Isolation of a chymotrypsinlike enzyme from Treponema denticola

Abstract

A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, al-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50°C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction. * Corresponding author.35405 that has the ability to hydrolyze many functionally important serum and tissue proteins. MATERIALS AND METHODSBacterial strains and culture conditions. T. denticola ATCC

Mutagenesis of outer membrane virulence determinants of the oral spirochete Treponema denticola

“…Chymotrypsin-like protease activity associated with T. denticola outer membranes was due to CTLP [17,18]. As expected, and as shown in Table 1, strain CKE had greatly reduced enzymatic activity against SAAPNA, con¢rming that CTLP accounted for most of the chymotrypsin-like protease activity of T. denticola.…”

“…Along with the increased total CTLP activity in MHE, its protein pro¢le was suggestive of a defect in localization of the enzyme. When prepared for SDS-PAGE in the absence of protease inhibitors active against CTLP, most of the cellular proteins visible on stained gels [17]. No proteins with signi¢cant homology to the deduced 43 kDa product of ORF2 have been reported [9], but it clearly was closely linked both genetically and functionally to CTLP activity.…”

“…Chymotrypsin-like activity of T. denticola cells and supernatants of cultures grown to 5U10 W ml 3I were assayed using the chromogenic substrate succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (SAAPNA), as described previously [17].…”

Purification and characterization of a 65‐kilodalton diisopropylfluorophosphate‐binding protein in the outer membrane of Fusobacterium nucleatum Fev1

“…to depend on the presence of peptides in the medium for growth (5), An amino acid and peptide supply can occur naturally in the gingival pockets through protease action, which is well known for several oral spirochetes (6,7) and bacteroides (8,9) including B. gingivalis (10), Thus binding of fusobacteria to such protease-producing bacteria would be very profitable from a metabolic point of view. No extracellular proteolytic activities has been found originating from the strains of/^ nucleatuni tested by us (5), On the other hand we found recently that some outer membrane proteins oi F. nucleatum bound covalently to diisopropylfluorophosphate (DFP) (11), a strong serine protease inhibitor.…”

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola

“…Of these, prolyl-phenylalanine speci¢c protease activity is well characterized and is considered to be associated with the virulence of this microorganism. This protease has been described as a chymotrypsin-like protease [20]. The native form has a molecular mass of approximately 100 kDa and consists of three proteins of molecular masses 72, 43, and 38 kDa (or 28 kDa) [13,20,21].…”