Inactivation of DltA Modulates Virulence Factor Expression in Streptococcus pyogenes

D-alanylated lipoteichoic acid is a virtually ubiquitous component of gram-positive cell walls. Mutations in the dltABCD operon of numerous species exhibit pleiotropic effects, including reduced virulence, which has been attributed to increased binding of cationic antimicrobial peptides to the more negatively charged cell surface. In this study, we have further investigated the effects that mutating dltA has on virulence factor expression in Streptococcus pyogenes.Isogenic Delta dltA mutants had previously been created in two distinct M1T1 isolates of S. pyogenes. Immunoblots, flow cytometry, and immunofluorescence were used to quantitate M protein levels in these strains, as well as to assess their ability to bind complement. Bacteria were tested for their ability to interact with human PMN and to grow in whole human blood. Message levels for emm, sic, and various regulatory elements were assessed by quantitative RT-PCR. Cell walls of Delta dltA mutants contained much less M protein than cell walls of parent strains and this correlated with reduced levels of emm transcripts, increased deposition of complement, increased association of bacteria with polymorphonuclear leukocytes, and reduced bacterial growth in whole human blood. Transcription of at least one other gene of the mga regulon, sic, which encodes a protein that inactivates antimicrobial peptides, was also dramatically reduced in Delta dltA mutants. Concomitantly, ccpA and rofA were unaffected, while rgg and arcA were up-regulated.This study has identified a novel mechanism for the reduced virulence of dltA mutants of Streptococcus pyogenes in which gene regulatory networks somehow sense and respond to the loss of DltA and lack of D-alanine esterification of lipoteichoic acid. The mechanism remains to be determined, but the data indicate that the status of D-alanine-lipoteichoic acid can significantly influence the expression of at least some streptococcal virulence factors and provide further impetus to targeting the dlt operon of gram-positive pathogens in the search for novel antimicrobial compounds

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or 'double' tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the 'double' tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy

Spa Contributes to the Virulence of Type 18 Group A Streptococci

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261–1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci

M protein of the group A streptococcus binds to the seventh short consensus repeat of human complement factor H

Streptococcus pyogenes evades complement by binding the complement-regulatory protein factor H (fH) via the central conserved C-repeat region of M protein. However, the corresponding binding region within fH has not previously been precisely localized. fH is composed of 20 conserved modules called short consensus repeats (SCRs), each of which contains approximately 60 amino acids. A series of fH truncated and deletion mutants were prepared, and their interaction with M6 protein was examined. The M protein binding site was initially localized to SCRs 6 to 15 as demonstrated by ligand dot blotting, chemical cross-linking, and enzyme-linked immunosorbent assay. SCR 7 was then shown to contain the M protein binding site, as a construct consisting of the first seven SCRs bound M protein but a construct containing the first six SCRs did not bind. In addition, deletion of SCR 7 from full-length fH abolished binding to M protein. SCR 7 is known to contain a heparin binding domain, and binding of fH to M6 protein was almost totally inhibited in the presence of 400 U of heparin per ml. These results localize the M6 protein binding site of fH to SCR 7 and indicate that it is in close proximity to the heparin binding site.Timothy K. Blackmore, Vincent A. Fischetti, Tania A. Sadlon, Helena M. Ward, and David L. Gordo

Binding of human plasma proteins to Streptococcus pyogenes M protein determines the location of opsonic and non-opsonic epitopes

Antibodies directed against a pathogenic microorganism may recognize either protective or non-protective epitopes. Because antibodies elicited by a vaccine must be directed against protective epitopes, it is essential to understand the molecular properties that distinguish the two types of epitope. Here we analyse this problem for the antiphagocytic M protein of Streptococcus pyogenes, using the opsonizing capacity of antibodies to estimate their ability to confer protection in vivo. Our studies were focused on the M5 protein, which has three surface-exposed regions: the amino-terminal hypervariable region (HVR) and the B- and C-repeat regions. We first analysed the role of different M5 regions in phagocytosis resistance under non-immune conditions, employing chromosomal mutants expressing M5 proteins with internal deletions, and demonstrate that only the B-repeat region is essential for phagocytosis resistance. However, only antibodies to the HVR were opsonic. This apparent paradox could be explained by the ability of fibrinogen and albumin to specifically bind to the B- and C-repeats, respectively, causing inhibition of antibody binding under physiological conditions, while antibodies to the HVR could bind and promote deposition of complement. These data indicate that binding of human plasma proteins plays an important role in determining the location of opsonic and non-opsonic epitopes in streptococcal M protein