# Biogenesis and functions of outer membrane vesicles in Bacteroidetes

> **NIH NIH R21** · WASHINGTON UNIVERSITY · 2022 · $236,250

## Abstract

Abstract
The human intestine is colonized with ~1014 microorganisms that make up the gut microbiota. Approximately
40% of the bacteria inhabiting the human gut belong to the phylum Bacteroidetes, which can promote both
healthy and diseased states. Long-term colonization of Bacteroidetes in the human gut is largely due to their
ability to utilize dietary polysaccharides that are indigestible by the host and endogenous host glycans.
Polysaccharide degradation in Bacteroidetes is controlled by a diverse array of multi-gene polysaccharide
utilization loci (PUL), which encode the receptors, glycosidases (GH), and transporters needed to sense and
digest various glycans. Through proteomic analyses, we determined that numerous GH are preferentially
packaged into outer membrane vesicles (OMV). OMV are spherical, membranous structures generated by
blebbing of the outer membrane (OM) of Gram-negative bacteria. Bacteroidetes OMV have been proposed to
play important roles in immune modulation, maintenance of intestinal homeostasis, and promotion of
interbacterial mutualistic interactions. Despite their increasing importance, no definitive mechanism for OMV
biogenesis has been established, and OMV biology remains one of the least studied fundamental processes in
microbiology. We have previously demonstrated that Bacteroides fragilis and B. thetaiotaomicron produce
significant amounts OMV that are homogenous in size and shape. The OMV-specific proteome showed a high
prevalence of GH and proteases. The GHs specifically packed into OMV are usually encoded within PUL, and
their cognate transporters are not detected in OMV, which suggests that PUL systems partition between OM and
OMV. We constructed fusions of OMV- and OM-specific proteins with fluorescent proteins and employed them
as markers for live fluorescence microscopy. For the first time, we visualized the formation of OMV. The use of
different fluorescent markers allowed us to differentiate between bona fide OMV and lysis by products. Our
preliminary data demonstrate that, in Bacteroidetes, OMV are the result of a highly orchestrated physiological
process. In aim 1 of this proposal we will employ biochemistry and mass spectrometry to investigate how OMV
cargo is regulated to maximize the digestion of dietary and endogenous host glycans. In aim 2, we will employ
fusions between OMV proteins and luciferase to investigate the molecular machinery required for OMV
biogenesis. Understanding these processes may lead, in the future, to manipulation of bacterial vesiculation for
innovative interventions to treat pathologies involving gut dysbiosis, such as Crohn’s disease and IBD.

## Key facts

- **NIH application ID:** 10431386
- **Project number:** 1R21AI168719-01
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Mario Feldman
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $236,250
- **Award type:** 1
- **Project period:** 2022-01-21 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10431386

## Citation

> US National Institutes of Health, RePORTER application 10431386, Biogenesis and functions of outer membrane vesicles in Bacteroidetes (1R21AI168719-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10431386. Licensed CC0.

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