Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases

Abstract

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

Extracellular vesicle-mediated export of fungal RNA

“…For EV isolation, yeast cells were transferred to Sabouraud dextrose broth and cultured for an additional 24 h at 25 °C, with shaking. Strain 11 of C. albicans was isolated from a male patient87 and kindly provided by Dr. Dornelas from the Institute of Hematology Arthur Siqueira Cavalcanti (Rio de Janeiro, Brazil). C. albicans cells were maintained at 36°C on Sabouraud dextrose plates.…”

Aspartic Protease Inhibitors as Potential Anti-Candida albicans Drugs: Impacts on Fungal Biology, Virulence and Pathogenesis

“…The arrowheads show the fragmentation of the proteinaceous substrate after proteolysis. For experimental details see Braga-Silva and co-workers [181,261]. PIs, were able to restrain the enzymatic activity of Sap1, Sap2 and Sap3 in a concentration-dependent fashion [175][176][177][178][179], in which ritonavir was by far the most effective inhibitor of these Sap isoenzymes [175].…”

“…Effects of HIV PIs on the Sap2 activity produced by Candida albicans. All the results presented herein were conducted using the strain 11 of C. albicans (originally isolated from the blood of a 46-year-old male), which presents the trailing phenomenon in terms of fluconazole/itraconazole susceptibility and able to produce elevated amounts of Sap2 [261]. (A) Sap2 was incubated for 2 h at 37°C in 20 mM sodium citrate, pH 4.0, in the absence (-) or in the presence (+) of ritonavir or amprenavir, both inhibitors at different concentrations ranging from 6.25 to 200 μM.…”

HIV aspartyl protease inhibitors as promising compounds againstCandida albicansAndré Luis Souza dos Santos

“…Conserved cysteine residues are probably implicated in maintaining the three-dimensional structure of the enzyme [31] . The SAP genes are differentially expressed depending on the C. albicans strain and environmental conditions [32][33][34][35][36] (Figure 1). For instance, eight of these proteases (Saps1-8) are secreted into the extracellular environment, while Sap9 and Sap10 are membrane GPIanchored proteins [37] .…”