ORCID

http://orcid.org/0000-0001-5464-722X

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Systems Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Asthma is a common respiratory disease with a highly heterogeneous pathophysiology. The human gut microbiota, comprising of all the microorganisms that inhabit the gastrointestinal tract, is linked to the development of asthma and can alter airway inflammation in animal models. The idea that the gut microbiota can have bidirectional cross-talk with the lung, such as gut dysbiosis affecting lung disease, is termed the gut-lung axis. While the gut microbiota of early life has been an area of particular interest for asthma pathogenesis research, the effect of its taxonomic and functional composition after asthma diagnosis is less clear. This dissertation employs both amplicon sequencing followed by gnotobiotic mouse models (Chapter 2) and whole metagenomic shotgun sequencing (Chapter 3) to characterize human fecal microbiomes from school-aged children and adults with asthma. Investigation of the gut-lung axis in asthma later in life requires clinical studies with well-defined asthma inclusion criteria and strategic gnotobiotic experiments guided by taxonomic profiling of properly handled human samples. In Chapter 2, amplicon sequencing was used to identify population shifts between allergic moderate-to-severe asthma and healthy cohorts. Taxonomic shifts associated with asthma were observed even when accounting for other microbiome-modifying covariates such as age and race. Additionally, statistical modeling and gnotobiotic mouse models were used to identify taxa that could affect lung inflammation in vivo. A Naïve Bayes Classifier fit to a mixture model that accounts for the sparsity inherent to compositional data was built to optimize selection of samples from the human cohorts that would best represent a asthma-associated microbial community differences. The selected stool samples were then used to inoculate, or “humanize”, germ-free mouse gastrointestinal tracts before administration of allergen sensitization and challenge. Immunophenotyping, IgA-Seq, gut permeability assays, and whole genome sequencing of human fecal bacterial isolates uncovered an enterotoxigenic Bacteroides fragilis that affected lung inflammation in the context of intact human fecal communities as well as on its own in a monocolonization experiment. A PCR screen for the B. fragilis toxin (bft) across all human participants revealed that bft was more prevalent in the stool of people with asthma compared to that of healthy individuals. These findings suggest that the gut microbiota affects lung inflammation even after the diagnosis of asthma. While discovery of disease-modifying taxa is invaluable, taxonomic profiling by amplicon sequencing skips the genetic material that encodes a wealth of functional information about gut microbes. In Chapter 3, whole metagenomic shotgun sequencing is utilized on the human fecal samples from Chapter 2 to describe the genetic content of the entire gut microbiota, also called the “metagenome”. Read-based annotation revealed a shift in genetic content attributable to asthma even when accounting for covariates such as age and race. Metabolic pathway annotation suggested that fatty acid metabolism pathways, particularly those that result in long-chain fatty acid synthesis, are differentially abundant in the asthma cohort. Antibiotic resistance is a growing concern among physicians who provide asthma care and patients with asthma tend to require more antibiotic prescriptions than usual, particularly macrolide antibiotics. Antibiotic usage was tallied for all participants in this study and a higher proportion of the asthma cohort was found to have taken antibiotics in the past year compared to the healthy cohort. Subsequent profiling of antibiotic resistance genes (ARGs) in the gut metagenomes revealed an increased richness of ARGs in the asthma cohort while the total abundance of ARGs was not increased. Additionally, macrolide resistance markers were differentially abundant in the asthma cohort. Interestingly, the B. fragilis toxin, found to be more prevalent in the same asthma cohort in Chapter 2, was more likely to co-occur in the samples with ermF in the asthma cohort compared to the healthy cohort. Co-occurrence analysis of all ARGs and all virulence factors revealed a unique set of VF-ARG pairs in the asthma cohort compared to the healthy, together suggesting that the asthma gut microbiota offers opportunities for virulence factors and ARGs to co-occur that do not co-occur in healthy gut microbiota. The ermF-bft pair is particularly concerning given that bft has the potential to affect airway inflammation and macrolide resistance is already becoming a clinical problem for patients with asthma. In summary, this work characterizes metagenomic shifts in the gut microbiota associated with asthma, identifies a gut pathobiont that can alter lung inflammation, and reveals accumulation of antibiotic resistance genes in populations suffering from asthma. These findings provide needed insights into the gut-lung axis of asthma beyond diagnosis, and will guide development of gut-directed therapy for a frustratingly common disease.

Language

English (en)

Chair and Committee

Andrew L. Kau S Swamidass

Committee Members

Megan T. Baldridge, Gautam Dantas, Makedonka Mitreva,

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