Enzyme-linked immunosorbent assay for group A Streptococcal anti-DNase B in human sera, using recombinant proteins - Comparison to the DNA methyl green micromethod.

Among the four known Streptococcal nucleases comprising of DNase A, B, C and D; DNase B is the most common, and determination of the levels of antibody to DNase B (ADB) is often used to confirm a clinical diagnosis of Streptococcus pyogenes/group A Streptococcal (GAS) infection. The commonly used assays for antibodies that neutralize DNase B or streptolysin O activity use partially purified antigens that often fail to detect antibody changes subsequent to culture documented infections. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed employing his-tagged recombinant DNase B as plate antigen for comparison to the commonly used DNA methyl green micromethod (DMGM). DNAs from various Streptococcal species were screened for presence of dnaseB gene by PCR. Measurements of ADB in sera collected from subjects belonging to different ages, and ethnic groups were used to compare the two methods. dnaseB was not detected by PCR in DNA samples isolated from different strains of group B (GBS), C (GCS) and G (GGS) Streptococci. The ADB based ELISA proved to be highly sensitive and more responsive to changes in antibody concentration than DMGM. Use of recombinant DNase B eliminates the variability associated with the enzyme, partially purified from Streptococcal culture supernatants from various commercial sources and may provide a more reliable source of antigen to a wider group of laboratories concerned with GAS diagnosis.

Human CD18 is the functional receptor for Aggregatibacter actinomycetemcomitans leukotoxin.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative organism of localized aggressive periodontitis, a rapidly progressing degenerative disease of the gingival and periodontal ligaments, and is also implicated in causing subacute infective endocarditis in humans. The bacterium produces a variety of virulence factors, including an exotoxic leukotoxin (LtxA) that is a member of the repeats-in-toxin (RTX) family of bacterial cytolysins. LtxA exhibits a unique specificity to macrophages and polymorphonuclear cells of humans and other primates. Human lymphocyte function-associated antigen 1 (LFA-1) has been implicated as the putative receptor for LtxA. Human LFA-1 comprises the CD11a and CD18 subunits. It is not clear, however, which of its subunits serves as the functional receptor that confers species-specific susceptibility to LtxA. Here we demonstrate that the human CD18 is the receptor for LtxA based on experiments performed with chimeric beta2-integrins recombinantly expressed in a cell line that is resistant to LtxA effects. In addition, we show that the cysteine-rich tandem repeats encompassing integrin-epidermal growth factor-like domains 2, 3, and 4 of the extracellular region of human CD18 are critical for conferring susceptibility to LtxA-induced biological effects.

Mapping of the binding site for Mannheimia haemolytica leukotoxin within bovine CD18.

To map the site involved in Mannheimia haemolytica leukotoxin (LktA) binding and biological activity within bovine CD18, bovine x human CD18 chimeric constructs were generated and coexpressed with bovine CD11a in K562 cells. Studies with the chimeric leukocyte function-associated antigen 1 transductants demonstrate that the site required for LktA binding and biological effects resides within amino acid residues 500 and 600 of the extracellular region of bovine CD18.