| Candida albicans is one of the most important opportunistic fungal pathogens. Its infections, especially its invasive infections, were being paid attention due to the increase of ICU and immunocompromised individuals and the abuse of antibiotics. C. albicans is a typical diomorphic fungus, having the ability of morphogenesis, such as hyphal development and biofilm formation. This ability is closely associated with the morphogenesis regulation network and the secretory pathway. Moreover, This fungus evolved an elaborate stress response system to sense environmental stimuli, such as antifungal drugs, oxidative agents and nutrient depletion, and to respond to them for survival and growth. Both morphogenesis and stress response are essential for this fungus to effectively colonize and infect towards the host. Abundant evidence showed that the calcium homeostasis system plays an important role in these physiological processes.Both the endoplasmic reticulum(ER) and the vacuole are of significance in C. albicans cells. The ER is the centre of the secretory pathway, and is the main organelle mediating stress response, while the vacuole is the main calcium store which is indispensible for calcium homeostasis. Although many members of the calcium homeostasis system have been elucidated, the ER pump and the vacuolar channel has not been well characterized. Furthermore, although the role of calcium channels in morphogenesis and stress response has been confirmed, there has not yet been any strategy to inhibit these channels. Therefore, in this study, we identified and characterized both the ER pump and the vacuolar channel Yvcl, and investigated the effect of calcium channel blockers on morphogenesis and stress response in C. albicans. The main results were demonstrated as follows.(1) By BLASTP analysis, we identified an ER pump and named it Spfl. This protein belongs to the V-type P-ATPase family. By nickel affinity chromatography and ATPase assays, Spfl is confirmed to have the ability of ATPases. The SPF1-disrupted strain spf1Δ/Δ showed decreased growth rate under normal growth conditions, reduced ability of hyphal development under hyphal-inducing conditions, and a defect in biofilm formation, flocculation and adhesion. Moreover, the mutant displayed hypersensitivity to such environmental stresses as antifungal drugs, low or high calcium levels, oxidative agents, alkaline pH and membrane damage. Systemic infection model further demonstrated that the mutant was severely attenuated in virulence. These results revealed that Spfl plays an important role in cell growth, morphogenesis and stress response in C. albicans. Therefore, it is an key virulence factor of this pathogen.(2) The effect of SPF1disruption on cytoplasmic and total calcium content was determined by fluo-3staining and atom absorption spectrophotometry. It was confirmed that Spfl functions as an key member of the calcium homestasis. Westerin blotting and EndoH analysis revealed that the disruption of SPF1led to the defect in N-glycosylation of Cdc101and secreted acid phosphatase (SAP). Moreover, Spfl is essential for cell wall integrity (CWI). The disruption caused hypersensitivity to cell wall stresses, decreased cell wall reconstruction, and abnormal cell wall compositions. Due to the important role of this protein, disruption of SPF1affected the expression of many genes, resulting in up-regulation of the calcium response gene CCH1, the CWI genes PGA13, ECM331, DFG5and the oxidative stress response gene OYE32, and down-regulation of the ER essential gene SEC61and the morphogenesis-related genes HWP1, ALS3and ECE1. These results revealed the close link between the calcium homeostasis system, the ER stress response system and the morphogenesis regulation network.(3) The vacuolar calcium channel Yvcl is also necessary for stress response, morphogenesis and pathogenicity of C. albicans. In terms of stress response, yvc1Δ/Δ displayed increased sensitivity to low or high calcium levels, alkaline pH and oxidative agents, defects in calcium fluctuation under alkaline or hypertonic stimulus, decreased levels of the antioxidases and down-regulation of oxidative stress response genes. As for, morphogenesis, the mutant had defects in hyphal development, flocculation, invasive growth and biofilm formation, tip-localization of the vacuole and the Spitzenkorper, and calcium accumulation at the tip, and displayed decreased expression of morphogenesis-related genes, indicating an essential role of Yvcl in morphogenesis of C. albicans, which is associated with the expression of morphogenesis-related genes and polarized growth. Moreover, the mutant showed decreased virulence during systemic infection and reduced ability of invading, damaging host epithelial cells and re-orienting to the cells, suggesting that Yvcl is another important virulence factor in C. albicans.(4) The calcium channel blockers verapamil and nifedipine could significantly inhibit calcium fluctuation of C. albicans cells under alkaline stimulus. However, while verapamil could also inhibit calcium influx under the stress caused by antifungal drugs, nifedipine showed no inhibitory effect under this stress. When used in combination with the antifungal drugs, fluconazole and tunicamycin, verapamil could enhance the inhibitory effect of antifungal drugs against this fungus, whereas nifedipine could not. Furthermore, verapamil inhibited hyphal development, biofilm formation and biofilm maintenance, and enhanced the inhibitory effect of antifungal drugs against biofilms. RT-PCR assays further revealed that verapamil alone or in combination with antifungal drugs led to decreased expression of the morphogenesis-related gene, ALS3, suggesting that the effect of verapamil on morphogenesis is associated with down-regulation of morphogenesis-related genes in C. albicans. These results revealed the inhibitory effect of verapamil on drug tolerance and morphogenesis, providing a novel strategy against C. albicans infections. |
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