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Characterization of emb, a gene encoding the major adhesin of Streptococcus defectivus.

ABSTRACT: Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins.

SUBMITTER: Manganelli R

PROVIDER: S-EPMC96276 | biostudies-literature | 1999 Jan

REPOSITORIES: biostudies-literature

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Characterization of emb, a gene encoding the major adhesin of Streptococcus defectivus.

Manganelli R R van de Rijn I I

Infection and immunity 19990101 1

Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis ...[more]

PMID: 9864195

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Project description:Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole-cell protein of S. suis type 2 and used to screen an S. suis gene library in an effort 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 45-kDa protein was present on a 1.6-kb pair DraI region on the cloned chromosomal fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 448 amino acid residues with a calculated molecular 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 sequence is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional domains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated the GDH of S. suis. Hybridization studies showed that the gene is conserved among the S. suis type 2 strains tested. Antiserum raised against the purified recombinant protein was reactive with a protein of the same molecular size 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 infected with a virulent strain of S. suis type 2, suggesting that the protein might serve as an antigen of diagnostic importance to detect S. suis infection. Activity staining showed that the S. suis GDH activity is NAD(P)H dependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of S. suis utilizes L-glutamate rather than alpha-ketoglutarate as the substrate. Highly virulent strains of S. suis type 2 could be distinguished from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We examined the cellular location of the protein using a whole-cell enzyme-linked immunosorbent assay and an immunogold-labeling technique. Results showed that the S. suis GDH protein is exposed at the surface of intact cells.

Dataset Information

Characterization of emb, a gene encoding the major adhesin of Streptococcus defectivus.

Publications

Characterization of emb, a gene encoding the major adhesin of Streptococcus defectivus.

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