Extracellular arginine aminopeptidase from Streptococcus gordonii FSS2

Abstract

Streptococcus gordonii is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), producing thrombus formation and tissue damage on the surfaces of heart valves. This is ironic, considering its normal role as a benign inhabitant of the oral microflora. However, strain FSS2 of S. gordonii has been found to produce several extracellular aminopeptidase- and fibrinogen-degrading activities during growth in a pH-controlled batch culture. In this report, we describe the purification, characterization, and partial cloning of a predicted serine class arginine aminopeptidase (RAP) with some cysteine class characteristics. Isolation of this enzyme by anion-exchange, gel filtration, and isoelectric focusing chromatography yielded a protein monomer of approximately 70 kDa, as shown by matrix-assisted laser desorption ionization, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Nested-PCR cloning enabled the isolation of a 324-bp-long DNA fragment encoding the 108-amino-acid N terminus of RAP. Culture activity profiles and N-terminal sequence analysis indicated the export of this protein from the cell surface. Homology was found with a putative dipeptidase from Streptococcus pyogenes and nonspecific dipeptidases from Lactobacillus helveticus and Lactococcus lactis. We believe that RAP may serve as a critical factor for arginine acquisition during nutrient stress in vivo and also in the proteolysis of host proteins and peptides during SBE pathology.

FIG. 1.

S. gordonii FSS2 growth and activity curve from a pH 7.0 controlled culture supplemented with 50 mM glucose and 3.5 mM arginine. Samples (1 ml) were removed from culture at fixed time points, and cells were removed from the medium by centrifugation (5 min; 4°C; 13,000 × g) followed by two washes and resuspension in the initial volume of unsupplemented medium. •, culture turbidity; ⧫, cell-free culture fluid; ▴, washed cells. Assays were performed as previously described using 30 μl of sample. OD₅₆₀, optical density at 560 nm; t, time.

FIG. 2.

SDS-PAGE of fractions from the purification of S. gordonii arginine aminopeptidase. Lanes 1 and 7, 15-μg molecular mass markers (phosphorylase b, 94 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa). The following lanes contained boiled, reduced samples: lane 2, 120-μg 80% ammonium sulfate precipitation; lane 3, 88 μg of flowthrough from DE52 anion exchange; lane 4, 42-μg peak from Superdex 75 gel filtration wash; lane 5, 34-μg eluted peak from Mono-Q; lane 6, 6 μg of purified RAP (arginine aminopeptidase) from Mono-P.

FIG. 3.

N-terminal sequence of S. gordonii arginine aminopeptidase (RAP) deduced from FSS2 genome. The single-underlined sequence represents the predicted transmembrane domain. The boldface letters mark a conserved signal peptide cleavage site for gram-positive bacteria. The double-underlined sequence represents the internal fragment used to search the electronic database and generate a 340-bp PCR product.