Release of biologically active kinin peptides, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin from human kininogens by two major secreted aspartic proteases of Candida parapsilosis

Abstract

In terms of infection incidence, the yeast Candida parapsilosis is the second after Candida albicans as causative agent of candidiases in humans. The major virulence factors of C. parapsilosis are secreted aspartic proteases (SAPPs) which help the pathogen to disseminate, acquire nutrients and dysregulate the mechanisms of innate immunity of the host. In the current work we characterized the action of two major extracellular proteases of C. parapsilosis, SAPP1 and SAPP2, on human kininogens, proteinaceous precursors of vasoactive and proinflammatory bradykinin-related peptides, collectively called the kinins. The kininogens, preferably the form with lower molecular mass, were effectively cleaved by SAPPs, with the release of two uncommon kinins, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin. While optimal at acidic pH (4-5), the kinin release yield was only 2-3-fold lower at neutral pH. These peptides were able to interact with cellular kinin receptors of B2 subtype and to stimulate the human endothelial cells HMEC-1 to increased secretion of proinflammatory interleukins (ILs), IL-1β and IL-6. The analysis of the stability of SAPP-generated kinins in plasma suggested that they are biologically equivalent to bradykinin, the best agonist of B2 receptor subtype and can be quickly converted to des-Arg(9)-bradykinin, the agonist of inflammation-inducible B1 receptors.

Keywords: 3,3′,5,5′-tetramethylbenzidine; ACE; ACN; B1 receptor; B1R; B2 receptor; B2R; BK; BSA; Bradykinin receptors; CBB; Candida parapsilosis; Candidiasis; Coomassie Brilliant Blue dye; DL-2-mercaptomethyl-3-guanidinoethylthiopropionic acid; DMEM; DTT; Dulbecco's modified Eagle's medium; EBM; EGF; ELISA; ESI; ESI-MS/MS; ESI-tandem mass spectrometry; ETD; FBS; FPLC; HC; HK; HKD4; HPLC; HRP; HRP-conjugated streptavidin; IL-1β; IL-6; ILs; IT; Interleukins; Kallikrein–kinin system; LC–MS; LK; LPS; MGTA; MS; NAC; PBS; RP-HPLC; SA-HRP; SAPs; SDS-PAGE; Secreted aspartic proteases; TFA; TMB; UHPLC; YCB; YPD; acetonitrile; angiotensin-converting enzyme; bovine serum albumin; bradykinin; dithiotreitol; domain 4 of high molecular mass kininogen; electron transfer dissociation; electrospray ionization; endothelial basal medium; enzyme-linked immunosorbent assay; epithelial growth factor; fast protein liquid chromatography; fetal bovine serum; high molecular mass kininogen; high-performance liquid chromatography; horseradish peroxidase; hydrocortizon; interleukin-1β; interleukin-6; interleukins; ion-trap; lipopolysaccharide; liquid chromatography–mass spectrometry; low molecular mass kininogen; mass spectrometry; phosphate-buffered saline; reversed-phase HPLC; secreted aspartic proteases; sodium dodecyl sulfate polyacrylamide gel electrophoresis; trifluoroacetic acid; ultra HPLC; yeast carbone base; yeast peptone dextrose medium; “non-albicans” Candida species.