A 46-kDa hemolytic protein referred to as cystalysin, from Treponema denticola ATCC 35404, was characterized and overexpressed in Escherichia coli LC-67. Cystalysin lysed erythrocytes, hemoxidized hemoglobin to sulfhemoglobin and methemoglobin, and removed the sulfhydryl and amino group from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. With L-cysteine as substrate, cystalysin obeys Michaelis-Menten kinetics. Cystathionine and s-aminoethyl-L-cysteine were also substrates. Several of the small alpha amino acids were found to be competitive inhibitors of cystalysin. The enzymatic activity was increased by beta-mercaptoethanol and was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme with the activity of an alphaC-N and betaC-S lyase (cystathionase). Since high amounts of H2S have been reported in deep periodontal pockets, this metabolic enzyme from T. denticola may also function in vivo as an important virulence molecule.
AgNP-PMMA demonstrated a tremendously broad-spectrum and long-intermediate-term antimicrobial effect with comparable mechanical properties to control PMMA. Current efforts are focused on further improving mechanical properties by reducing AgNP loading and assessing fatigue properties.
A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H 2 S, NH 3 , and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: K m ؍ 3.6 mM and k cat ؍ 12 s ؊1. Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH 3 , pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by -mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N␣-ptosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5phosphate-containing enzyme, with activity of an ␣C-N and C-S lyase (cystathionase) type. Since large amounts of H 2 S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.
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...
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