The periodontopathogen Porphyromonas gingivalis is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that P. gingivalis contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from P. gingivalis displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of P. gingivalis to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene oxyR. The Dps-deficient mutants, including the dps single mutant and the ftn dps double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the dps single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.Atmospheric oxygen is metabolically converted to reactive oxygen species (ROS), including superoxide anion radical, hydrogen peroxide, hydroxy radical, and singlet oxygen, in bacterial cells. ROS are also generated by phagocytic host cells such as polymorphonuclear leukocytes and macrophages and attack invading bacterial cells. It is widely recognized that two cellular systems function to protect organisms from oxidative stresses (15,33). One is regulated by antioxidant enzymes in which molecular oxygen and ROS are diminished or eliminated (42). Superoxide dismutase (SOD), catalase, peroxidase, and oxidase are involved in this reaction. The other is catalyzed by endonucleases by which oxidatively damaged nucleic acids are repaired. This includes Escherichia coli exonuclease III and endonuclease IV (51). These two systems cooperatively function to minimize the detrimental effects of ROS upon cells, as evidenced by the presence of common regulatory genes such as oxyR (43).Porphyromonas gingivalis is a gram-negative obligate anaerobe belonging to the division Cytophagales (23). This bacterium is one of the organisms that is most strongly associated with chronic adult periodontitis and expresses numerous potential virulence factors, such as fimbriae, hemagglutinins, lipopolysaccharides, and various proteases that are capable of hydrolyzing collagen, immunoglobulins, iron-binding proteins, and complement factors (21, 27). P. gingivalis, by definition, cannot grow in aerobic conditions but exhibits a high degree of aerotolerance. This aerotolerance enables the organism to survive in periodontal pockets that are ...

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1. Three rhesus monkeys were trained to perform a rapid (greater than 100 degrees/s) and a slow (less than 100 degrees/s) wrist movement guided by a visual cue. While the monkey performed wrist flexion or extension from a neutral position, Purkinje cell (P-cell) discharges were recorded from intermediate and lateral parts of lobules IV--VI of the cerebellum. 2. By the visually guided movement, we could control the direction of the wrist movement; the holding position at three different angles of the wrist joint: neutral, about 30 degrees flexed, and extended; and the velocity in four ranges: a) 10--30, b) 30--100, c) 100--300, and d) 300-650 degrees/s. 3. From 92 P-cells that significantly increased or decreased the discharge rate of simple spikes with task performance, we selected 45 P-cells ("response-locked" cells) as related to the wrist movement by statistical analyses of temporal correlation of P-cell activities to wrist movement. The direction of the frequency modulation (increase or decrease) was in a nonreciprocal fashion with oppositely directed wrist movements (flexion or extension) in 90% of the response-locked P-cells. The maintained frequencies at three holding positions did not significantly differ. 4. Nineteen P-cells changed their spike frequencies temporally locked to both rapid and slow wrist movements. By the discharge pattern in relation to the rapid and slow movements, these cells were classified into two groups. Discharge pattern in group I P-cells (n = 5) conformed very well to that of velocity, and a linear correlation between the instantaneous increase of the discharge rate and velocity was observed in analyses of individual trials. Group II cells showed increase (n = 9) or decrease (n = 5) of firing rate (20--50 spikes/s) larger than group I cells (less than 10 spikes/s) as long as the wrist was moving, even with very slow velocity (less than 30 degrees/s. The correlations between the increase of the discharge rate and the velocity in individual trials were less clear in group II than in group I cells. 5. The present study suggests the importance of the cerebellar cortex in controlling the slow limb movement as well as the rapid movement. The selected P-cells in this study also suggested that the velocity or some dynamic aspect related to the velocity of limb movement is the major information among the dissociated motion parameters coded by the simple-spike frequencies of the P-cells in the cerebellar hemisphere. Whether the latter suggestion represents an essential characteristic of all limb movement-related P-cells or reflects only a feature of a special subgroup among the movement-related cells should be clarified in future experiments.

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.

SummaryEvidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet-rich plasma (PRP). Mechanism of P. gingivalis -induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg-gingipain (Rgp) and Lys-gingipain (Kgp) did not suppress P. gingivalis -induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis -induced platelet aggregation using washed platelets. Mutant analysis revealed that P. gingivalis -induced platelet aggregation in PRP depended on Rgp-, Kgp-and haemagglutinin A (HagA)-encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalisinduced platelet aggregation in PRP. P. gingivalis cellreactive IgG in plasma, and Fc γ γ γ γ RIIa receptor and to a lesser extent GPIb α α α α receptor on platelets were found to be a prerequisite for P. gingivalis -induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis .