A novel role for pro-coagulant microvesicles in the early host defense against streptococcus pyogenes.

Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a KD value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.

Modulation of the coagulation system during severe streptococcal disease.

Haemostasis is maintained by a tightly regulated coagulation system that comprises platelets, procoagulant proteins, and anticoagulant proteins. During the local and systemic response to bacterial infection, the coagulation system becomes activated, and contributes to the pathophysiological response to infection. The significant human pathogen, Streptococcus pyogenes has multiple strategies to modulate coagulation. This can range from systemic activation of the intrinsic and extrinsic pathway of coagulation to local stimulation of fibrinolysis. Such diverse effects on this host system imply a finely tuned host-bacteria interaction. The molecular mechanisms that underlie this modulation of the coagulation system are discussed in this review.

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Coagulation, an ancestral serine protease cascade, exerts a novel function in early immune defense.

Phylogenetically conserved serine protease cascades play an important role in invertebrate and vertebrate immunity. The mammalian coagulation system can be traced back some 400 million years and shares homology with ancestral serine proteinase cascades that are involved in, for example, Toll receptor signaling in insects and release of antimicrobial peptides during hemolymph clotting. In the present study, we show that the induction of coagulation by bacteria leads to immobilization and killing of Streptococcus pyogenes bacteria inside the clot. The entrapment is mediated via cross-linking of bacteria to fibrin fibers by the action of coagulation factor XIII (fXIII), an evolutionarily conserved transglutaminase. In a streptococcal skin infection model, fXIII(-/-) mice developed severe signs of pathologic inflammation at the local site of infection, and fXIII treatment of wild-type animals dampened bacterial dissemination during early infection. Bacterial killing and cross-linking to fibrin networks was also detected in tissue biopsies from patients with streptococcal necrotizing fasciitis, supporting the concept that coagulation is part of the early innate immune system.

Stimulation of blood mononuclear cells with bacterial virulence factors leads to the release of pro-coagulant and pro-inflammatory microparticles.

Severe infectious diseases remain a major and life-threatening health problem. In serious cases a systemic activation of the coagulation cascade and hypovolemic shock are critical complications that are associated with high mortality rates. Here we report that blood mononuclear cells, stimulated with M1 protein of Streptococcus pyogenes or other bacterial virulence factors, produce not only pro-coagulant, but also pro-inflammatory microparticles (MPs). Our results also show that activation of the contact system on MPs contributes to these two effects. Phosphatidylserine (PS) plays an important role in these processes as its upregulation on MPs allows an assembly and activation of the contact system. This in turn results in stabilization of the tissue factor-induced clot and a processing of high-molecular-weight kininogen by plasma kallikrein followed by the release of bradykinin, a potent vascular mediator. Pro-coagulant monocyte-derived MPs were identified in plasma samples from septic patients and further analysis of MPs from these patients revealed that their pro-coagulant activity is dependent on the tissue factor- and contact system-driven pathway.

Treatment of invasive streptococcal infection with a peptide derived from human high-molecular weight kininogen.

Sepsis and septic shock remain an important medical problem, emphasizing the need to identify novel therapeutic opportunities. Hypovolemic hypotension, coagulation dysfunction, disturbed microcirculation, and multiorgan failure resulting from vascular leakage are often observed in these severe conditions. In the present study, we find that HKH20, a peptide derived from human high molecular weight kininogen (HK), down-regulates inflammatory reactions caused by Streptococcus pyogenes in a mouse model of sepsis. HK is a component of the pro-inflammatory and pro-coagulant contact system. Activation of the contact system in the bloodstream by S pyogenes leads to massive tissue damage in the lungs of the infected mice, which eventually results in the death of the animals. HKH20 inhibits activation of the contact system and protects mice with invasive S pyogenes infection from lung damage. In combination with clindamycin treatment, the peptide also significantly prolongs the survival of infected mice.

Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.

