C5a peptidase alters clearance and trafficking of group A streptococci by infected mice

Abstract

Group A streptococcal C5a peptidase (SCPA) specifically cleaves the human serum chemotaxin C5a at the polymorphonuclear leukocyte (PMNL) binding site. This study tested the proposal that SCPA contributes to virulence by retarding the influx of inflammatory cells and clearance of streptococci during the first few hours after infection. To investigate the specific contribution of SCPA to the virulence of group A streptococci, scpA insertion and deletion mutants were created by directed plasmid insertion into scpA and gene replacement. The precise locations of insertion and deletion mutations were confirmed by PCR and DNA sequence analysis. The impact of mutation on virulence was investigated with a mouse air sac model of inflammation. Experiments evaluated clearance of streptococci from the air sac within 4 h after infection. SCPA ؊ streptococci were cleared more efficiently than wild-type bacteria. Localization of streptococci in lymph nodes and spleens of infected mice revealed a significant difference between mutant and wild-type streptococci. PMNLs and other granulocytes that infiltrated the air sac were quantitated by single-color flow cytometry. The total cellular infiltrate was greater and PMNLs dominated the granulocytic infiltrates of air sacs inoculated with SCPA ؊ mutant bacteria. The data obtained are consistent with the possibility that SCPA ؊ streptococci are initially cleared from the site of infection primarily by PMNLs. Moreover, mutant and wild-type streptococci followed different paths of dissemination. SCPA ؊ bacteria were transported to lymph nodes, whereas wild-type streptococci avoided transport to the lymph nodes and rapidly spread to the spleen.

Dual modes of membrane binding direct pore formation by Streptolysin O

“…The Q476A/N and W503A/Y mutations in SLO were constructed in a similar manner, except the SLO domain 4 chromosomal sequence was inserted into the modified temperature-sensitive shuttle vector pGCP213 (Nielsen et al, 2012). Electroporation was used to transform S. pyogenes (Caparon and Scott, 1989), and the wild-type allele in JRS4 or SPN1 was replaced with the mutant versions as previously described (Ji et al, 1996). All primers are listed in Table S2, with M13 forward and reverse sequences (for insertion into shuttle vectors) underlined.…”

Group A streptococcal infections in children

“…C5a peptidase limits recruitment of phagocytes. 8 Streptococcal inhibitor of complement aids in evading complement mediated killing. 9 Streptococcal pyrogenic exotoxins are responsible for the clinical features of STSS and scarlet fever by acting as superantigens, which stimulate around 20% of the T-cell population by binding directly to the T-cell receptor rather than having to be presented in the MHC II binding groove.…”

Comparative functional analysis of the lac operons in Streptococcus pyogenes

“…Unless otherwise indicated, all references to genomic loci are based on the genome of S. pyogenes SF370 (Ferretti et al, 2001). The construction of mutants containing in-frame deletions in lacR.1 (spy1712) or lacR.2 (spy1924) was performed by allelic replacement (Ji et al, 1996) as previously described (Ruiz et al, 1998;Loughman and Caparon, 2006b) using the primers in Table 2. The strains are listed in Table 1 and as follows: JL320 (HSC5 lacR.1D2-248) (Loughman and Caparon, 2006a), and JL431 (HSC5 lacR.…”