FbsA, a fibrinogen-binding protein from Streptococcus agalactiae, mediates platelet aggregation

The bacterium Streptococcus agalactiae is an etiologic agent in the pathogenesis of endocarditis in humans. FbsA, a fibrinogen-binding protein produced by this pathogen, is considered an important virulence factor. In the present study we provide evidence that S agalactiae clinical isolates bearing FbsA attach to fibrinogen and elicit a fibrinogen-dependent aggregation of platelets. Mutants of S agalactiae lacking the fbsA gene lost the ability to attach to fibrinogen and to aggregate platelets. Plasmid-mediated expression of fbsA restored the capability for fibrinogen binding and platelet aggregation in S agalactiae fbsA mutants, and allowed Lactococcus lactis to interact with fibrinogen and to aggregate human platelets. Moreover, a monoclonal anti-FbsA antibody inhibited bacterial adherence to fibrinogen and S agalactiae-induced platelet aggregation. Platelet aggregation was inhibited by aspirin, prostaglandin E(1,) the peptide RGDS, and the antibody abciximab, demonstrating the specificity of platelet aggregation by S agalactiae and indicating an involvement of integrin glycoprotein IIb/IIIa in the induction of platelet aggregation. Aggregation was also dependent on anti-FbsA IgG and could be inhibited by an antibody against the platelet FcgammaRIIA receptor. These findings indicate that FbsA is a crucial factor in S agalactiae-induced platelet aggregation and may therefore play an important role in S agalactiae-induced endocarditis

Analysis of RogB-Controlled Virulence Mechanisms and Gene Expression in Streptococcus agalactiae

Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis in neonates and also the causative agent of different serious infections in immunocompromised adults. The wide range of diseases that are caused by S. agalactiae suggests regulatory mechanisms that control the formation of specific virulence factors in these bacteria. The present study describes a gene from S. agalactiae, designated rogB, encoding a protein with significant similarity to members of the RofA-like protein (RALP) family of transcriptional regulators. Disruption of the rogB gene in the genome of S. agalactiae resulted in mutant strain RGB1, which was impaired in its ability to bind to fibrinogen and fibronectin. Mutant RGB1 also exhibited a reduced adherence to human epithelial cells but did not show an altered invasion of eukaryotic cells. By real-time PCR analysis, mutant RGB1 revealed an increased expression of the cpsA gene, encoding a regulator of capsule gene expression. However, strain RGB1 exhibited a reduced expression of the rogB gene and of two adjacent genes, encoding putative virulence factors in S. agalactiae. Furthermore, mutant RGB1 was impaired in the expression of the fbsA gene, coding for a fibrinogen receptor from S. agalactiae. The altered gene expression in mutant RGB1 could be restored by plasmid-mediated expression of rogB, confirming a RogB deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. Reporter gene studies with a promotorless luciferase gene fused to fbsA allowed a growth-dependent analysis of fbsA expression in S. agalactiae. These reporter gene studies also suggest that RogB exerts a positive effect on fbsA expression in S. agalactiae

Malate synthase from Corynebacterium glutamicum: sequence analysis of the gene and biochemical characterization of the enzyme

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The Novel Fibrinogen-Binding Protein FbsB Promotes Streptococcus agalactiae Invasion into Epithelial Cells

Streptococcus agalactiae is a major cause of bacterial sepsis and meningitis in human newborns. The interaction of S. agalactiae with host proteins and the entry into host cells thereby represent important virulence traits of these bacteria. The present report describes the identification of the fbsB gene, encoding a novel fibrinogen-binding protein that plays a crucial role in the invasion of S. agalactiae into human cells. In Western blots and enzyme-linked immunosorbent assay (ELISA) experiments, the FbsB protein was demonstrated to interact with soluble and immobilized fibrinogen. Binding studies showed the N-terminal 388 residues of FbsB and the Aα-subunit of human fibrinogen to recognize each other. By reverse transcription (RT)-PCR, the fbsB gene was shown to be cotranscribed with the gbs0851 gene in S. agalactiae. Deletion of the fbsB gene in the genome of S. agalactiae did not influence the binding of the bacteria to fibrinogen, suggesting that FbsB does not participate in the attachment of S. agalactiae to fibrinogen. In tissue culture experiments, however, the fbsB deletion mutant was severely impaired in its invasion into lung epithelial cells. Bacterial invasion could be reestablished by introducing the fbsB gene on a shuttle plasmid into the fbsB deletion mutant. Furthermore, treatment of lung epithelial cells with FbsB fusion protein blocked S. agalactiae invasion of epithelial cells in a dose-dependent fashion. These results suggest an important role of the FbsB protein in the overall process of host cell entry by S. agalactiae

