Johnsson, E

Johnsson, E. of activates all three pathways of complement (33). The CP is usually activated via mannan-specific Protirelin immunoglobulin G antibodies (54), which are commonly found in human serum, and more than 70% of healthy blood donors have IB1 these antibodies (17). Mannan, one of the major components around the yeast cell surface (46), activates the LP. Furthermore, C3b molecules bind directly to the surface of and cause AP activation (34, 35). Although activates all three pathways, it is unclear how this yeast inactivates toxic complement activation products and how the yeast inhibits subsequent opsonization and phagocytosis. C4b-binding protein (C4BP) is the major fluid-phase inhibitor of the CP and LP (1). This human serum protein consists of one -chain and seven identical -chains, all of which are composed of complement control protein (CCP) domains. The -chain consists of eight CCPs, and the -chain consists of three CCPs (10, 29, 49). Disulfide bonds between the – and -chains and hydrophobic interactions keep the 570-kDa molecule together (32). C4BP regulates complement by binding to C4b (31) via the N terminus of each -chain (6, 15), thereby making it susceptible to degradation by a plasma serine proteinase factor I and by accelerating the decay of the CP C3-convertase C4b2a (25). C4BP also inhibits the activity of the AP C3-convertase in a fluid phase (23) and acts as a cofactor in factor I-mediated cleavage of C3b (5). The number of pathogenic microbes which are able to bind and utilize human complement inhibitors is increasing. So far, two pathogens, i.e., (31, Protirelin 39, 50) and (46, 47), are known to bind the CP inhibitor C4BP and also the AP inhibitors factor H and FHL-1. The binding is mediated by the streptococcal M-proteins (31, 39, 50) and the porins of (46, 47), both of which have multiple, partially overlapping binding domains for the three host regulators. We have recently shown that evades the AP of complement by binding factor H and FHL-1 via two different binding moieties on the surface (38). Given the activation of all three pathways on the surface of candida, it was of interest to analyze the evasion mechanisms of this pathogenic yeast for the CP and LP. In this study, we report a novel mechanism for to control and inhibit CP activation. The cellular and hyphal forms bind C4BP from human serum. Surface attachment was confirmed with purified native and recombinant proteins. Using a novel enzyme-linked immunosorbent assay (ELISA), the binding site of C4BP Protirelin was localized within the -chain to CCP1-2. As FHL-1 competes binding of C4BP to the surface of strains and growth conditions. The wild-type SC5314 (19) strain of was used in experiments. In addition, strains ATCC 18804 and EBP, SEY6210 were used (strain collection of the Hans Kn?ll Institut). Yeasts were grown at 28C on a shaker for 16 h in 5% Sabouraud agar (Biokar; Diffchamb, Gothenburg, Sweden). After centrifugation (5 min at 3,800 cells and induced hyphal forms (107) were incubated at room temperature (RT) for 60 min with NHS treated with EDTA (1:3 dilution), and hyphae were incubated with purified C4BP (10 g/ml). After incubation, the samples were washed three times with ice-cold Protirelin PBS supplemented with 1% bovine serum albumin (BSA; 1% BSA-PBS), and nonspecific binding sites were blocked with the same buffer for 30 min at RT. The samples were incubated with MAb70 overnight at 4C (20 g/ml). After three washes with 1% BSAPBS, rabbit anti-mouse immunoglobulin G, labeled with Alexa 647 or FITC, was added at a dilution of 1 1:50 in 1% BSA-PBS at RT. Factor H was visualized with a polyclonal antiserum specific for the N-terminal domains of the protein (anti-CCP1-4 and a secondary goat anti-rabbit antibody labeled with Alexa 488). The samples were washed three times with 1% BSA-PBS and examined with a laser scanning microscope (LSM 510 META; Zeiss, Jena, Germany). The stained cells were also examined by flow cytometry (FACScan; Becton-Dickinson, Heidelberg, Germany). Forward scatters were used to define the cell population, and 10,000 events were routinely counted. Cells were also treated with trypsin (50 mg/ml in PBS, 60 min at 37C) to cleave the proteins from the outer surface. Afterwards, cells were incubated in NHS-EDTA, and binding of C4BP was measured in a flow cytometer. Serum absorption experiments. Cells and hyphal forms of strain SC5314 and cells of strains EBP and ATCC 18804 and (5 109) were incubated in NHS-EDTA (1:3 dilution) for 60 min at 37C. The cells were washed five times with a washing buffer (100 mM NaCl, 50 mM Tris-HCl, 0.05% Tween 20 Protirelin [pH 7.4]). Proteins bound to the surface were eluted with 3 M KSCN, and the supernatants were collected. Aliquots of the.