manipulate the microbiome.In Chapter 2, I focus on the question of what genetic factors affect microbiome composition. To do so, I focus on a specific gut microbe known as Phocaeicola (Previously Bacteroides) vulgatus, or Pvu. Pvu is among the most abundant Bacteroidaceae species. Pvu also has myriad health associations in human studies, is an early life colonizer, and an efficient long-term colonizer in both humans and mice. However, the genes required for Pvu to establish itself in a complex microbiome are unknown. To address this gap in knowledge, I present experiments using transposon mutagenesis and insertion sequencing (INSeq) to understand Pvu colonization of the mammalian gut. This reverse genetics approach identifies several potential pathways that Pvu might use to colonize and persist in a complex microbiome. I further elucidate the functions of a hypothetical secreted protein, Pvu777, that is required for competition in vivo in a complex microbiome. In vivo competition experiments using genetically engineered Pvu strains recapitulate these findings in Pvu777 as well as the downstream putative fatty acid transporter Pvu776. Comparative genomics suggests that the operon containing Pvu777, which consists of the predicted DNA Binding/Histone-like protein Pvu778, Pvu777, and Pvu776 may be unique to Pvu and closely related gut Bacteroides and Phocaeicola species. RNA Seq approaches link Pvu777 to outer membrane and envelope functions. In conclusion, we identify a variety of pathways required for Pvu to colonize and persist in a complex microbiome using an INSeq screen, and elucidate the potential functions of one of the genes emerging from this screen using a range of experimental approaches. These findings could be used to inform further fitness-based studies of Pvu, but could also be used to inform methods to control its in vivo abundance, in addition to suggesting mechanisms that could be used to design efficiently colonizing engineered gut bacteria. In Chapter 3, I focus on the question of how nutritional challenges affect the gut microbiome using a zebrafish model of starvation. Starvation is a widespread nutritional challenge for which animals possess many physiological adaptations. However, current research into animal starvation has focused mainly on tissue histopathologies associated with starvation, excluding the physiological changes in the GI tract as well as the gut microbiome. In Chapter 3, we used RNA sequencing and 16S rRNA gene sequencing to uncover changes in the intestinal transcriptome and microbiome of zebrafish subjected to long-term starvation and refeeding compared to continuously fed controls. Starvation over 21 days led to increased diversity and altered composition in the intestinal microbiome compared to fed controls, including relative increases in Vibrio and reductions in Plesiomonas bacteria. Starvation also led to significant alterations in host gene expression in the intestine, with distinct pathways affected at early and late stages of starvation. This included increases in the expression of ribosome biogenesis genes early in starvation, followed by decreased expression of genes involved in antiviral immunity and lipid transport at later stages. These effects of starvation on the host transcriptome and microbiome were almost completely restored within 3 days after refeeding. Comparison with published datasets identified host genes responsive to starvation as well as high-fat feeding or microbiome colonization, and predicted host transcription factors that may be involved in starvation response. Overall, the results presented in Chapter 3 demonstrate that there may be distinct stages of starvation that lead to specific changes in gut microbial ecology and host GI tract transcriptome. These stages of starvation are largely reversible upon refeeding and the ensuing changes in host gene expression and microbiome composition may be an adaptive response to recover from starvation. This work could thus inform future research investigating the roles of specific bacterial taxa in host starvation, as well as mechanistic studies looking at the roles of specific host genes in starvation and refeeding using genetically modified hosts. In Chapter 4, I suggest studies that could extend from the work presented in Chapter 2, focusing on the role of individual genes in the Pvu 777 operon in vivo as well as within Pvu. I also suggest potential roles for the predicted DNA-Binding/Histone-like protein Pvu778 in the regulation of Pvu gene expression and Pvu fitness. I conclude by considering the evolutionary conservation of the 777 operon among Pvu and close relatives, and methods to investigate the fitness requirements of the 777 operon in these related bacterial species as well. Structural studies of Pvu777 are also proposed, which would help clarify the function and potential binding partners for the Pvu777 hypothetical protein. Thus, the studies proposed in Chapter 4 would help provide a clearer picture of the functions, regulation, and evolutionary history of the 777 operon, which would underscore its importance as a potentially conserved operon involved in Pvu fitness in vivo.