Effect Of Pyrvinium Pamoate Alone And In Combination With Azole On Exophiala Dermatitidis And Aspergillus Fumigatus

Objective1.The effect of Pyrvinium Pamoate(PP)on ExtrR in Exophiala dermatitidis was determined.To clarify the effect of ExtrR on the growth,virulence and drug sensitivity of Exophiala dermatitidis.To elucidate the regulatory mechanism of ExtrR in PP alone and in combination with azole for anti Exophiala dermatitidis.2.The related targets affecting the synergistic effect of PP and azole in the knockout strain bank of Aspergillus fumigatus were screened.3.The effects of the new ABC transporter ROA1 in Aspergillus fumigatus knockout bacteria bank on the sensitivity,growth and oxidative stress of azole drugs were further analyzedMethod1.The E.dermatitidis knockout strain △ ExtrR was constructed by electroporation.The in vitro drug sensitivity of PP combined with azoles was detected by microdilution method.E-test was used to detect the sensitivity of the isolates to azoles.The role of ExtrR in the growth and virulence of E.dermatitidis was determined by growth curve measurement,colony diameter measurement,and fungal morphology observation(small culture).2.With reference to CLSI M38-A2 and M44-A2 protocols of the American Institute of Clinical Laboratory Standardization,the in vitro synergic antibacterial effect of PP combined with itraconazole(ITR),Voriconazole(VOR)and Posaconazole(POS)was detected by using the microliquid-based dilution checkerboard method,and the reversed PP synergic azole strains in the knockout bacteria bank of Aspergus fumigatus were screened.To further clarify the antifungal mechanism of PP in collaboration with azole.3.A mutant strain of Aspergillus fumigatus △ROA1 was constructed using a high-throughput gene knockout protoplasts method,and its sensitivity to azole drugs was tested in vitro.The role of △ROA1 in the growth and virulence of Aspergillus fumigatus was determined by fungal morphology observation(small culture),osmotic pressure measurement,intracellular reactive oxygen species test and oxidative stress test.RT-PCR was used to detect the expression of cyp51,MDR1,CDR1 b,Srb A and Atr R genes to determine the resistance mechanism of △ROA1.Results1.Compared with the E.dermatitidis 34 wild strain(WT),the △ExtrR knockout strain grew slower than the E.dermatitidis 34 wild strain WT.There was no significant difference in colony diameter.E-test drug sensitivity test showed that the MIC values of ITR,VOR and POS to △ExtrR were lower than those of WT,and the sensitivity of △ExtrR to VOR was significantly higher than that of WT(P < 0.05).The sensitivity of△ExtrR to ITR and POS was not significantly different from that of the wild type strain(P > 0.05).The combined PP and azole susceptibility test showed that the △ ExtrR knockout strain and the wild type strain had a synergistic antifungal effect of POS in vitro.2.Oxidoreductase,The short chain dehydrogenase/reductase family superfamily oxidase-related gene AFUA＿8G01550,the MFS transporter related gene AFUA＿2G05840,SET,and the WW domain protein AFUA＿5G06000 and FAD-dependent oxidoreductase-related gene AFUA＿7G05070 was screened from Aspergillus fumigatus knockout library by microliquid-based checkerboard method.Their deletion mutants could reverse the synergistic antifungal effect of PP and POS.3.Fungal morphological observation(small culture)showed that the sporulation rate of △ROA1 was lower than that of the wild type strain WT.The drug sensitivity test of microliquid-based dilution method showed that the sensitivity of △ROA1 to ITR and VOR was significantly decreased(P < 0.05).E-test drug sensitivity test showed that the MIC values of △ROA1 to ITR,VOR and POS were higher than those of WT,but the difference was not statistically significant(P > 0.05).Osmotic pressure measurement showed that there was no significant difference in colony diameter between △ROA1and WT after Na Cl addition(P >0.05).After the addition of D-sorbitol,the colony diameter of △ROA1 was significantly larger than that of the wild strain,and the sensitivity of △ROA1 to D-sorbitol decreased,with significant differences(P< 0.05).Intracellular ROS assay showed that there was no significant difference in ROS production between △ROA1 and WT(P >0.05).The results of oxidative stress test showed that the colony diameters of △ROA1 and WT were significantly different at0 m M,0.2m M,2.5m M(P < 0.05).The colony diameter of △ROA1 was significantly larger than that of WT.The colony diameter of 0μM,5μM and 15μM △ROA1 in menadione group was significantly larger than that in WT(P < 0.05).RT-PCR showed that cyp51 B,MDR4,Srb A gene expression of △ROA1 was significantly increased(P <0.05).Conclusion1.In E.dermatitidis,the ExtrR deletion mutant △ExtrR is more sensitive to VOR,and ExtrR may affect the growth rate and morphology of E.dermatitidis.The current experimental results are not sufficient to prove that ExtrR is involved in the mechanism of PP-azole synergism against E.dermatitidis,and the specific regulatory mechanism of PP-azole synergism against E.dermatitidis needs to be further studied.2.The short chain dehydrogenase/reductase family superfamily oxidase-related gene AFUA＿8G01550,the MFS transporter related gene AFUA＿2G05840,SET and the WW domain protein AFUA＿5G06000 and FAD-dependent oxidoreductase-related gene AFUA＿7G05070 may be the targets of PP and POS against Aspergillus fumigatus.3.The absence of ROA1 gene in Aspergillus fumigatus leads to decreased sensitivity to ITR and VOR,and the mechanism may be related to the significant increase in cyp51 B,MDR4 and Srb A gene expressions of ROA1.ROA1 may also be involved in the regulation of mycelium sporulation rate,oxidant and osmotic pressure stability.