Characterization of a basic serine proteinase (pI ∼ 9.5) secreted by virulent strains of Dichelobacter nodosus and identification of a distinct, but closely related, proteinase secreted by benign strains

Abstract

An extracellular serine proteinase with a PI approximately 9.5 (referred to as 'basic proteinase') was purified to homogeneity, from strains of Dichelobacter nodosus that cause virulent foot-rot, by gel filtration of concentrated culture supernatant on Sephadex G-100 and chromatography on sulphopropyl-Sephadex C-25 at pH 8.6 D. nodosus strains that cause benign foot-rot do not secrete a corresponding basic proteinase with a pI of approximately 9.5. Benign strains secrete a closely related, but distinct, proteinase which has the same molecular mass and N-terminal sequences as the 'virulent' basic proteinase, but a lower pI of approximately 8.6. The basic proteinases from both strains appear to interact with other proteins present in the culture medium, which results in anomalous behavior on gel filtration. Pure D. nodosus 'virulent' basic proteinase has a molecular mass of 36 kDa and showed a low solubility at I < 0.05 precipitating quantitatively from solution as microcrystals. The proteinase shows optimal activity at pH 8.0 and is stable to heating to 55 degrees C for 30 min, but at higher temperatures activity is rapidly lost. Bivalent-metal ions (e.g. Ca2+) are required to maintain the structural integrity and stability of the proteinase; in the presence of EDTA or conditions that cause protein unfolding, the proteinase undergoes rapid and complete autolysis. Cleavage of oxidized insulin A- and B-chain showed that the basic proteinase has a broad specificity, including cleavage at lysine and arginine bonds.

S1 Pocket of a Bacterially Derived Subtilisin-like Protease Underpins Effective Tissue Destruction

“…Consistent with previous studies (10), these data reveal that the two proteases have similar specificity for the P1 residue in the peptide substrates tested and prefer cleaving after Leu over Phe. We also observed that BprV was more efficient than BprB at cleaving leucine-containing substrates (LAY, LAF, and LVY).…”

“…Proteolytic processing, a general feature of the biosynthesis of functional subtilisin-like proteases (9), is required to yield the final mature D. nodosus subtilase enzymes. This involves the removal of a signal sequence, a separate proregion, and the C-terminal domain (10,11).…”

Cloning, Expression, and Immunological Evaluation of Two Putative Secreted Serine Protease Antigens ofMycobacterium tuberculosis

“…Similarly, Plasmodium falciparum is thought to use secretory forms of serine proteases in erythrocyte invasion during the blood-borne stage of malaria (4,6). A secreted serine proteinase identified in the culture supernatant of virulent strains of Dichelobacter nodosus has also been associated with virulent foot-rot disease (26). Perhaps of most significance for a potential role of M. tuberculosis serine protease as a virulence factor is the recent demonstration that, in Aspergillus-related lung disease, a secreted serine proteinase was shown to be capable of hydrolyzing the major structural barriers of the lung (22).…”

Expression and secretion of heterologous proteases by Corynebacterium glutamicum

“…The proteolytic activity of the culture supernatants was initially assessed by hide powder azure assays (8). The protease in culture supernatants was quantitated by spectrophotometrically measuring the esterase activity of the culture supernatants with ␣-N-benzoyl-L-tyrosine ethyl ester (BTEE) as previously described (7). The amount of protease produced was estimated from a standard curve constructed by measuring the hydrolysis of BTEE at different concentrations of purified D. nodosus basic protease in a standard assay.…”