Kinetic and thermodynamic characterization of the interactions between the components of human plasma kinin-forming system and isolated and purified cell wall proteins of Candida albicans

Abstract

Cell wall proteins of Candida albicans, besides their best known role in the adhesion of this fungal pathogen to host's tissues, also bind some soluble proteins, present in body fluids and involved in maintaining the biochemical homeostasis of the human organism. In particular, three plasma factors - high-molecular-mass kininogen (HK), factor XII (FXII) and prekallikrein (PPK) - have been shown to adhere to candidal cells. These proteins are involved in the surface-contact-catalyzed production of bradykinin-related peptides (kinins) that contribute to inflammatory states associated with microbial infections. We recently identified several proteins, associated with the candidal cell walls, and probably involved in the binding of HK. In our present study, a list of potential FXII- and PPK-binding proteins was proposed, using an affinity selection (on agarose-coupled FXII or PPK) from a whole mixture of β-1,3-glucanase-extrated cell wall-associated proteins and the mass-spectrometry protein identification. Five of these fungal proteins, including agglutinin-like sequence protein 3 (Als3), triosephosphate isomerase 1 (Tpi1), enolase 1 (Eno1), phosphoglycerate mutase 1 (Gpm1) and glucose-6-phosphate isomerase 1 (Gpi1), were purified and characterized in terms of affinities to the human contact factors, using the surface plasmon resonance measurements. Except Gpm1 that bound only PPK, and Als3 that exhibited an affinity to HK and FXII, the other isolated proteins interacted with all three contact factors. The determined dissociation constants for the identified protein complexes were of 10(-7) M order, and the association rate constants were in a range of 10(4)-10(5) M(-1)s(-1). The identified fungal pathogen-host protein interactions are potential targets for novel anticandidal therapeutic approaches.

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen

“…As described in a previous report [73], these bacteria have the ability to bind HK and other components of the contact system on the cell surface mainly through the surface-exposed gingipains, thereby facilitating the production of kinins as a result of this adsorption. This phenomenon has been also described in detail for C. albicans, and several HK-binding proteins present at the surface of the fungal cells have been indicated [59,60], some of which have been identified by our present analysis as susceptible to citrullination by PPAD, including Als3 and Eno1. However, these modifications did not affect the level of HK binding to the modified fungal surface, nor did HK citrullination play a role in this process, unlike the gingipains which significantly reduced the binding of HK to fungal cells.…”

“…Such abilities to entrain plasma cascades and thus evade host innate immunity were previously described for the opportunistic pathogen C. albicans [55,56]. A number of C. albicans surface-exposed proteins responsible for binding of human HPG or HK have been identified to date [57][58][59][60]. The consequence of HK binding may be the production of biologically active kinins capable to interact with B2 and B1 receptors, and also to stimulate human cells to produce proinflammatory cytokines [45,46,61].…”

Plasma contact factors as therapeutic targets

“…Its capacity to activate FXI could reflect the importance of thrombin generation and coagulation to one or more functions not directly tied to hemostasis at a site of vascular injury. For example, contact activation-induced coagulation may limit microorganism spread [67,68], a process that falls under the broad umbrella of immunothrombosis [69,70]. Inappropriate triggering of contact-initiated coagulation could contribute to pathologic thrombus formation.…”

Characterization of the interactions between human high-molecular-mass kininogen and cell wall proteins of pathogenic yeasts, Candida tropicalis

“…The mixture of proteins extracted from the cell wall of C. tropicalis pseudohyphae with β-1,3-glucanase was dialyzed against 20 mM Tris-HCl buffer pH 8.0, centrifuged for 15 minutes at 15 000 × g, and applied to a pre-equilibrated Resource TM Q 1 ml column (GE Healthcare, Uppsala, Sweden). Protein separation was carried out with a Knauer (Berlin, Germany) chromatograph (equipped with an HPLC Pump K-1001 with a solvent organizer/proportioning valve K-1500, a UV detector 2600 with a control unit IF2, and EuroChrom software for pump control, data acquisition, and analysis); the elution was performed with a 20 ml linear gradient from 20 mM Tris-HCl buffer pH 8.0 without NaCl to the same buffer with 0.5 M NaCl, at a flow rate of 1 ml/min, with detection based on absorbance measurements at 280 nm, in accordance with the protocols described previously (Bras et al, 2012;Seweryn et al, 2015). The homogeneity of eluted proteins was confirmed with SDS-PAGE electrophoresis and identification thereof was carried out with mass spectrometry (Method 2) as described below.…”

“…Protein identification with mass spectrometry. Particular fungal proteins were identified after electrophoretic separation by SDS-PAGE in the Laemmli system and manual excision of selected bands from gel as described in detail elsewhere (Seweryn et al, 2015). Two methods based on liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) were used for the analysis of peptides obtained after protein digestion with trypsin.…”