Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library

There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine

The GBS PI-2a pilus is required for virulence in mice neonates.

BACKGROUND:Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone. METHODOLOGY/PRINCIPAL FINDINGS:In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity. CONCLUSIONS/SIGNIFICANCE:These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context

Analysis of the Streptococcus agalactiae exoproteome

International audienceThe two-component regulatory system CovRS is the main regulator of virulence gene expression in Group B Streptococcus (GBS), the leading cause of invasive infections in neonates. In this study we analyzed by mass spectrometry the GBS extracellular protein complex (i.e. the exoproteome) of NEM316 wild-type (WT) strain and its isogenic covRS deletion mutant (ΔcovRS). A total of 53 proteins, 49 of which had classical secretion signals, were identified: 12 were released by both strains while 21 and 20 were released exclusively by WT and ΔcovRS strains, respectively. In addition to known surface proteins, we detected here unstudied cell-wall associated proteins and/or orthologs of putative virulence factors present in other pathogenic streptococci. While the functional role of these proteins remains to be elucidated, our data suggest that the analysis of the exoproteome of bacterial pathogens under different gene expression conditions may be a powerful tool for the rapid identification of novel virulence factors and vaccine candidates

Immunobiological characterization of genetic products, identified from whole genome lambda-display libraries of Pneumococcus

Objectives: Streptococcus pneumoniae is an important cause of morbidity and mortality worldwide, including meningitis. Here, we employed the isogenic Δspr0075-, Δspr1370- and Δspr1875-knock-out mutants of the DP1004 pneumococcal strain to investigate the role of these proteins in the interaction with microglia, the resident brain macrophages. Methods: By screening a whole genome phage display library with sera from patients, we identified clones carrying pneumococcal B-cell epitopes. Among these, we isolated six antigenic fragments of sequences matching three previously unidentified proteins, encoded by the ORFs spr0075, spr1370 and spr1875 of R6 strain genomic sequence. By using an in vitro infection model and a gentamicin protection assay we evaluated, respectively, the ability of microglial BV2 cells to phagocytose and kill the isogenic mutants and the survival of these bacteria inside the microglia. Results: All strains were efficiently internalized by BV2 cells; yet the levels of phagocytosis obtained with Δspr0075 strain were lower than those observed with the other groups. The survival of the deleted strains inside the microglia was significantly different. The residual CFU of Δspr1370 and Δspr1875 mutants were, indeed, respectively, higher and lower, than those observed with parental DP1004. Moreover, preliminary results indicate that a similar trend is also observed in a bactericidal assay using BV2 as effector cells. Conclusion: These findings indicate that the proteins encoded by spr1370 and spr1875 loci are involved in interaction between S. pneumoniae and microglia

FbsC, a Novel Fibrinogen-binding Protein, Promotes Streptococcus agalactiae-Host Cell Interactions

International audienceStreptococcus agalactiae (group B Streptococcus or GBS) is a common cause of invasive infections in newborn infants and adults. The ability of GBS to bind human fibrinogen is of crucial importance in promoting colonization and invasion of host barriers. We characterized here a novel fibrinogen-binding protein of GBS, designated FbsC (Gbs0791), which is encoded by the prototype GBS strain NEM316. FbsC, which bears two bacterial immunoglobulin-like tandem repeat domains and a C-terminal cell wall-anchoring motif (LPXTG), was found to be covalently linked to the cell wall by the housekeeping sortase A. Studies using recombinant FbsC indicated that it binds fibrinogen in a dose-dependent and saturable manner, and with moderate affinity. Expression of FbsC was detected in all clinical GBS isolates, except those belonging to the hypervirulent lineage ST17. Deletion of fbsC decreases NEM316 abilities to adhere to and invade human epithelial and endothelial cells, and to form biofilm in vitro. Notably, bacterial adhesion to fibrinogen and fibrinogen binding to bacterial cells were abolished following fbsC deletion in NEM316. Moreover, the virulence of the fbsC deletion mutant and its ability to colonize the brain were impaired in murine models of infection. Finally, immunization with recombinant FbsC significantly protected mice from lethal GBS challenge. In conclusion, FbsC is a novel fibrinogen-binding protein expressed by most GBS isolates that functions as a virulence factor by promoting invasion of epithelial and endothelial barriers. In addition, the protein has significant immunoprotective activity and may be a useful component of an anti-GBS vaccine

IDENTIFICATION OF FIBRINOGEN BINDING PROTEIN FRAGMENTS USING GENOMIC PHAGE DISPLAY LIBRARIES OF GROUP B STREPTOCOCCUS

Obiective: The ability to bind fibrinogen (Fng) is a virulence determinant for a diverse group of bacterial pathogens. Group B streptococcus (GBS) is an important cause of sepsis and invasive infections. The objective of the present work was to obtain novel insights regarding the mechanism of Fng binding by GBS. Methods: We constructed two genomic libraries of GBS using, respectively, the COH-1 and 2603 R/V strains. These libraries were selected using Fng-coated beads as bait. For virulence studies neonatal (24-hour-old) BALB/c mice (Charles River) were inoculated s.c. with a LD90 (60 CFU/pup) of the wild type serotype III GBS strain or with a fbsA defective mutant. Results: After one round of selection of the COH-1 library approximately 10% of the clones strongly reacted against Fng by immunoblot of phage plaques. The percentage of Fng-binding clones rose to 100% after 3 rounds of selection. All reactive clones contained 2-5 repeats of fibrinogen binding protein A (FbsA), a previously described factor shown to mediate adherence and invasion of human epithelial cells. We further show here that FbsA is an important virulence factor, as evidenced by a decreased ability of the fbsA mutant to cause infection. Experiments are underway to characterize the protective activities of FbsA after active immunization with the protein fragments identified by phage display libraries. Moreover we have developed an anti-FbsA neutralizing monoclonal antibody that is being tested for its ability to passively protect neonatal mice from GBS infection. Conclusion:Our data confirm and extend previous studies indicating that FbsA is an important Fng binding factor. Moreover we show that FbsA is an essential virulence factor in vivo