Friday, May 16, 2025
5/16/2025
Structural studies of the OrfX-type progenitor toxin complex of botulinum neurotoxin - PROJECT SUMMARY Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus Clostridium, which are the causative agents of the neuroparalytic disease botulism. Nevertheless, type A and type B BoNTs (BoNT/A and BoNT/B) have been successfully used in clinic for a variety of therapeutic and aesthetic applications, while type E BoNT (BoNT/E) is currently in clinic trials. BoNTs are naturally produced by bacteria alongside several non- toxic neurotoxin-associated proteins (NAPs). These NAPs are believed to engage with BoNTs in the framework of progenitor toxin complexes (PTCs), which are high molecular weight multi-protein complexes. Accidental BoNT poisoning mainly occurs through oral ingestion of tainted foods, and NAPs are thought to protect BoNTs and facilitate their absorption during oral intoxication. There are two different types of PTC depending on the neurotoxin gene clusters: the HA-type PTC has four NAPs, including non-toxic non-hemagglutinin (NTNH) and three hemagglutinin proteins (HA17, HA33, and HA70), while the OrfX-type PTC has a different set of NAPs, including NTNH and four proteins with unknown function (OrfX1, OrfX2, OrfX3, and P47). Extensive structural and functional studies of the HA-type PTC, such as that of BoNT/A, demonstrate that the NAPs not only protect the inherently fragile BoNT/A against the hostile environment of the gastrointestinal (GI) tract, but also interact with host receptors to help BoNT/A pass through the intestinal epithelial barrier before being released into the general circulation. However, the OrfX-type PTC remains largely uninvestigated. The goal of this proposal is to elucidate the structure and function of the OrfX-type PTCs, including mapping the detailed protein-protein interaction network and understanding the specificity and regulatory mechanisms underlying PTC assembly. We will exploit a combination of cryogenic electron microscopy (cryo-EM) and X-ray crystallography, complemented with other biochemical and biophysical methods. To comply with the CDC Select Agent regulations, we will carry out all our studies using genetically inactivated BoNTs, and we will focus on three aims: (1) characterize the mechanisms underlying the assembly of the OrfXs/P47 complex; (2) determine the structures of the OrfXs/P47 complex and sub-complexes; and (3) characterize the potential regulatory factors and receptors of the OrfXs/P47 complex. Together, these proposed studies will advance our structural and mechanistic understanding of the OrfX-type BoNT complex, lay the knowledge basis for the development of new therapeutic approaches to treat and/or prevent botulism, and potentially shed light on the pathogenesis of other OrfXs/P47-associated oral bacterial toxins beyond BoNTs.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).