Atsuyo Baba

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

ABSTRACT Arg-gingipain (Rgp) and Lys-gingipain (Kgp) are Porphyromonas gingivalis cysteine proteinases implicated as major virulence factors in pathologies of periodontitis. We purified a 660-kDa cell-associated gingipain complex existing as a homodimer of two catalytically active monomers which comprises their catalytic and adhesin domains. Electron microscopy revealed that the complex was composed of a globular particle with a 10-nm external diameter possessing one or two electron-dense hole-like structures. Two-dimensional gel electrophoresis and immunoblot analyses revealed the association of lipopolysaccharide (LPS) with the catalytic domains and a hemagglutinin domain, Hgp44, of Rgp and Kgp in the complex. The complex significantly degraded human type I collagen and elastin and strongly disrupted viability of human gingival fibroblasts and umbilical vein endotherial cells with an efficiency which was higher than that of the monomeric gingipains. The native complex produced only a small amount of nitrogen dioxide, tumor necrosis factor alpha, and interleukin-6 by macrophages, whereas the heat-denatured complex resulted in increased production. Inhibition of the proteolytic activities of the gingipain complex did not up-regulate the cytokine production, indicating that the functional domains in LPS are structurally masked by the complex proteins. These results indicate the importance of the complex in evasion of host defense mechanisms as well as in host tissue breakdown.

Roles for Arg- and Lys-gingipains in the disruption of cytokine responses and loss of viability of human endothelial cells by Porphyromonas gingivalis infection.

Abstract Accumulating evidence indicates that periodontal disease is associated with human cardiovascular diseases. The periodontal pathogen Porphyromonas gingivalis was shown to be present in atherosclerotic plaques in addition to periodontal pockets. This bacterium is known to produce two individual cysteine proteinases, Arggingipain (Rgp) and Lysgingipain (Kgp). Here we show that these two enzymes are responsible for either the disruption of cytokine responses in human umbilical vein endothelial cells (HUVEC) to the bacterium infection or the loss of cell viability. The expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA in HUVEC was greatly induced when infected with the wildtype strain, nevertheless, their protein levels in the culture medium were markedly decreased. This decrease was completely abolished in the cells infected with the Rgp/Kgpnull mutant, but not in either the Rgp or Kgpnull mutants. Loss of the adhesion activity and viability of HUVEC were greatly induced by the culture supernatant of the wildtype strain and strongly inhibited by either a combination of the Rgp and the Kgpspecific inhibitors or the deficiency of the Rgp and Kgpencoding genes. These findings indicate that P. gingivalis modulates the cytokine response in the cells and disrupts the adhesion activity and the viability through the cooperative action of Rgp and Kgp and thereby may contribute to pathogenesis of cardiovascular diseases as well as periodontal disease.

Arg-Gingipain Is Responsible for the Degradation of Cell Adhesion Molecules of Human Gingival Fibroblasts and Their Death Induced by Porphyromonas gingivalis

Abstract Arggingipain (Rgp) and Lysgingipain (Kgp) are two major cysteine proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis, which has been shown to act as major pathogen in the development and progression of periodontal diseases. These enzymes are also important for this organism to proliferate and survive in periodontal pockets. Here we show that Rgp is responsible for the disruption of fibronectinintegrin interactions in human gingival fibroblasts by P. gingivalis. Fibroblasts incubated with the culture supernatant of P. gingivalis showed a timedependent loss of the adhesion activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting revealed that fibronectin and integrin subunits ?2, ?1 and ?3 in the fibroblast culture largely disappeared with the treatment. The detached cells became committed to death by disruption of contacts between adhesion molecules. In contrast, the culture supernatants from the Rgpdeficient mutants produced no significant changes in either cell adhesion or viability. Prior treatment of the culture supernatant of P. gingivalis with an Rgp inhibitor, but not a Kgp inhibitor, strongly inhibited the detachment of fibroblasts followed by cell death. These results suggest that Rgp disrupts the integrinfibronectin interactions in fibroblasts, thereby contributing to the damage of periodontal tissues in periodontal diseases caused by P. gingivalis.

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: Identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes

Abstract Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis, an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA, kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA-deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus, whereas the kgp-null and rgpA rgpB-deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA, kgp, and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

A novel, potent dual inhibitor of Arg-gingipains and Lys-gingipain as a promising agent for periodontal disease therapy

The periodontal pathogen Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg‐gingipain (Rgp) and Lys‐gingipain (Kgp). Growing evidence indicates that these 2 types of gingipains synergistically contribute to the entire virulence of the organism and increase the risk of periodontal disease (PD) by disrupting the host immune system and degrading the host tissue and plasma proteins. Therefore, a dual inhibitor of both gingipains would have attractive clinical potential for PD therapy. In this study, a novel, potent, dual inhibitor of Rgp and Kgp was developed through structure‐based drug design, and its biological potency was evaluated in vitro and in vivo. This inhibitor had low nanomolar inhibitory potency (Ki=40 nM for Rgp, Ki=0.27 nM for Kgp) and good selectivity for host proteases and exhibited potent antibacterial activity against P. gingivalis by abrogating its manifold pathophysiological functions. The therapeutic potential of this inhibitor in vivo was also verified by suppressing the vascular permeability that was enhanced in guinea pigs by the organism and the gingival inflammation in beagle dog PD models. These findings suggest that a dual inhibitor of Rgp and Kgp would exhibit noteworthy anti‐inflammatory activity in the treatment of PD.—Kataoka, S., Baba, A., Suda, Y., Takii, R., Hashimoto, M., Kawakubo, T., Asao, T., Kadowaki, T., Yamamoto, K. A novel, potent dual inhibitor of Arg‐gingipains and Lys‐gingipain as a promising agent for periodontal disease therapy. FASEB J. 28, 3564–3578 (2014). www.fasebj.org

Isolation and characterization of a novel and potent inhibitor of Arg-gingipain from Streptomyces sp. strain FA-70.

Abstract Arg-gingipain (Rgp) is a major cysteine proteinase produced by the oral bacterium Porphyromonas gingivalis, which is a major pathogen of advanced periodontal diseases. This enzyme is important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. The development of Rgp inhibitors thus provides new therapeutic approaches to periodontal diseases. In this study, we first isolated and purified a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. This compound was found to be an antipain analog composed of phenylalanyl-ureido-citrullinyl-valinyl-cycloarginal (C27H43N9O7). The Ki value was calculated to be 4.510 9 M when benzyloxycarbonyl-phenylalanylarginine-4-methly-coumaryl-7-amide was used as a substrate. This compound also inhibited cathepsins B, L, and H, though their Ki values were much higher than that of Rgp. FA-70C1 had little or no inhibitory activity on Lys-gingipain, another cysteine proteinase of P. gingivalis. The Rgp-induced degradation of various human proteins was completely blocked by this inhibitor. Disruption of both the bactericidal activity of polymorphonuclear leukocytes and the viability of human fibroblasts and umbilical vein endothelial cells induced by the culture supernatant of P. gingivalis was suppressed by the inhibitor in a dose-dependent manner. The enhancement of vascular permeability induced by in vivo administration of the culture supernatant of P. gingivalis was strongly inhibited by the inhibitor. Furthermore, the growth of P. gingivalis was suppressed by FA-70C1 in a dose-dependent manner. These results strongly suggest that FA-70C1 is a useful tool to prevent the virulence of P. gingivalis.