# The mechanistic basis for toxin secretion in Clostridioides difficile

> **NIH NIH F32** · VANDERBILT UNIVERSITY MEDICAL CENTER · 2022 · $72,302

## Abstract

Project Summary/Abstract
Understanding how large macromolecules are transported across a cell wall is a complex and poorly
understood biological process. The nosocomial pathogen Clostridioides difficile produces and secretes two
large toxins that are responsible for causing disease. Although much is known about the effector functions of
the toxins, very little is known about how the toxins are secreted. The toxins are encoded on a pathogenicity
locus which also encodes TcdE, a holin-like protein and TcdL, the N-terminal remnant of an endolysin. While
bacteriophages use holin/endolysin systems to trigger bacterial cell lysis and escape, multiple reports now
suggest that TcdE is used for the secretion of the toxins. This proposal will address the outstanding questions
of how TcdE and TcdL interact with C. difficile toxins to create a pore and secrete toxins without causing cell
lysis. TcdE and TcdL will be fluorescently tagged, and their localization and oligomerization will be determined
in vivo using structured illumination microscopy and stepwise photobleaching. The oligomerization state(s) of
TcdE will also be determined in vitro using cryo-transmission electron microscopy (cryo-TEM). TcdL has
recently been discovered and its role in toxin secretion has not been fully explored. TcdL will be deleted and
overexpressed to determine what, if any, role it has during toxin secretion. Direct binding assays will assess if
TcdL can interact with the toxins or TcdE to facilitate toxin secretion. Finally, the cell wall architecture of C.
difficile will be assessed during toxin secretion using correlative light and electron microscopy, and focused
ion-beam scanning electron microscopy. The 3D structure of TcdE and the cell wall will be determined in situ
using cryo-electron tomography. This proposal will shed new light on a basic biological process: the transport
of macromolecules across the cell membrane. The experiments proposed here will build upon my strong
foundation in bacterial genetics and physiology. I will be training in the laboratory of Dr. D. Borden Lacy, an
established leader in the field of structural biology with a specialty in solving high-resolution toxin structures.
Her guidance will allow me to learn cutting edge techniques in structural biology and high-resolution light
microscopy. The techniques that I will learn and the research program that I will build over the course of this
training will allow me to transition into an independent investigator at a R1 research institution.

## Key facts

- **NIH application ID:** 10445004
- **Project number:** 5F32GM139303-03
- **Recipient organization:** VANDERBILT UNIVERSITY MEDICAL CENTER
- **Principal Investigator:** Shannon L Kordus
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $72,302
- **Award type:** 5
- **Project period:** 2020-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10445004

## Citation

> US National Institutes of Health, RePORTER application 10445004, The mechanistic basis for toxin secretion in Clostridioides difficile (5F32GM139303-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10445004. Licensed CC0.

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