Date of Award

2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

First Advisor

Gary M. Mawe

Abstract

A growing body of research on the gut microbiome highlights the pivotal role of enteric microbes as a dynamic interface with the host in healthy and pathological conditions. As such, the gut microbiome represents a promising novel target to treat a variety of clinical symptoms, including the alleviation of gastrointestinal (GI) symptoms. It is known that gut bacteria alter GI function through a variety of mechanisms including signaling through microbial metabolites. One notable metabolite is tryptophan (Trp), an essential amino acid. Trp can markedly impact signaling pathways in the gut through its role as the precursor to serotonin (5-hydroxytryptamine; 5-HT). 5-HT is a critical signaling molecule that regulates many important GI functions, including intestinal motility and secretion, and consequently has been implicated as a therapeutic target for the treatment of constipation and diarrhea.

An emerging, transient approach to manipulate the gut microbiota for therapeutic outcomes is through the oral administration of isolated bacterial strains. However, the underlying mechanisms of action of these bacteria are not well established, making it challenging to ascertain predictive treatment strategies. Here, we elucidate a novel strategy to manipulate 5-HT signaling and 5-HT-mediated functions by exploiting the biochemical capacity of specific bacteria to alter luminal Trp availability. We utilized the isolated bacterial strain Bacillus (B.) subtilis R0179, which expresses the key enzyme in Trp synthesis from indole, tryptophan synthase.

To test our hypothesis that oral administration of the Trp-synthesizing strain B. subtilis R0179 alters intestinal 5-HT signaling, we treated C57BL/6J mice daily via oral gavage with B. subtilis R0179 spores dissolved in phosphate buffered saline at a dose of 109 CFU/ml per day. We assessed colonic tissue levels of Trp, 5-HT, and the 5-HT break down product 5-hydroxyindoleacetic acid (5-HIAA) using high performance liquid chromatography. Following one week of treatment, mice that received B. subtilis R0179 exhibited greater levels of Trp and increased 5-HT signaling, as indicated by an elevated 5-HIAA/5-HT ratio, compared to vehicle-treated mice. We further demonstrate that these effects are dependent on spore viability as well as the ability to synthesize Trp. Mice treated with either heat inactivated B. subtilis R0179 or with a B. subtilis strain that does not express tryptophan synthase did not display altered colonic Trp or 5-HT signaling.

To test our hypothesis that B. subtilis R0179 treatment alters 5-HT-mediated gut function, we assessed changes in intestinal function in response to bacteria treatment using two validated motility assays: whole gut transit and colonic motility. We found that one week of treatment with B. subtilis R0179 accelerated colonic motility in healthy mice. Additionally, using a mouse model of constipation, we found that B. subtilis R0179 treatment restores deficits in colonic motility in constipated animals. Lastly, we found that the prokinetic effects of B. subtilis R0179 were blocked by co-administration of a 5-HT4 receptor (5-HT4R) antagonist and were absent in 5-HT4R knockout mice, demonstrating the involvement of the 5-HT4R in mediating the bacteria’s prokinetic actions.

Taken together, these data demonstrate that serotonergic signaling and resulting intestinal motility can be augmented by treatment with bacteria that synthesize Trp. Our findings provide mechanistic insight into a transient and predictable strategy to alter microbiota-gut dynamics with the goal of promoting GI motility.

Language

en

Number of Pages

157 p.

Download

Share

COinS