# Development of probiotic-based and Clostridioides difficile-targeted therapeutics

> **NIH NIH R21** · UNIVERSITY OF SOUTH FLORIDA · 2024 · $240,875

## Abstract

Abstract
Clostridioides difficile is a Gram-positive, spore-forming and toxin-producing anaerobic bacterium. It is the most
common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of life-threatening
pseudomembranous colitis. Central to predisposition to C. difficile infection (CDI) is the disruption of the gut
microbiota by broad-spectrum antibiotics. Currently, standard treatment of CDI is the administraion of
vancomycin, fidaomicin or metronidazole, but none of these is fully effective as the antibiotic treatment results
in an estimated 15-35% of recurrence. Treatment of recurrent CDI (rCDI) is one of the major challenges in the
field. Alarmingly, significantly decreased susceptibility to metronidazole and vancomycin has been reported. As
such, metronidazole is no longer recommeded as the first-line drug for CDI treatment. Novel non-antibiotic
therapeutics that specifically target C. difficile are desperately needed to control CDI and recurrence.
We have recently identified a C. difficile phage lysin lytic domain, designated as LCD, which potently and broadly
lyse different ribotypes of C. difficile clinical isolates. We have also identified a panel of single-domain variable
fragments of heavy-chain only antibodies (VHHs) against the unique and conserved C. difficile protein Cwp84.
Cwp84 is a surface-associated cysteine protease and plays a critical role in the maturation of surface-layer
proteins that are important for bacterial colonization, as antibodies against Cwp84 protected hamsters from lethal
CDI by delaying C. difficile colonization. To generate a targeted therapy specifically against C. difficile, we have
fused LCD with one anti-Cwp84 VHH, generating a novel fusion protein LCD-VHH27. LCD-VHH27 is significantly
(p<0.0001) more potent than LCD in lysing different C. difficile strains including 2 hypervirulent epidemic RT027
strains. In this R21, we will generate more LCD-VHH fusions and use our novel antibiotic-free probiotic systems
Saccharomyces boulardii and Lactococcus lactis to deliver the fusion proteins to the site of C. difficile colonization
and infection in animal models of CDI. We hypothesize that engineered probiotics that secrete LCD-VHH fusions
at the lower intestines where C. difficile colonizes will lead to a potent therapeutic efficacy against CDI.

## Key facts

- **NIH application ID:** 10876669
- **Project number:** 1R21AI183094-01
- **Recipient organization:** UNIVERSITY OF SOUTH FLORIDA
- **Principal Investigator:** Xingmin Sun
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $240,875
- **Award type:** 1
- **Project period:** 2024-05-28 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10876669

## Citation

> US National Institutes of Health, RePORTER application 10876669, Development of probiotic-based and Clostridioides difficile-targeted therapeutics (1R21AI183094-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10876669. Licensed CC0.

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