Structural-functional studies of gingipain biogenesis and localization

The National Institute of Dental and Craniofacial Research (NIDCR), a component of the Department of Health and Human Services National Institutes of Health, awarded Loma Linda University $631,170 on July 21, 2026, under the Oral Diseases and Disorders Research program (CFDA 93.121) to conduct structural-functional studies of gingipain biogenesis and localization in the context of periodontitis and Porphyromonas gingivalis pathogenesis.

The project will expand understanding of gingipains—cysteine proteases secreted by P. gingivalis via a Type IV secretion system—by conducting comprehensive assessment of gingipain structure-function properties, including biogenesis and specific bacterial surface localization. Research will determine post-translational sequential processing steps leading to formation of gingipain complexes on the bacterial surface, conduct comparative analysis of proteolytic activity and biological functions of single-chain pro-gingipains, and examine assembly of native non-covalent complexes composed of catalytic domains and hemagglutinin domains. The work addresses a significant gap in mechanistic understanding of virulence factors essential for therapeutic development, given that periodontitis is prevalent, currently lacks effective treatment, and has been linked to systemic diseases including atherosclerosis, rheumatoid arthritis, some cancers, diabetes, and Alzheimer's disease.

Work is performed at Loma Linda, California. The period of performance extends from July 21, 2026, to April 30, 2030.

Abstract

There is an urgent need to explore new therapeutic approaches in the management of periodontitis, a prevalent and chronic disease that currently lacks effective treatment. Further, a growing number of studies have linked periodontitis to systemic diseases such as atherosclerosis or rheumatoid arthritis, as well as some cancers, diabetes, and alzheimer's disease. Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is a keystone pathogen in microbial-induced periodontal disease. A comprehensive mechanistic understanding of some of its virulence factors is essential as the first step for therapeutic development. The bacterium uses a type IV secretion system to secrete an array of virulence factors, including gingipains [cysteine proteases - ARG-specific rgpa and rgpb, and LYS-specific KGP], which are essential for its pathogenicity. There is still a significant GAP in our understanding of the mechanism of post- translational proteolytic processing of progingipains and membrane surface assembly of non- covalent complexes composed of a catalytic domain and hemagglutinin (HA) domains. In addition, the role of HA domains in p. Gingivalis pathogenesis is unclear. Our objective is to expand the understanding of the gingipains' hemagglutinating/adhesive properties and the formation of their surface complexes. The specific aims of the current application are designed for a comprehensive assessment of the gingipain structure-function properties, including its biogenesis and specific localization. Experiments are outlined to determine the post-translational sequential processing steps leading to the formation of gingipain complexes on the bacterial surface. We will conduct a comparative analysis of proteolytic activity, independent biological functions of single-chain pro gingipains, and assembly of native non-covalent complexes of the catalytic domain with HA domains. We will determine the structure of non-covalent gingipain surface complexes. Finally, we will dissect the functional roles of distinct hemagglutinin/adhesin domains in p. Gingivalis adhesion and pathogenicity. A conceptual innovation is characterizing the structures of full-length gingipains and native rgpa-KGP membrane complexes. This will shed new light on the cooperative mechanism of proteolytic cleavage and adhesion/hemagglutination activity exerted by gingipains. Given their specific bacterial surface localization, we posit that only simultaneous inhibition of both properties will lead to the development of effective drugs against periodontitis and p. Gingivalis-driven systemic diseases.