# Mechanistic analysis and engineering probiotic bacteria with SagA

> **NIH NIH F32** · ROCKEFELLER UNIVERSITY · 2020 · $67,446

## Abstract

Project Summary
Multidrug-resistant bacteria are increasingly the cause of infectious disease fatalities worldwide. Because
civilization is at the precipice of a post-antibiotic era, alternative methods to treat infectious diseases need to
be utilized. One especially insidious bacterial pathogen, Clostridium difficile, thrives in the human gut and
releases its toxin after antibiotic treatment diminishes the commensal intestinal microbiota. C. difficile infection
(CDI) can cause profuse diarrhea and colitis and is one of the leading causes of gastroenteritis related
fatalities. CDI can be difficult to treat, as C. difficile can form dormant spores during treatment with antibiotics
and cause relapse after treatment has ended. There is evidence fecal microbiota transplantation or treatments
with probiotic bacteria can prevent or treat CDI. However, the mechanistic basis for probiotic-based protection
is not fully understood. Recent work from the Hang Lab at Rockefeller University has identified secreted
antigen A (SagA), a putative peptidoglycan hydrolase secreted by the commensal bacterium Enterococcus
faecium that increased host immunity to CDI in mice. Remarkably, recombinant expression of SagA in other
bacterial species also increased mouse tolerance against CDI implicating SagA as a potential therapeutic
agent that can be delivered by a probiotic. In order to understand how SagA activity increases host protection,
peptidoglycan hydrolase activity of SagA will be characterized. Additionally, stable expression strains of SagA
will be engineered in probiotic bacteria. Finally, host protection against CDI will be investigated using the stably
engineered strains of probiotic-SagA in a mouse model. The long-term goals of this project are to elucidate the
protective mechanism of SagA and engineer probiotics to stably express and secrete SagA for future design of
novel therapeutics against enteric bacterial infections.
Aim 1: Biochemical characterization of SagA peptidoglycan hydrolase activity
Aim 2: Engineer stable expression strains of SagA in probiotic bacteria
Aim 3: Investigate protection of SagA-probiotic against Clostridium difficile

## Key facts

- **NIH application ID:** 9920591
- **Project number:** 5F32AT010087-02
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Charles W Hespen
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $67,446
- **Award type:** 5
- **Project period:** 2019-04-16 → 2022-04-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9920591

## Citation

> US National Institutes of Health, RePORTER application 9920591, Mechanistic analysis and engineering probiotic bacteria with SagA (5F32AT010087-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9920591. Licensed CC0.

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