An investigation into the use of SDS-PAGE of cell surface extracts and proteolytic activity to differentiate Prevotella nigrescens and Prevotella intermedia

Abstract

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species (Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAPPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32-149 and 3-16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.

Proteolysis and utilization of albumin by enrichment cultures of subgingival microbiota

“…That most of the proteinase activity occurred cell‐associated is a common observation for periodontal microflora ( 6, 11). Most of the proteolytic activity in the rumen and the large intestine also resides in the bacterial fraction ( 15, 22).…”

Carious Dentine Provides a Habitat for a Complex Array of Novel Prevotella -Like Bacteria

“…The genus Prevotella was separated from the Bacteroidiaceae in 1990 (23) and presently comprises diverse organisms that constitute the largest group of medically important gram-negative anaerobes. Cultured Prevotella organisms have been implicated in a wide range of infections, and some members of the genus express marked proteolytic activity and hemolysins (3)(4)(5)8). Typically, anaerobic culture significantly underestimates the abundance of Prevotella and other anaerobes at sites of infection.…”

Genetic heterogeneity in Prevotella intermedia, Prevotella nigrescens, Prevotella corporis and related species isolated from oral and nonoral sites

“…Previous studies have found no consistent differences between P. intermedia and P. nigrescens and observable phenotypic properties ( 4, 6–8, 10, 11, 19, 39). Nor did this study reveal any relationship between phenotypic characteristics and ribotype of either P. intermedia or P. nigrescens : there is no relationship between fibril type ( 7), coaggregating ability ( 8), adhesion to epithelial cells ( 10 and unpublished results) or hemagglutinating ability (unpublished results), and particular ribotypes.…”