Relevance of Peptide Uptake Systems to the Physiology and Virulence of Streptococcus agalactiae

Streptococcus agalactiae is a major cause of invasive infections in human newborns. To satisfy its growth requirements, S. agalactiae takes up 9 of the 20 proteinogenic amino acids from the environment. Defined S. agalactiae mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits. Oligopeptide uptake by S. agalactiae was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins (OppA1 and OppA2) with overlapping substrate specificities. Dipeptides were found to be taken up in parallel by the oligopeptide permease OppA1-F, by the dipeptide ABC transporter DppA-E, and by the dipeptide symporter DpsA. Reverse transcription-PCR analysis revealed a polycistronic organization of the genes oppA1-F and dppA-E and a monocistronic organization of dpsA in S. agalactiae. The results of quantitative real-time PCR revealed a medium-dependent expression of the operons dppA-E and oppA1-F in S. agalactiae. Growth of S. agalactiae in human amniotic fluid was shown to require an intact dpsA gene, indicating an important role of DpsA during the infection of the amniotic cavity by S. agalactiae. Deletion of the oppB gene reduced the adherence of S. agalactiae to epithelial cells by 26%, impaired its adherence to fibrinogen and fibronectin by 42 and 33%, respectively, and caused a 35% reduction in expression of the fbsA gene, which encodes a fibrinogen-binding protein in S. agalactiae. These data indicate that the oligopeptide permease is involved in modulating virulence traits and virulence gene expression in S. agalactiae

The Surface Protein Srr-1 of Streptococcus agalactiae Binds Human Keratin 4 and Promotes Adherence to Epithelial HEp-2 Cells▿

Streptococcus agalactiae is frequently the cause of bacterial sepsis and meningitis in neonates. In addition, it is a commensal bacterium that colonizes the mammalian gastrointestinal tract. During its commensal and pathogenic lifestyles, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present study, the serine-rich repeat protein Srr-1 from S. agalactiae was functionally investigated. Immunofluorescence microscopy showed that Srr-1 was localized on the surface of streptococcal cells. The Srr-1 protein was shown to interact with a 62-kDa protein in human saliva, which was identified by matrix-assisted laser desorption ionization-time-of-flight analysis as human keratin 4 (K4). Immunoblot and enzyme-linked immunosorbent assay experiments allowed us to narrow down the K4 binding domain in Srr-1 to a region of 157 amino acids (aa). Furthermore, the Srr-1 binding domain of K4 was identified in the C-terminal 255 aa of human K4. Deletion of the srr-1 gene in the genome of S. agalactiae revealed that this gene plays a role in bacterial binding to human K4 and that it is involved in adherence to epithelial HEp-2 cells. Binding to immobilized K4 and adherence to HEp-2 cells were restored by introducing the srr-1 gene on a shuttle plasmid into the srr-1 mutant. Furthermore, incubation of HEp-2 cells with the K4 binding domain of Srr-1 blocked S. agalactiae adherence to epithelial cells in a dose-dependent fashion. This is the first report describing the interaction of a bacterial protein with human K4

Adherence to and Invasion of Human Brain Microvascular Endothelial Cells Are Promoted by Fibrinogen-Binding Protein FbsA of Streptococcus agalactiae

Streptococcus agalactiae is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host tissues. Deletion of the fbsA gene, encoding the fibrinogen protein FbsA, significantly impaired the adherence and invasion of human brain microvascular endothelial cells (HBMEC) by S. agalactiae. The adherence and invasiveness of an fbsA deletion mutant were restored by reintroducing the fbsA gene on an expression vector. Heterologous expression of fbsA in Lactococcus lactis enabled this bacterium to adhere to but not to invade HBMEC, suggesting that FbsA is a streptococcal adhesin. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA fusion protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. The S. agalactiae fbsA mutant induced a release of the neutrophil chemoattractant interleukin-8 (IL-8) equal to that induced by the wild type. Taken together, our studies demonstrate that FbsA promotes the adherence of S. agalactiae to HBMEC but that FbsA neither mediates the bacterial invasion into host cells nor plays a role in IL-8 release for HBMEC