Host genetic variations play a significant role in conferring predisposition to infection. In this study, we examined the immune mechanisms underlying the host genetic predisposition to severe Staphylococcus aureus infection in different mouse strains. Whereas C57BL/6 mice were the most resistant in terms of control of bacterial growth and survival, A/J, DBA/2, and BALB/c mice were highly susceptible and succumbed to infection shortly after bacterial inoculation. Other strains (C3H/HeN, CBA, and C57BL/10) exhibited intermediate susceptibility levels. Susceptibility of mice to S. aureus was associated with an inability to limit bacterial growth in the kidneys and development of pathology. Resistance to S. aureus in C57BL/6 mice was dependent on innate immune mechanisms because Rag2-IL2Rgamma(-/-) C57BL/6 mice, which are deficient in B, T, and NK cells, were also resistant to infection. Indeed, neutrophil depletion or inhibition of neutrophil recruitment rendered C57BL/6 mice completely susceptible to S. aureus, indicating that neutrophils are essential for the observed resistance. Although neutrophil function is not inhibited in A/J mice, expression of neutrophil chemoattractants KC and MIP-2 peaked earlier in the kidneys of C57BL/6 mice than in A/J mice, indicating that a delay in neutrophil recruitment to the site of infection may underlie the increased susceptibility of A/J mice to S. aureus.

Contact system activation in severe infectious diseases.

Hemostasis is a sensitive and tightly regulated process, involving vascular endothelium and blood cells, as well as factors of the coagulation and fibrinolytic cascades. In severe and invasive infectious diseases, the equilibrium between the procoagulant and anticoagulant status of the host may change dramatically and can induce life-threatening complications. A growing body of evidence suggests that the contact system, also known as the intrinsic pathway of coagulation or kallikrein/kinin system, participate in these processes. Contact activation leads to the release of the highly potent proinflammatory peptide bradykinin and initiates the intrinsic pathway of coagulation. Several studies have shown a systemic activation of the contact system in animal models of severe bacterial infections, and similar findings were also reported when monitoring patients suffering from sepsis, severe sepsis, or septic shock. Complications resulting from a systemic activation of the contact system are pathologically high levels of bradykinin, consumption of contact factors, and a subsequent induction of inflammatory reactions. These conditions may contribute to serious complications such as hypotension and vascular leakage. Here, we summarize the state of the art in this field of research with a focus on the contact system, and we also discuss a potential role for the contact system as a target for the development of novel antimicrobial strategies.

Streptococcal M proteins and their role as virulence determinants.

Group A streptococci (GAS, Streptococcus pyogenes) are exclusive human pathogens that have been extensively studied for many decades. The spectrum of diseases caused by these bacteria ranges from uncomplicated and superficial to severe and invasive infections. In order to give rise to these complications, GAS have evolved a number of surface-bound and secreted virulence factors, of which the M proteins are probably the best characterized. Evidence has emerged that M proteins are multifunctional pathogenic determinants, and over the years many interactions between M proteins and the human host have been reported. The present review article aims to present a state-of-the-art overview of the most important virulence mechanisms employed by M proteins to trigger disease.

Activation of the human contact system on neutrophil extracellular traps.

Pattern recognition is an integral part of the innate immune system. The human contact system has been shown to interact with the surface of many bacterial and fungal pathogens, and once activated leads to the generation of antimicrobial peptides and the proinflammatory mediator bradykinin. Here we show that apart from these surfaces also neutrophil extracellular traps (NETs) provide a surface that allows the binding and activation of the contact system. In addition, we present evidence that M1 protein, a streptococcal surface protein, in concert with human fibrinogen triggers polymorphonuclear neutrophils to form NETs.

Transcription of the pst operon of Clostridium acetobutylicum is dependent on phosphate concentration and pH.

