Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization.
Porphyromonas gingivalis W50, W83, A7A1-28, and ATCC 33277 were investigated for their abilities to lyse sheep, human, and rabbit erythrocytes. All of the P. gingivalis strains studied produced an active hemolytic activity during growth, with maximum activity occurring in late-exponential-early-stationary growth phase. The enzyme was cell bound and associated with the outer membrane. Fractionation of P. gingivalis W50 localized the putative hemolysin almost exclusively in the outer membrane fraction, with significant hemolytic activity concentrated in the outer membrane vesicles. Ca2+ and Mg2+ ions significantly increased the expression of hemolytic activity. Hemolytic activity was inhibited by proteinase K, trypsin, the proteinase inhibitors Na-P-tosyl-L-lysine chloromethyl ketone and benzamidine, the metabolic inhibitor M-chlorophenyl-hydrazone, and iodoacetate. KCN and sodium azide (NaN3) only partially inhibited P. gingivalis hemolytic activity, while antiserum to whole cells of each of the P. gingivalis strains had a significant inhibitory effect on hemolytic activity. The P. gingivalis W50 hemolysin was inhibited by cysteine, dithiothreitol, and glutathione at concentrations of at least 10 mM; at low concentrations (i.e., 2 mM), dithiothreitol did not completely inhibit hemolytic activity. Heating to temperatures above 55 degrees C resulted in an almost complete inhibition of hemolytic activity. The effect of heme limitation (i.e., iron) on hemolysin production indicated that either limitation or starvation for heme resulted in significantly increased hemolysin production compared with that of P. gingivalis grown in the presence of excess heme.
Chu
L
L
Department of Periodontics, University of Texas Health Science Center, San Antonio 78284.
Bramanti
T E
TE
Ebersole
J L
JL
Holt
S C
SC
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001439:Bacteroides / Q000201:enzymology* / Q000502:physiology; D006418:Heme / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000378:metabolism* / Q000502:physiology; D006461:Hemolysis / Q000187:drug effects*; D006462:Hemolytic Plaque Technique; D006801:Humans; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D011817:Rabbits; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012756:Sheep; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D006460:Hemolysin Proteins; D009842:Oligopeptides; D015842:Serine Proteinase Inhibitors; D013439:Sulfhydryl Reagents; D006418:Heme