Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2

Given the lack of effective vaccines to control Streptococcus suis infectio n and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole cell pro tein of S. suis type 2 and used to screen an S. suis gene library in an eff ort to identify protective antigen(s) and antigens of diagnostic importance . A clone that produced a 45-kDa S. suis-specific protein was identified by Western blotting. Restriction analysis showed that the gene encoding the 4 5-kDa protein was present on a 1.6-kb pair DraI region on the cloned chromo somal fragment. The nucleotide sequence contained an open reading frame tha t encoded a polypeptide of 448 amino acid residues with a calculated molecu lar mass of 48.8 kDa, in close agreement with the size observed on Western blots. A GenBank database search revealed that the derived amino acid seque nce is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional do mains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated t he GDH of S. suis. Hybridization studies showed that the gene is conserved among the S. suis type 2 strains tested. Antiserum raised against the purif ied recombinant protein was reactive with a protein of the same molecular s ize as the recombinant protein in S. suis strains, suggesting expression of the gene in all of the isolates and antigenic conservation of the protein. The recombinant protein was reactive with serum from pigs experimentally i nfected,vith a virulent strain of S. suis type 2, suggesting that the prote in might serve as an antigen of diagnostic importance to detect S. suis inf ection. Activity staining showed that the S. suis GDH activity is NAD(P)H d ependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of S. suis utilizes L-glutamate rather than alpha -ketoglutarate as th e substrate. Highly virulent strains of S. suis type 2 could be distinguish ed from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We exa mined the cellular location of the protein using a whole-cell enzyme linked immunosorbent assay and an immunogold-labeling technique. Results showed t hat the S. suis GDH protein is exposed at the surface of intact cells.