Genotype, exercise, and diet mediate host-microbiota interactions: the hunt for improved taxonomic resolution using nanopore sequencing
Creators: name: Dowden, Robert A.
Publisher: name: No Publisher Supplied
Year: 2021

description: The mammalian gut is home to a dense community of 100 trillion microorganisms collectively known as the microbiome, where a wide-body of evidence demonstrates the microbiome is involved in numerous metabolic and immune processes for the host. Unfortunately, the optimum microbiome for health and longevity remains ill-defined. To date, the gut microbiome is typically surveyed by short-read next-generation sequencing (NGS) of amplicons constructed from the 16S ribosomal RNA (rRNA) gene (~500 bp), with many host genetic and environmental factors impacting community structure and function. In this dissertation, I show how host genotype, exercise, and diet can each alter the gut microbiota composition using different mouse models and third-generation sequencing (TGS) of rRNA operons (~4.2kb), whereby improving the taxonomic resolution of community surveys. In Aim 1, we tested if genotype and activity impacted the selection of individual bacterial species and strains in the guts of a longevity model adenylyl cyclase type 5 knock out (AC5KO) and wild type (WT) mice as a proof of concept. Our results provide evidence that certain novel bacterial species (Muribaculum intestinale and Parasutterella excrementihominis) inhabit AC5KO and WT mice depending upon activity status, while other taxa (Turicibacter sanguinis and Turicimonas muris) did not demonstrate such patterning. Aim 1 supports the need for improved taxonomic resolution when probing microbiomes. In Aim 2, we investigated the role of gut microbes on exercise-induced training adaptations to select aspects of metabolism; cardiac/skeletal muscle blood flow; and, beneficial microbial derived short chain fatty acid (SCFA) profiles in AC5KO and WT mice prior to, following exercise training, and after antibiotic (ABX) treatment. Our results demonstrate improvements to exercise capacity were eliminated with ABX treatment, which also improved glucose uptake in both genotypes. Furthermore, we found ABX treatment significantly decreased myocardial and hind-limb (gastrocnemius) blood flow in AC5KO, but not WT, mice. Additionally, significant increases in acetic and corresponding decreases in butyric acid percentage were seen following ABX treatment in both genotypes. The results of Aim 2 support an intact gut microbiota is critical to AC5KO’s metabolism impacting exercise capacity, central and peripheral blood flow, and beneficial microbial metabolite profile. Lastly, Aim 3 explored sex differences and temporal dynamics in community structure in response to very high fat feeding and physical activity over 12-weeks. Aim 3 expands upon our work from Aim 1 to improve our strain level detection by utilizing a newly constructed rRNA operon database (rOpDB) and shows the influence of physical activity, diet, and sex to alter community β-diversity. Phylogenetic analysis of long-read consensus (LRC) rRNA operons constructed from Muribaculum_intestinale_YL27, four Candidatus_Arthromitus spp., and Turicibacter_sanguinis_BIOML-A124 demonstrates strain-level clustering based upon physical activity, diet, and sex. Aim 3 establishes rRNA operon profiling as a rapid, affordable, and computationally simplistic alternative to typical NGS of 16S rRNA genes used to survey microbiomes. This dissertation provides further evidence for the role of nature and nurture in modulating host-microbiome interactions and highlights our labs novel use rRNA operon profiling to survey community composition. Our long-read approach allows for an affordable and rapid pipeline that improves the taxonomic resolution of 16S rRNA gene profiles widely used in metagenomics.