The effects of secreted aspartyl proteinase inhibitor ritonavir on azoles-resistant strains of Candida albicans as well as regulatory role of SAP2 and ERG11

Abstract

Background: Candida albicans, the main human fungal pathogen, can cause fungal infection and seriously affect people's health and life. This study aimed to investigate the effects of ritonavir (RIT) on C. albicans and the correlation between SAP2 as well as ERG11 and drug resistance.

Results: Secreted aspartyl proteinases (Saps) activities and pathogenicity of C. albicans with different drug resistance were measured. M27-A4 broth microdilution method was used to analyze the drug sensitivity of RIT combined with fluconazole (FCA) on C. albicans. After that, SAP2 and ERG11 mutations were examined by polymerase chain reaction (PCR) and sequencing, and quantitative real-time PCR was utilized to determine the expression of the two genes. By analyzing pz values, the Saps activity of cross-resistant strains was the highest, followed by voriconazole (VRC)-resistant strains, FCA-resistant strains, itraconazole (ITR)-resistant strains, and sensitive strains. The pathogenicity of C. albicans in descending order was as follows: cross-resistant strains, VRC-resistant strains, ITR-resistant strains, FCA-resistant strains, and sensitive strains. With the increase of RIT concentrations, the Saps activity was gradually inhibited. Drug sensitivity results showed that there was no synergistic effect between RIT and FCA. Additionally, no gene mutation sites were found in SAP2 sequencing, and 17 synonymous mutations and 6 missense mutations occurred in ERG11 sequencing. Finally, the expression of SAP2 and ERG11 was significantly higher in the resistant strains compared with the sensitive strains, and there was a positive liner correlation between SAP2 and ERG11 messenger RNA expression (r = .6655, p < .001).

Conclusion: These findings may help to improve our understanding of azole-resistant mechanisms of C. albicans and provide a novel direction for clinical therapeutics of C. albicans infection.

Keywords: Candida albicans; ERG11; SAP2; secreted aspartyl proteinases.

Figure 1

(A) pz values of Candida albicans with different drug resistance. a: p < .05, compared with the cross‐resistant strains. b: p < .05, compared with the FCA‐resistant strains. c: p < .05, compared with the ITR‐resistant strains. (B) Secreted aspartyl proteinases (Saps) activity of C. albicans treated with different concentrations of ritonavir (RIT). Upper: The formation of colony ring and transparent ring in C. albicans with different concentrations of RIT. Lower: pz value of C. albicans with different concentrations of RIT. a: p < .05, compared with the strains treated with 0 μg/ml RIT. b: p < .05, compared with the strains treated with 2 μg/ml RIT. c: p < .05, compared with the strains treated with 8 μg/ml RIT. FCA, fluconazole; ITR, itraconazole; VRC, voriconazole

Figure 2

(A) The blood culture result of mice treated with C. albicans on Sabouraud dextrose agar medium at 37°C for 48 h. (B) Survival curves of mice treated with C. albicans with different drug resistance. FCA, fluconazole; ITR, itraconazole; VRC, voriconazole

Figure 3

The mRNA expression of SAP2 (A) and ERG11 (B) in the sensitive, cross‐resistant, FCA‐resistant, ITR‐resistant and VRC‐resistant strains. *p < .05, compared with the sensitive strains; ^# p < .05, compared with the cross‐resistant strains. FCA, fluconazole; ITR, itraconazole; mRNA, messenger RNA; VRC, voriconazole