In Vitro Antifungal Effects And Mechanism Of Polyoxometalates Against Candida Species

Purpose:The incidence of invasive candida infection increases significantly with the increasing number of immunocompromised syndrome（e.g.AIDS）,organ transplant patients,and patients receiving chemotherapeutic agents for cancer treatment.The mortality among patients with invasive candidiasis is as high as 40%.Candida infection remains the most common etiological agent of hospital-acquired bloodstream infections.The antifungal agents for pathogenic fungi are polyenes,echinocandins and triazoles.However,with the wide-spread use of antifungal agents,the incidence of various adverse effects or resistance to antifungal agents derivatives emerged.Therefore,there is an urgent need for developing novel efficient antifungals.In this study,we synthesized a new compound and a nano-dosage compound,which structure,composition,and morphology are characterized.We evaluated the antifungal effect of compounds against different species of Candida fungus,which are C.tropicalis,C.parapsilosis,C.krusei and C.glabratas.The ATCC 22019,ATCC 90028 and ATCC 750 are tested as quality control and standard strains.Antifungal effect of the polyoxometalates against clinical isolates of C.albicans and other yeast fungi（C.tropicalis,C.parapsilosis,C.krusei and C.glabratas）and the possible mechanisms were investigated.Method:The structure,composition and morphology of the test compounds were determined by Fourier transform infrared（FT-IR）and X-ray diffraction（XRD）,scanning electron microscopy（SEM）,transmission electron microscopy（TEM）and elemental analysis and single-crystal X-ray diffraction.We evaluated the antifungal effect of compounds against different species of Candida fungus by microwell dilute assay,growth inhibitor curves.To investigate the antifungal mechanism of compounds,the ergosterol contents were determined by high performance liquid chromatography（HPLC）and real-time PCR.Result:1.Ag₃PW₁₂O₄₀ nanoparticls was successfully synthesized.It has a symmetric dodecahedral shape with average sizes about 800nm.2.Zn₃（FLC）₆V₁₀O₂₈8 was successfully synthesized.The single crystal X-ray diffraction of Zn₃（FLC）₆V₁₀O₂₈8 shows that the compound belongs to monoclinic system and space group is P2_1/c.The unit cell parameter can be divided into three groups:a=16.983（2）?,b=17.773（2）?,c=20.033（3）?,α=90^o,β=110.3（4）^o,γ=90^o.3.15 Candida albicans,1 Candida glabrata,1 Candida krusei,1 Candida parapsilosis,and 1 Cryptococcus tropicalis strains had been screened.The MIC₅₀and MIC₈₀ values of Ag₃PW₁₂O₄₀0 nanoparticles are 2-32μg/m L and 4-128μg/m L,respectively.The MIC₅₀ and MIC₈₀ values of Zn₃（FLC）₆V₁₀O₂₈·10H₂O are 0.5-32μg/m L and 1-128μg/mL,respectively.4.The antifungal activities of Ag₃PW₁₂O₄₀0 and Zn₃（FLC）₆V₁₀O₂₈·10H₂O are time-and dose-depedened.The inhibition ratio of C.albicans HL963 cells at 48 h with the concentrations of 16μg/mL,32μg/m L and 64μg/mL of Ag₃PW₁₂O₄₀reached the peak value 88.53%±0.05%,90.27%±0.12%and 90.40%±0.25%respectively.The inhibition ratio of C.albicans HL973 cells at 48h with the concentrations of 8,16,32μg/mL of Zn（flc）reached the peak value94.81%±2.49%,97.27%±0.99%和98.17%±0.38%,respectively.In comparison among the delay curves,the C.albicans receiving Ag₃PW₁₂O₄₀0 and Zn₃（FLC）₆V₁₀O₂₈·10H₂O had a significant higher inhibition rate than that in the FLC groups（P<0.05）.5.The ergosterol contents of HL 963 in the control,FLC and Ag₃PW₁₂O₄₀extract were 2.20±0.153 mg/m L,0.79±0.118 mg/mL and 0.10±0.003 mg/m L,respectively（P<0.05）.The inhibition rate of ergosterol of the Ag₃PW₁₂O₄₀ group is59.08%±7.23%,which significantly inhibited the biosynthesis of ergosterol on the cell biofilm.The ergosterol contents of HL 973 in the control,FLC and ZnFLC extract were 7.46±0.11,4.29±0.10 and 0.60±0.03 mg/m L,respectively（P<0.05）.The inhibitory rate of ergosterol of Zn₃（FLC）₆V₁₀O₂₈·10H₂O was 92.02%±0.22%,which significantly inhibited the biosynthesis of ergosterol on the cell biofilm.6.The results showed that the expression of ERG1,ERG7,ERG11 of Ag₃PW₁₂O₄₀0 treated-HL963 were significantly upregulated with the fold change relative to control of 1.91±0.19,2.08±0.29 and 1.45±0.17,respectively.The expressions of ERG1,ERG7,ERG11,ERG27 and ERG28 of the Zn₃（FLC）₆V₁₀O₂₈treated-HL 973 were significantly upregulated with the fold change relative to control of 18.11±0.96,11.19±0.47,14.39±3.06,8.07±1.19 and 9.19±0.28,respectively.Conclusion:1.Ag₃PW₁₂O₄₀0 and Zn₃（FLC）₆V₁₀O₂₈ are well-defined and chemically stable.2.Ag₃PW₁₂O₄₀ and Zn₃（FLC）₆V₁₀O₂₈ have anti-candida effects in vitro.3.The mechanism of Ag₃PW₁₂O₄₀0 and Zn₃（FLC）₆V₁₀O₂₈ against C.albicans showed that Ag₃PW₁₂O₄₀0 and Zn₃（FLC）₆V₁₀O₂₈ damaged the fungal cell membrane and reduced the ergosterol content.The expression of ERG which have effects on the synthesis of ergosterol were all significantly upregulated by Ag₃PW₁₂O₄₀0 and Zn₃（FLC）₆V₁₀O₂₈.