Heterogeneity of Hemolysin Genes in Prevotella intermedia

Prevotella intermedia is a gram-negative anaerobe that has been implicated in the pathogenesis of periodontal diseases. The organism possesses hemolytic activity that has been associated with its virulence. Two putative hemolysin genes, phlA and phlB, have been identified in some P. intermedia strains. Objectives: (a) To express the putative hemolysin genes, phlA and phlB, in Escherichia coli; (b) To determine the distribution of hemolysin genes and their associated hemolytic activity among different oral isolates of P. intermedia. Methods: PhlA and phlB of P. intermedia ATCC 25611 were amplified by PCR using primers designed from the database of P. intermedia 17 (TIGR). These amplified genes, singly or in combination, were cloned into pQE plasmids and transformed into E. coli M15. Zymogram analysis for the presence of hemolytic activity was performed by SDS-PAGE. The hemolytic activity of oral isolates of P. intermedia was determined by the hemolysin tube assay. Results: Both putative hemolysin genes phlA and phlB, which encode a 38.7 and 29.9 kDa proteins respectively, were over-expressed upon induction by IPTG. Transformants containing these genes showed hemolytic activity. Based on the intensity of hemolytic activity expressed and sequence homology to phlA and phlB, P. intermedia strains were classified into four groups (Types 1, 2, 3, and 4) with the ranking order of Type 2>3>1= 4, in hemolytic activity. Of the 46 oral isolates tested in this study, 31 isolates belonged to Type 1 whereas the rest was classified as Type 2. The hemolytic activity among all test P. intermedia oral isolates was enhanced by the addition of 20 mM DTT or EDTA. Conclusion: Two putative hemolysin genes (phlA and phlB) of P. intermedia were expressed in E. coli. These recombinant proteins exhibited hemolytic activity in vitro. There were at least 4 genotypes of phl found in P. intermedia isolates.