Structural-functional studies of gingipain biogenesis and localization

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 examine the pathogenic potential of different HA variants of Kgp expressed by P. gingivalis strains. 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. Project Number: 1R56DE034451-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Dental and Craniofacial Research (NIDCR) | Principal Investigator: Hansel Fletcher (+1 co-PI) | Institution: LOMA LINDA UNIVERSITY, Loma Linda, CA | Award Amount: $358,823 | Activity Code: R56 | Study Section: Oral, Dental and Craniofacial Sciences Study Section[ODCS] View on NIH RePORTER: https://reporter.nih.gov/project-details/11409335