Depolarization of polymorphonuclear leukocytes by Porphyromonas (Bacteroides) gingivalis 381 in the absence of respiratory burst activation

Abstract

Bacteroides spp. may contribute to the chronicity of mixed infections by affecting the normal functions of polymorphonuclear leukocytes (PMN). This study evaluated the physiologic and biochemical responses of human peripheral blood PMN to a variety of strains of the oral periodontal pathogen Porphyromonas (Bacteroides) gingivalis. Strain 381 and ATCC type strain 33277 caused rapid and lasting depolarization of the electrochemical potential that exists across the PMN membrane by a mechanism that was independent of activation of the pertussis toxin-sensitive G protein or protein kinase C. Membrane depolarization did not initiate increases in intracellular calcium or respiratory burst activation, and activity was not inhibited by surface proteolysis or sugars. However, membrane depolarization was associated with inhibition of PMN responses to the chemotactic peptide N-formylmethionyl leucyl phenylalanine. Membrane-depolarizing activity was isolated with the outer membrane of strain 381 by surface extraction of the bacteria by using Zwittergent 3,14, followed by Sephacryl S-200 gel filtration chromatography. The partially purified outer membrane components were heat stable, were not inhibited by tosyl-lysine chloromethyl ketone, and inhibited N-formylmethionyl leucyl phenylalanine-stimulated superoxide production. The results suggest that outer membrane components of P. gingivalis 381 and 33277 have porinlike activity that can depolarize PMN membranes and immobilize PMN responses to chemotactic peptides. This may prove to be an important virulence characteristic of these strains. * Corresponding author. t Present Address: Department of Periodontics, University of Texas Health Sciences Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7894.cidal respiratory burst. The purpose of this study was to characterize the physiological and biochemical responses of human PMN to selected strains of the periodontal pathogen P. gingivalis to gain an insight into how the presence of this microorganism may affect PMN and, subsequently, periodontal disease progression. MATERIALS AND METHODSBacterial strains and growth conditions. P. gingivalis 381, ATCC 33277, W83, and W50 and rifampin-resistant 3079.03-Rl were grown to the late-logarithmic-early-stationary phase in prereduced Schaedler broth (Oxoid, Basingstoke, England) containing hemin at 10 ,ug-ml-'. The purity of cultures was ascertained by phase-contrast microscopy and Gram staining and confirmed by aerobic and anaerobic subculturing on blood agar plates, followed by biochemical analysis using API ZYM chromogenic substrates (Analytab Products, Plainview, N.Y.). Cells were harvested by centrifugation, washed twice in sterile, isotonic saline, lyophilized, and stored at -20°C. Immediately prior to use, bacterial samples were suspended at 1 mg (dry weight) ml-' in Kreb-Ringer phosphate, pH 7.4 (KRP) and dispersed by vortexing and intermittent 30-s bursts of sonication at 50 W at 4°C in a Braun-sonic 1510 homogenizer (B. Braun, AG, Melsungen, Germany) until a sing...

Cytopathic Effects of the Major Surface Protein and the Chymotrypsinlike Protease ofTreponema denticola

“…The porin activity of rMsp was essentially identical to that of native Msp in the black lipid bilayer system. When tested in HeLa cells, the high conductance events associated with rMsp incorporation into the cell membrane were of shorter duration than those reported previously for native Msp (56). We have not detected rMsp in the oligomeric form typical of native Msp (17).…”

“…Cytopathic effects on eukaryotic cells due to translocation of bacterial porin-like molecules to the cell membrane have been reported for Neisseria gonorrhoeae (28), Salmonella typhimurium (19), Porphyromonas gingivalis (56), and Eikenella corrodens (62). A recent study implicated bacterial porins in bone resorption (50), presumably through modulation of proinflammatory cytokines (31).…”

“…Compared with the purified native and recombinant molecules, higher concentrations of the Msp complex were required for cytotoxicity. This apparently modulated effect could be due to one or more factors, including the formation of inactive Msp aggregates or some other artifact of the lengthy incubation protocol used during preparation of the Msp complex (56).…”

Life Below the Gum Line: Pathogenic Mechanisms ofPorphyromonas gingivalis

“…Succinate may act by reducing the intracellular pH of neutrophils (216). P. gingivalis can also immobilize PMN responses to chemotactic peptides by depolarizing PMN membranes (176). This activity is associated with an outer membrane protein of 31.5 kDa, which may be a porin.…”

Pathogenicity and virulence of black-pigmented Gram-negative anaerobes

“…The trypsin-like enzymatic activity of P. gingivalis has been shown to interfere with these morphological changes involved in chemotaxis [85,86]. The avirulent P. gingivalis strains 381 and ATCC 33277, but not the virulent strains W83 and W50, have a porin-like activity that can depolarize PMN membranes and immobilize PMN responses to chemotactic peptides [87]. P. gingivalis also affects chemotaxis induced by complement activation.…”