The pst operon of Clostridium acetobutylicum ATCC 824 comprises five genes, pstS, pstC, pstA, pstB, and phoU, and shows a gene architecture identical to that of Escherichia coli. Deduced proteins are predicted to represent a high-affinity phosphate-specific ABC (ATP-binding cassette) transport system (Pst) and a protein homologous to PhoU, a negative phosphate regulon regulator. We analyzed the expression patterns of the pst operon in P(i)-limited chemostat cultures during acid production at pH 5.8 or solvent production at pH 4.5 and in response to P(i) pulses. Specific mRNA transcripts were found only when external P(i) concentrations had dropped below 0.2 mM. Two specific transcripts were detected, a 4.7-kb polycistronic mRNA spanning the whole operon and a quantitatively dominating 1.2-kb mRNA representing the first gene, pstS. The mRNA levels clearly differed depending on the external pH. The amounts of the full-length mRNA detected were about two times higher at pH 5.8 than at pH 4.5. The level of pstS mRNA increased by a factor of at least 8 at pH 5.8 compared to pH 4.5 results. Primer extension experiments revealed only one putative transcription start point 80 nucleotides upstream of pstS. Thus, additional regulatory sites are proposed in the promoter region, integrating two different extracellular signals, namely, depletion of inorganic phosphate and the pH of the environment. After phosphate pulses were applied to a phosphate-limited chemostat we observed faster phosphate consumption at pH 5.8 than at pH 4.5, although higher optical densities were recorded at pH 4.5.

Streptococcus pyogenes collagen type I-binding Cpa surface protein. Expression profile, binding characteristics, biological functions, and potential clinical impact.

The Streptococcus pyogenes collagen type I-binding protein Cpa (collagen-binding protein of group A streptococci) expressed by 28 serotypes of group A streptococci has been extensively characterized at the gene and protein levels. Evidence for three distinct families of cpa genes was found, all of which shared a common sequence encoding a 60-amino acid domain that accounted for selective binding to type I collagen. Surface plasmon resonance-based affinity measurements and functional studies indicated that the expression of Cpa was consistent with an attachment role for bacteria to tissue containing collagen type I. A cpa mutant displayed a significantly decreased internalization rate when incubated with HEp-2 cells but had no effect on the host cell viability. By utilizing serum from patients with a positive titer for streptolysin/DNase antibody, an increased anti-Cpa antibody titer was noted for patients with a clinical history of arthritis or osteomyelitis. Taken together, these results suggest Cpa may be a relevant matrix adhesin contributing to the pathogenesis of S. pyogenes infection of bones and joints.

Function of the fibronectin-binding serum opacity factor of Streptococcus pyogenes in adherence to epithelial cells.

The serum opacity factor (SOF) of Streptococcus pyogenes is a serotyping tool and pathogenesis factor. Using SOF-coated latex beads in cell adherence assays and antiserum directed against SOF in S. pyogenes-HEp-2 cell adherence inhibition experiments, we demonstrate SOF involvement in the fibronectin-mediated adherence of S. pyogenes to epithelial cells. SOF exclusively targets the 30-kDa N-terminal region of fibronectin. The interaction revealed association and dissociation constants 1 order of magnitude lower than those of other S. pyogenes fibronectin-binding proteins.

Streptococcus pyogenes fibronectin-binding protein F2: expression profile, binding characteristics, and impact on eukaryotic cell interactions.

Some Streptococcus pyogenes (group A streptococci, GAS) strains have previously been shown to express the fibronectin-binding protein F2 instead of the functionally related but structurally dissimilar protein F1/SfbI. In this study, recombinant N-terminal and C-terminal portions and the two fibronectin-binding domains of protein F2 were used to assess affinity parameters of the interaction with fibronectin and its N-terminal 70-, 30-, and 45-kDa fragments. The association and dissociation equilibrium constants for both binding domains were in the nanomolar range, although the repeat domain of protein F2 exceeded the affinity of the unique domain by up to one order magnitude. Both domains primarily interacted with the 30-kDa fibronectin fragment. Using a prtF2 gene isogenic mutant of a serotype M49 GAS strain that does not harbor the protein F1/SfbI gene, the attachment values of whole bacteria to immobilized fibronectin and to HEp-2 epithelial cells were found to be 6- and 2-fold decreased, respectively. Reduction of prtF2 mutant internalization rates for eukaryotic cells exceeded the reduction of attachment rates, indicating an independent contribution of protein F2 to both processes. The prtF2 transcription and protein F2 expression profiles documented maximum expression at the transition to the stationary phase especially under aerobic growth condition. The protein F2 function as the major fibronectin-binding adhesin in a subset of GAS strains, its expression pattern, and highly specific interaction with fibronectin would be consistent with a status as an indispensable virulence factor for both earlier and later pathogenetic stages of GAS superficial infections.