Group B streptococci (GBS) and group A streptococci (GAS) are known to have surface-associated peptidase activity which specifically cleaves C5a (C5a-ase), the primary chemotaxin for polymorphonuclear leukocytes. Amplification products were obtained from GBS genomic DNA template by using the polymerase chain reaction and primers corresponding to the C5a peptidase gene of GAS (scpA12). A restriction map of the GBS full-length amplified gene (scpB) was developed. The scpB restriction map was found to be highly similar to that of the analogous gene in GAS. A 50-bp deletion was located near the 5' end of scpB in a region where repeat sequences are found in scpA12. Hybridization experiments confirmed that other serotypes of GBS also carry an scpB-like gene. These results show that GBS contain a gene, scpB, which is very similar to that harbored by M12 GAS. The probability that scpB encodes the C5a-ase protein is discussed. * Corresponding author. an scpA12-like gene (scpB) in GBS DNA. The results show that the GBS gene is strikingly similar to that of GAS.
Immunofluorescent staining was used to determine that the streptococcal C5a peptidase (SCP) exists as a cell surface antigen on group A streptococci. The ability of hyperimmune serum to neutralize cell-associated SCP activity provided further evidence for the location of SCP. Quantification of SCP during growth in vitro by indirect enzyme-linked immunosorbent assay showed that approximately 90% of the measurable antigen is cell bound.
Infection with group B streptococci (GBS) is associated with a poor acute inflammatory response in which neutrophils fail to localize at the site of invasion. In the present studies, we have examined the effects of group B streptococci on C-derived chemotactic activity in human serum. Fresh human serum was activated to form C5a and C5adesarg by incubation with zymosan. The activated serum was then incubated with group B organisms, centrifuged, and the supernatants tested for chemotactic activity for human polymorphonuclear leukocytes. Group B organisms caused a dose-dependent decrease in C-dependent chemotactic activity. The degree of inhibition was profound with 1 X 10(9) bacteria/ml (10% of control). Experiments indicated that significant chemotactic factor inactivation occurred within 2 min of exposure to GBS organisms, while maximal inhibition occurred after 30 min incubation. A number of different strains of GBS of types I, II, and III possessed inhibitory activity. In contrast, group D streptococci, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae failed to inhibit the C-derived chemotactic activity in human serum. Group A streptococci that were M protein positive also inactivated C-dependent chemotactic activity in serum, as previously reported. The inhibitory activity of the GBS strains could be abolished by heat or trypsin treatment but not by neuraminidase, pronase, or pepsin. C5a levels in zymosan-activated serum as measured by RIA were not decreased after incubation with an inhibitory strain suggesting that absorption was not involved. SDS-PAGE analysis revealed that group B streptococci degrade the C5a molecule, increasing its electrophoretic mobility by removing a fragment with a m.w. of approximately 650 Da. Thus, one of the reasons for the poor inflammatory response at the site of GBS infection may reside in the ability of these pathogens to inactivate C-derived inflammatory mediators. The GBS C5a-ase activity probably serves as an additional virulence factor for these organisms contributing to the poor inflammatory response characteristic of group B streptococcal infection.
The previous finding that phagocytosis-resistant M+ group A streptococci bear quantities of C3 which are sufficient for phagocytosis of their M- derivatives was investigated at two levels. It was first established that the C3 associated with M+ streptococci was not able to promote adherence to cells bearing the complement receptors CR1 and CR3 under conditions in which M- streptococci readily attached. The molecular form of C3 bound to M+ and M- streptococci was then defined by adding 125I-C3 to serum used for opsonization. C3 eluted from the bacteria by chaotropic and hydrolytic agents was analyzed by SDS-PAGE, and revealed that both cell types bound the opsonic forms of C3, C3b, and iC3b. Furthermore, approximately 80% of the C3b and iC3b associated with both cell types was covalently bound to a surface component, although most of the C3 bound to M+ streptococci was detergent-extractable, whereas greater than 50% of that bound to M- streptococci was not. These findings demonstrate that the M+ surface is interfering with the receptor binding of deposited C3b and iC3b, and that this contributes to resistance to phagocytosis by these organisms.
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