Impaired Platelet Attack by fibronectin Binding Partner Msb2 of Candida Albicans

Platelets can bridge innate and adaptive immunity. To attack microorganisms, platelets store microbicidal and immune stimulatory proteins in their α-granules. Platelet integrins and their plasma ligands can govern both binding of the pathogens and activation of platelets with subsequent secretion of their granular constituents. Fibronectin, a large dimeric glycoprotein, with specific binding sites for platelet integrins (α5β1, αvβ3, and αIIbβ3) and collagens, circulates in plasma and is a component of the extracellular matrix. C. albicans can also bind fibronectin. The ability to germinate and grow in hyphae is a relevant factor of virulence in vivo. The integrity of the C. albicans cell surface during filamentation is sensed by Msb2, a transmembrane protein. Recently, it has been shown that Msb2 can be shedded, thus providing a putative mechanism of C. albicans to escape from the host attack. We now hypothesized that Msb2 has a binding site for plasma fibronectin and that shedded (soluble) Msb2 can protect C. albicans from opsonization by fibronectin.

Fibronectin purified from human fresh frozen plasma by gelatin-sepharose affinity column was labeled with alexa fluor 488 according the manufacturer's instructions. To induce germination, C. albicans was cultured in complete medium (yeast extract, peptone, and dextrose) at 30°C, washed and starved for 1 h at room temperature before dilution in PBS supplemented with fetal calf serum (10 %) to a final optical density of 0.4 at 37°C. Msb2 heme agglutinin (HA)-tagged purified by affinity chromatography using anti-HA antibody-coupled agarose beads was kindly provided by J. Ernst (Szafranski-Schneider E, PLoS Pathog. 2012;8(2):e1002501). C. albicans yeast and hyphae forms or plates (24 wells) coated with 10 μg/ml purified HA-tagged Msb2 or 10μg/ml unlabeled fibronectin, and blocked with 1% BSA were incubated with various concentrations fibronectin-alexa fluor 488 for 60 min or 120 min. Flow cytometry (FACS, Beckman Coulter), confocal laser scanning microscope (LSM, Zeiss) and fluorometer (Fluoroskan Ascent, Thermo Scientific) were used to measure the fluorescent fibronectin.

Binding of 5, 10, 50, and 100 μg/ml fibronectin to 10 μg/ml immobilized Msb2 revealed saturation at 10 μg/ml fibronectin. Immobilized Msb2 bound 2.6 fold more fluorescent fibronectin than BSA (p=0.016). Immobilized fibronectin bound 30 % more fluorescent fibronectin than Msb2 and significantly more than BSA (p<0.01). C. albicans germination over time at 30 min, 60 min, and 120 min generated 5–10 %, 40 %, and 90 % hyphae, as determined by microscopy and confirmed by flow cytometry. Induction of C. albicans hyphae enhanced binding of fibronectin (40 μg/ml) from 1.7 % positive yeast cells (baseline), 6 % at 30 min, 11.7 % at 60 min, and 12.6 % at 120 min (p<0.05). As shown by LSM, C. albicans germ tubes are a main target for fluorescent fibronectin. Germination of C. albicans yeast form increased binding of platelets up to fivefold after 30 min and twentyfold after 60 min (p<0.05). Moreover, pre-treatment of C. albicans hyphae with fibronectin (40 μg/ml) enhanced the binding of platelets caused by integrin αIIbβ3 fibronectin interaction. Abciximab (2 μg/ml), a specific αIIbβ3 antagonist, blocked binding of platelets to C. albicans opsonized by fibronectin. Binding of fluorescent fibronectin was not homogeneously distributed on the cell surface of C. albicans and upon addition of soluble Msb2 binding of fluorescent fibronectin diminished as documented by LSM.

Hyphae of C. albicans, a relevant factor of virulence, are attacked by platelets. Fibronectin, a ligand of the platelet integrins_, opsonizes the hyphae of C. albicans and fosters platelet binding to C. albicans. Hence, platelets and fibronectin, with its intrinsic multivalent binding sites to collagen and platelet integrins (α5β1, αvβ3, and αIIbβ3) can support the platelet-mediated host defense against C. albicans. Msb2 is a novel fibronectin binding protein. Binding of fibronectin to C. albicans is impaired in the presence of soluble Msb2. Hence, shedding of Msb2 in vivo can protect C. albicans against the platelet-mediated host attack.

No relevant conflicts of interest to declare.