PXD070923

PXD070923 is an original dataset announced via ProteomeXchange.

Title	Nanobodies against Clostridioides difficile CDTb provide a toolkit for potent toxin neutralization and highly sensitive quantitation
Description	Clostridioides difficile is a pathogenic bacterium and a leading cause of antibiotic-associated diarrhea. Symptoms of the infection arise due to production of large clostridial toxins that disrupt the intestinal barrier and cause an acute host inflammatory response. Epidemic C. difficile strains also produce the C. difficile transferase toxin (CDT), a binary toxin consisting of separate enzymatically active (CDTa) and cell-binding (CDTb) components. However, the role of CDT during C. difficile pathogenesis remains poorly understood. We created a CDTb nanobody clone library and identified and purified five clones with promising CDTb-binding properties. Studies using the Carterra LSAXT platform revealed high affinity binding interactions between the nanobodies and three distinct CDTb epitopes. Functionally, these nanobodies potently neutralize cellular cytopathic effects of CDT at equimolar concentrations in vitro. We further identified two distinct neutralization mechanisms – inhibition of CDTb heptamer formation and inhibition of cell surface binding, both of which are crucial for CDTa delivery into the host cell. We also uncovered a neutralization mechanism whereby nanobodies non-canonically stack ‘in parallel’ to the D3’ domain of CDTb via β-sheet interactions. These nanobodies were used in a sandwich ELISA assay to monitor CDTb levels between 1- and 7- days post R20291 infection in the cecal material of infected mice. Notably, levels of CDTb spiked during days 3 and 4, with monomers constituting the majority of CDTb. We anticipate that these reagents will allow researchers to further expand toxin intervention and monitoring strategies to obtain a deeper understanding of the CDT mechanism of action.
HostingRepository	PRIDE
AnnounceDate	2026-02-02
AnnouncementXML	Submission_2026-02-01_16:10:33.884.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	carla.oneale@vanderbilt.edu carla.oneale@vanderbilt.edu
SpeciesList	scientific name: Escherichia coli; NCBI TaxID: NEWT:562;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Exploris 240

Revision	Datetime	Status	ChangeLog Entry
0	2025-11-18 18:15:48	ID requested
⏵ 1	2026-02-01 16:10:34	announced

10.1016/j.jbc.2025.111082;

Nabukhotna K, Kroh HK, Anderson DM, Cano Rodr, í, guez R, Shupe JA, McGresham M, O'Neale CVT, Shrem RA, Wadzinski BE, Schey KL, Spiller BW, Lacy DB, Nanobodies against Clostridioides difficile CDTb provide a toolkit for potent toxin neutralization and highly sensitive quantitation. J Biol Chem, 302(2):111082(2025) [pubmed]

submitter keyword: LC-MSMS

Borden Lacy
contact affiliation	Department of Biochemistry, Vanderbilt University
contact email	borden.lacy@vanderbilt.edu
lab head
carla.oneale@vanderbilt.edu carla.oneale@vanderbilt.edu
contact affiliation	Vanderbilt University
contact email	carla.oneale@vanderbilt.edu
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD070923

PRIDE project URI

• PRIDE
  1. PXD070923
     1. Label: PRIDE project
     2. Name: Nanobodies against Clostridioides difficile CDTb provide a toolkit for potent toxin neutralization and highly sensitive quantitation