Genes Impacting Fitness and Protection of Gut Bacteroidales during Stress

The human gut microbiome is a complex microbial community containing diverse microbes that affect host health. Bacteroidales is the most predominant bacterial order of the human gut and includes highly prevalent species from the genera Bacteroides, Phocaeicola, Parabacteroides, and Segatella. Phocaeicola vulgatus (P. vulgatus) is one of the most ubiquitous and abundant members of the healthy human gut, but less studied than other gut Bacteroidales species. P. vulgatus, like other gut Bacteroidales, persists in the face of numerous stressors in the gut including those of host and microbial origin; however, mechanisms of this resilience are unknown. In this thesis, I aim to characterize how P. vulgatus withstands stressors found in the mammalian gut first using an unbiased, high-throughput genetic screening method, randomly-barcoded transposon insertion sequencing (RB-TnSeq), and secondly through a targeted investigation of a unique protective locus. RB-TnSeq allows for the calculation of fitness scores for all genes in the bacterial genome under specific conditions and identification of genes that are conditionally essential. I have constructed and characterized an RB-TnSeq library in P. vulgatus strain PvCL10T00C06 (PvCL10), which was isolated in our lab from a healthy human donor and performed RB-TnSeq experiments in vivo, in a mouse model of colitis, and in the presence of relevant gut stressors in vitro to identify genes important for bacterial resistance to stress. We have established a bioinformatic pipeline to identify each gene's contribution to fitness under each stressor condition of interest and further investigated mechanisms of protection for the most relevant fitness determinant genes identified by making targeted deletions and combining transcriptomic and biochemical analyses. To our knowledge, this work presents the first RB-TnSeq screen during DSS-induced colitis and our transcriptomic analyses during colitis have identified a previously uncharacterized sigma/anti-sigma factor pair that are strongly upregulated. For my second project, previous work had identified a sigma/anti-sigma factor pair in PvCL10 that are activated by antibacterial toxins which target the outer membrane. I present work which identifies a genetic locus regulated directly by the anti-sigma factor, and investigate the functions of the encoded proteins. These analyses uncovered a novel mechanism of bacterial regulation and additional protective features of PvCL10 against stress. Altogether, this thesis provides a comprehensive analysis of how this abundant species of bacteria copes with numerous stressors that it encounters in the gut. This understanding can ultimately improve the design of live biotherapeutics to allow strains to better engraft and survive in the gastrointestinal tract.