BackgroundStaphylococci belong to the most important pathogens causing implant-associated infections. Colonization of the implanted medical devices by the formation of a three-dimensional structure made of bacteria and host material called biofilm is considered the most critical factor in these infections. To form a biofilm, bacteria first attach to the surface of the medical device, and then proliferate and accumulate into multilayered cell clusters. Biofilm accumulation may be mediated by polysaccharide and protein factors.Methology/Principal FindingsThe information on Staphylococcus aureus protein factors involved in biofilm accumulation is limited, therefore, we searched the S. aureus Col genome for LPXTG-motif containing potential surface proteins and chose the so far uncharacterized S. aureus surface protein C (SasC) for further investigation. The deduced SasC sequence consists of 2186 amino acids with a molecular mass of 238 kDa and has features typical of Gram-positive surface proteins, such as an N-terminal signal peptide, a C-terminal LPXTG cell wall anchorage motif, and a repeat region consisting of 17 repeats similar to the domain of unknown function 1542 (DUF1542). We heterologously expressed sasC in Staphylococcus carnosus, which led to the formation of huge cell aggregates indicative of intercellular adhesion and biofilm accumulation. To localize the domain conferring cell aggregation, we expressed two subclones of sasC encoding either the N-terminal domain including a motif that is found in various architectures (FIVAR) or 8 of the DUF1542 repeats. SasC or its N-terminal domain, but not the DUF1542 repeat region conferred production of huge cell aggregates, higher attachment to polystyrene, and enhanced biofilm formation to S. carnosus and S. aureus. SasC does not mediate binding to fibrinogen, thrombospondin-1, von Willebrand factor, or platelets as determined by flow cytometry.Conclusions/SignificanceThus, SasC represents a novel S. aureus protein factor involved in cell aggregation and biofilm formation, which may play an important role in colonization during infection with this important pathogen.

Summary.  Background:  Human neutrophil α‐defensins (HNPs) are important constituents of the innate immune system. Beyond their antimicrobial properties, HNPs also have pro‐inflammatory features. While HNPs in plasma from healthy individuals are barely detectable, their level is strongly elevated in septic plasma and plasma from patients with acute coronary syndromes. Objectives:  As thrombosis and inflammation are intertwined processes and activation of human polymorphonuclear leukocytes (PMNL) and subsequent degranulation is associated with full activation of surrounding platelets, we studied the effect of HNPs on platelet function. Methods:  The effect of HNPs on platelet activation parameters and apoptosis was investigated via aggregometry, flow cytometry, confocal microscopy and the ELISA technique. Results:  It was found that HNPs activate platelets in pathophysiologically relevant doses, inducing fibrinogen and thrombospondin‐1 binding, aggregation, granule secretion, sCD40L shedding, and procoagulant activity. HNPs bound directly to the platelet membrane, induced membrane pore formation, microparticle formation, mitochondrial membrane depolarization and caspase‐3‐activity. Confocal microscopy revealed the HNP‐induced formation of polymeric fibrinogen and thrombospondin‐1 amyloid‐like structures, which bound microorganisms. Platelets adhered to these structures and formed aggregates. Blocking of glycoprotein IIb/IIIa (GPIIb/IIIa) markedly inhibited HNP‐induced platelet activation. In addition, heparin, heparinoid, serpins and α2‐macroglobulin, which all bind to HNPs, blocked HNP‐1‐induced platelet activation in contrast to direct thrombin inhibitors such as hirudin. Conclusions:  HNPs activate platelets and induce platelet apoptosis by formation of amyloid‐like proteins. As these structures entrapped bacteria and fungi, they might reflect an additional function of HNPs in host defense. The described mechanism links again thrombosis and infection.

Globalization and migration promote the spread of Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus strains. The toxin PVL is linked to the development of thrombosis in association with osteomyelitis. The mechanisms by which PVL drives thrombosis development are however still unknown. We demonstrate that PVL-damaged neutrophils activate platelets via neutrophil secretion products, such as α-defensins and the myeloperoxidase product HOCl, as well as the formation of HOCl-modified proteins. Neutrophil damage by PVL is blocked by anti-PVL-antibodies, explaining why especially young osteomyelitis patients with a low antibody titre against PVL suffer from thrombotic complications. Platelet activation in the presence of PVL-damaged neutrophils is prevented by α-defensin inhibitors and by glutathione and resveratrol, which are both inhibitors of HOCl-modified protein-induced platelet activation. Remarkably, intravenously infused glutathione also prevents activation of human platelets in an ex vivo assay. We here describe a new mechanism of PVL-neutrophil-platelet interactions, which might be extrapolated to other toxins that act on neutrophils. Our observations may make us think about new approaches to treat and/or prevent thrombotic complications in the course of infections with PVL-producing S. aureus strains.

Platelets bind to Candida albicans, the major cause of candidiasis. But in contrast to other microorganisms the fungus does not aggregate platelets. Gliotoxin (GT), which possesses immunosuppressive properties, is produced by various fungi, including the opportunistic pathogens Aspergillus fumigatus and C. albicans . Its mode of action involves the formation of mixed disulfides with host proteins. Disulfide exchanges play an important role in platelet activation. Therefore, the effect of C. albicans and GT on platelet function was tested. C. albicans yeast cells (5,000-10,000 cells/microl) and GT, in pathophysiologically relevant concentrations (0.05-0.5 microM), inhibited platelet fibrinogen binding, anti gp IIb/IIIa antibody PAC-1 binding, aggregation and procoagulant activity in a dose-dependent manner. Alpha granule release, measured via CD62P surface expression, was not affected. Addition of reduced glutathione partially counteracted the effect of C. albicans and GT on platelet fibrinogen binding and platelet aggregation. The C. albicans metabolite GT features antithrombotic properties in addition to its immunosuppressive functions. Since treatment with reduced glutathione partially counteracted the inhibitory effect of C. albicans yeast cells and GT on platelet fibrinogen binding, the antithrombotic activity is likely to depend on the disulfide bridge of this mycotoxin. GT production by C. albicans could contribute to its survival in the blood stream during vascular infections. The knowledge of the underlying mechanisms of the antithrombotic properties might help to treat fungal infections as well as thrombosis.

Objective-Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation.