Activities and Partial Purification of Extracellular Proteases of Bacteroides nodosus from Virulent and Benign Footrot

Abstract

In an attempt to differentiate virulent and benign strains of B. nodosus, the extracellular proteolytic activity of these cultures was assayed with elastin, casein and hide powder azure, and the stability to heating at 55°C was determined. Broth cultures of both strains hydrolysed 125I-labelled elastin, indicating that this activity is not a unique marker of virulence. When cultures were grown in Trypticase-arginine-serine broth medium modified by omitting Na2C03 and thioglycollic acid, the total proteolytic activity and its stability at 55°C could be used to differentiate isolates causing virulent or benign footrot lesions. However, when other broth cultures were used, these parameters could no longer be used to make such a distinction. The proteases of a virulent and benign strain of B. nodosus were partially purified and characterized. Four to five closely related proteases were detected by polyacrylamide gel electrophoresis at pH 8·8 in both types of isolates. The proteases are serine-type enzymes requiring a divalent metal ion such as calcium for activity. The proteases of the benign strain were somewhat less stable to heat than the enzymes of the virulent strain. Differences in the relative mobilities of the proteases of virulent and benign strains of B. nodosus, on electrophoresis at pH 8'8, suggest that this property may be used to distinguish virulent and benign strains.

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

“…The tissue damage which accompanies the infection indicates the involvement of extracellular proteinases in the degradation of host tissue [5]. D. nodosus secretes a number of extracellular proteinases which readily degrade the proteins present in skin and hoof of sheep, including elastin [6][7][8], keratin, fibrinogen and collagen [8]. Furthermore, virulence of D. nodosus correlates with differences in the properties of the proteinases, and these have been used in diagnostic tests to distinguish virulent and benign strains of the organism [7,[9][10][11].…”

“…Furthermore, virulence of D. nodosus correlates with differences in the properties of the proteinases, and these have been used in diagnostic tests to distinguish virulent and benign strains of the organism [7,[9][10][11]. Electrophoretic analyses of D. nodusus culture supernatants of virulent and benign strains showed the presence of four or five proteinases with pl values ranging from 4.9 to 6.0 [12] and apparent molecular masses of 40-42 kDa [6]. The partial purification of these 'acidic' proteinases has been described [6,13].…”

Variation in the structural subunit and basal protein antigens of Bacteroides nodosus fimbriae

“…4). B. nodosus is known to produce a wide range of extracellular proteases (7,15,25,26) isolates from serotypes Al and Fl. In some cases, however, the differences may be quite pronounced, as in isolates VCS 1133 (serotype A2) and VCS 1071 (serotype E2) (Fig.…”

Expression and secretion of heterologous proteases by Corynebacterium glutamicum

“…Measurement of proteolytic activity in culture supernatants. 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).…”