Biochemical and Functional Properties of Lysine-Specific Cysteine Proteinase (Lys-Gingipain) as a Virulence Factor of Porphyromonas gingivalis in Periodontal Disease

Abstract

The oral anaerobic bacterium Porphyromonas gingivalis has been implicated as a major etiologic agent of progressive periodontal disease. A novel lysine-specific cysteine proteinase, termed "Lys-gingipain," was purified from the culture supernatant of the Arg-gingipain-deficient mutant of P. gingivalis (KDP112) by a simple method including immunoaffinity chromatography. The purified enzyme was found to be composed of a single polypeptide of Mr=51,000. Analysis of the enzymatic properties revealed several distinctive features of this enzyme. The proteolytic activity was remarkably activated by thiol-reducing agents and inhibited by idoacetamide, idoacetic acid, and leupeptin. The enzyme was also inhibited by the chloromethyl ketones of tosyl-L-lysine and tosyl-L-phenylalanine. However, internal protease inhibitors, such as cystatins and alpha1-antichymotrypsin, had no effect on the activity, suggesting its resistance to normal host defense systems in vivo. Despite its narrow specificity for synthetic substrates containing Lys in the P1 site, the enzyme extensively degraded human type I collagen and immunoglobulins G and A (both serum and secretory types). Most important, the enzyme was able to disrupt the functions of polymorphonuclear leukocytes, as shown by its inhibitory effect on the generation of active oxygen species from the activated cells. These results suggest that Lys-gingipain, like Arg-gingipain, plays a crucial role as a virulence factor from P. gingivalis in the development of periodontal disease via the direct destruction of periodontal tissue components and the disruption of normal host defense mechanisms.

Metabolic Network Model of a Human Oral Pathogen

“…A preference for peptides is probably a consequence of the organism's gingipain enzymes, known to be highly proteolytic (93). As a result, the organism can take full advantage of the degradation of host tissues by metabolizing the peptide fragments (1). Some by-products of amino acid catabolism, such as isobutyrate, are thought to be mediating the interaction with other species.…”

A Functional Virulence Complex Composed of Gingipains, Adhesins, and Lipopolysaccharide Shows High Affinity to Host Cells and Matrix Proteins and Escapes Recognition by Host Immune Systems

“…Interestingly, the C-terminal adhesin domains of rgpA and kgp are highly homologous, although the propeptide and proteinase domains have no sequence similarity (21,33,36). Using various Rgp-and/or Kgp-deficient mutants as well as soluble gingipains purified from the culture supernatant of P. gingivalis strains, the virulence of the bacterium has been shown to be exclusively attributable to gingipains (1,20,(26)(27)(28). These include extensive degradation of various host proteins including collagen, fibronectin, and fibrinogen (1,20,34), cytokines such as interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-␣) (7,10,31), complement factors C3 and C5 (47), and immunoglobulins (1,20); disruption of the bactericidal activity of polymorphonuclear leukocytes (1,20,26); and strong induction of human fibroblast (3,4) and human umbilical vein endothelial cell (HUVEC) (5) death.…”

vimA Gene Downstream of recA Is Involved in Virulence Modulation in Porphyromonas gingivalis W83

“…Several reports (reviewed in reference 26) have documented the multiple effects of proteases, which, in addition to being essential for growth, play a role in complement and immunoglobulin degradation, inactivation of cytokines and their receptors, platelet aggregation, attenuation of neutrophil antibacterial activities, increase in vascular permeability, and prevention of blood clotting. Recently, the proteases which are not coordinately regulated (63) have been shown to act as major processing enzymes for various cell surface proteins, including individual proteases (2,4,21). …”