Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization

Abstract

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 NaP -tosyl-L-lysine chloromethyl ketone and benzamidine, the metabolic inhibitor M-chlorophenylhydrazone, 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°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. * Corresponding author. and Gharbia (45) clearly demonstrate that heme modulates P. gingivalis virulence. Kay et al. (23) have demonstrated that their P. gingivalis W50 strain also possessed hemolytic activity, which appeared to be concentrated in the extracellular vesicles. The report presented here describes a putative hemolysin which is sensitive to-SH-containing molecules and appears to have a close association with the outer membrane and outer membrane vesicles. MATERIALS AND METHODS Culture conditions. P. gingivalis W50, W83, ATCC 33277, and A7A1-28 were used in this study. For growth, the cells were plated to enriched Trypticase soy agar or were grown in 2.1% (wt/vol) Mycoplasma broth (BBL, Becton Dickinson, Cockeysville, Md.) supplemented with 5 ptg (wt/vol) of heme per ml. All cultures were incubated in a Coy anaerobic chamber (85% N2, 10% H2, and 5% C02) maintained at 37°C. Cultures were incubated for 24 h or for times appropriate for the experimental design and were harvested and fractionated as described below. Escherichia coli HB101 was used as a hemolysin-negative control. Cell fractionation. For localization of the P. gingivalis hemolysin, cultures were grown, harvested by centrifugation, and washed twice with 3 mM sodium citrate-0.9% NaCl buffer (pH 6.8) (NCN buffer). Cell envelope, outer membrane vesicles, spent growth supernatant, and solublevesicle supernat...

Purification and characterization of hemolysin from Porphyromonas gingivalis A7436

“…These observations point to the fact that hemolytic activity and RGP activity are distinct from each other. The expression of P. gingivalis hemolytic activity has shown to be regulated by iron [3]. Excess iron has shown to depress hemolytic activity [3,6], while addition of the iron-chelator BPD increases hemolytic activity [3,6].…”

“…The expression of P. gingivalis hemolytic activity has shown to be regulated by iron [3]. Excess iron has shown to depress hemolytic activity [3,6], while addition of the iron-chelator BPD increases hemolytic activity [3,6]. Genco et al [13] have reported a higher hemolytic activity in a hemin uptake de¢cient mutant of P. gingivalis as compared to the wild-type strain.…”

Isolation and characterization of hemolysin activated by reductant from Prevotella intermedia

“…The gingival pocket is normally at neutral pH, however P. intermedia grown on agar is in an acidic environment generated by main end products such as acetic acid and succinic acid. Iron limitation resulted in signi¢cantly higher hemolysin production of P. gingivalis [9]. A similar e¡ect of iron is known for diphtheria toxin, Pseudomonas aeruginosa exotoxin A, and other toxins [24].…”

“…Hoshi et al [13] reported that the mode of action of the hemolysin from P. gingivalis was 'hotcold' lysis, similar to hemolysin from Leptospira canicola [11], Staphylococcus aureus [23] or Clostridium welchii [12]. However, hemolytic activity by prevolysin O did not increase with chilled treatment after incubation at 37 ‡C for 1 h. Some hemolytic substances from Gram-negative bacteria are activated by divalent cations [9]. The present hemolysin from P. intermedia did not possess such a property.…”

“…Hemolysin is produced by a large number and variety of Gram-positive and -negative bacteria and has been implicated as a virulence factor [7^12]. There are several studies that reported that oral bacteria related to periodontitis such as P. gingivalis [9,13], Treponema denticola [10], Actinobacillus actinomycetemcomitans [14] and Prevotella spp. [15,16] have some hemolytic substances.…”