Platelets Can Attack Candida Albicans Hypha Opsonized by Fibronectin
Authors: Stoldt Volker R. , Abtmeier Matthias , Huynh Khon C. , Scharf Rudiger E. 
Published: October 2019

Abstract
Abstract 2210

Background and objectives:
Candida albicans is a dimorphic fungus that can change between its yeast and hypha form. The ability to germinate and grow in hypha is a relevant factor of virulence. 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 to the pathogen and activation of platelets with subsequent secretion of their granular constituents. Fibronectin (Fn), a large dimeric glycoprotein, with specific binding sites for integrins and collagens, circulates in plasma and is part of extracellular matrix of the host. Both platelets and C. albicans are known to interact with Fn. Platelet can bind Fn specifically by their integrins α5β1, αIIbβ3, and αvβ3. Several protein species have been identified on the surface of C. albicans also interacting with Fn. Here, we examined whether or not Fn can support the platelet-mediated host defense against C. albicans.

Material and methods:
Platelet-rich plasma from citrated anticoagulated human blood was washed with phosphate-buffered saline (PBS), pH 6.5, containing 2 U/ml apyrase. The platelet pellet was carefully resuspended in Tyrode buffer, pH 7.3, containing 2 mM MgCl2, 2 mM CaCl2, and 10 μM of the cell tracker dye CMFDA (5-chloromethylfluorescein diacetate, Invitrogen). CMFDA did not affect platelet function, as documented by intact activation and normal adhesion, aggregation, secretion, and thromboxyne A2 formation. Washed and stained platelets were adjusted at a concentration of 2.5 × 108 plt/ml. Platelet aggregation was induced by 40 nM phorbol 12-myristate 13-acetate (PMA) or 10 μg/ml collagen and recorded by changes in light transmission. Fn was purified from human fresh-frozen plasma by gelatine sepharose affinity chromatography and subsequent elution with 3 M urea. The isolated adhesive protein was conjugated with alexa fluor 488 according the manufacturer's instruction (Invitrogen). C. albicans, cultured over night in yeast extract pepton glucose medium at 30°C, was washed twice in PBS, pH 7.3, and starved for 1 h at room temperature in PBS. C. albicans was diluted in PBS supplemented with fetal calf serum (10 %) to adjust a final optical density of 0.4 as a measure of hypha formation and to induce germination at 37°C. Samples were taken at various times of germination. For flow cytometric binding studies, serum was washed away and C. albicans yeast and hypha forms were incubated for 1h with 40 μg/ml Fn-alexa fluor 488 or 1×107 fluorescently CMFDA-stained platelets were added under static conditions.

Results:
C. albicans germination (mean + SD) at 30 min, 60 min, and 120 min of incubation revealed 5+4 %, 40+9 %, and 90+12 % hypha, as determined by light microscopy and confirmed by flow cytometry, as quantified by their characteristic forward and side scatter profiles. C. albicans hypha induction increased binding of Fn-alexa fluor 488 (40 μg/ml) from 1.2+0.3 % positive yeast cells (baseline), to 9.4+2.9 % at 30 min, 23.0+3.4 % at 60 min, or 45.0+5.1 % at 120 min, respectively (p<0.05, each). As observed by microscopy, C. albicans germination tubes promoted the binding Fn. Upon incubation, germation of C. albicans, obtained after 120 min, increased its binding to washed platelets 5-fold, as compared to the yeast form (p<0.05). Moreover, pretreatment of C. albicans hypha with Fn (40 μg/ml) enhanced their specific binding to platelet integrin αIIbβ3, as abciximab (2 μg/ml) blocked Fn binding to C. albicans completely.

Conclusion:
Hypha of C. albicans, a relevant factor of virulence, can be attacked by platelets. Fibronectin, a ligand of the β1 and β3 platelet integrins, opsonizes the hypha forms of C. albicans and supports platelet binding. The multiple binding sites in this adhesive protein for collagen and integrins can support the platelet-mediated host defense and may modify adaptive immune responses against C. albicans.

Disclosures:
No relevant conflicts of interest to declare.