The isolation, characterization and cloning of three novel peptidases from streptococcus gordonii
Goldstein, Jason Marc
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Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora yet is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Colonization and adherence mechanisms have been identified, yet factors necessary to sustain growth remain unidentified. Strain FSS2 produced three extracellular aminopeptidase activities during growth in neutral pHcontrolled batch cultures. The first included a serine-class dipeptidyl-aminopeptidase, an x-Pro DPP (Sg-xPDPP) found as an 85 kDa monomer by SDS-PAGE while appearing as a homodimer under native conditions. Kinetic studies indicated a unique and stringent x- Pro specificity comparable to the DPPIV/CD26 and lactococcal x-Pro DPP families. Isolation of the full-length gene uncovered a 759-amino acid polypeptide with a mass of 87,115 Da and theoretical pI of 5.6. Significant homology was found with PepX gene family members from Lactobacillus ssp. and Lactococcus ssp., and putative streptococcal x-Pro DPPs. The second activity was a putative serine-class arginine aminopeptidase (Sg- RAP) with some cysteine-class characteristics. It was found as a protein monomer of 70 kDa under denaturing conditions. Nested PCR cloning enabled the isolation of a 324 bplong DNA fragment encoding the protein’s 108 amino acid N-terminus. Culture activity profiles and N-terminal sequence analysis indicated the release of this protein from the cell surface. Homology was found with a putative dipeptidase from Streptococcus pyogenes and non-specific dipeptidases from Lactobacillus heleveticus and Lactococcus lactis. The third peptidase belonged to an extracellular, metallo-class dipeptidase found as a 55 kDa monomer as determined by SDS-PAGE and gel filtration analysis. Kinetic studies indicated degradation of various hydrophobic dipeptides except for an x-Pro sequence. Lesser activity was detected against the N-terminus of hydrophobic tripeptides. Isolation and sequence analysis of the full-length gene indicated a 467-amino acid polypeptide with a mass of 51,114 Da and theoretical pI of 4.8. Homology was found with the PepV gene family coding for cytoplasmic, non-specific dipeptidases from Lactobacillus and Lactococcus ssp. Collectively, these aminopeptidases may serve as critical factors during arginine acquisition, degradation of proline peptides, hydrolysis of di-, tri- and oligopeptides and maintenance of amino acid pools. During SBE pathology this peptidase system would serve to sustain growth in vivo, functioning in proteolysis of host protein and potentially modulating biologically active peptides.