The Mechanism Of Phenylbutyric Acid Combined With Fluconazole Against Resistant Candia Albicans

The pathogenic fungus Candida albicans(C.albicans)is one of the most commonly clinically isolated fungal species,and its resistance to the antifungal drug fluconazole is increasing.Combination therapy is an effective approach against drug-resistance.Candida species are common opportunistic fungal pathogens,C.albicans is the most common.They can cause mucosal(oropharyngeal,vaginal,urinary tract)infections and less commonly skin,nail,and systemic infections.Azole antifungal drugs are the first-line drugs for the treatment of Candida infection.However,along with the wide application of antifungal drugs,Candida species are gradually become resistant to antifungals,especially to fluconazole(FLC).A number of physiological attributes of yeast were associated with azoles resistance,such as transport alterations(CDR,MDR and FLU),target alterations by mutations and gene up-regulation(ERG),biofilms formation and abnormal expression of heat shock protein.Biofilm formation is one of the important mechanism of the fungal resistance,and it result in a great challenge to clinical treatment.A large amount of research has been conducted to elucidate the underlying biofilm resistance mechanisms in C.albicans cells.Similar to bacterial biofilms,compared to planktonic cells,fungal biofilms exhibit up to 20,000-fold increase in antifungal MICs(minimum inhibitory concentrations).In addition,it has been shown that cells within a biofilm structure are also less sensitive to our immune system.Sodium phenylbutyrate(PBA)is a derivative of the short-chain fatty acid butyrate and is approved for treatment of urea cycle disorders and progressive familial intrahepatic cholestasis type 2.PBA has pleiotropic functions,as a stress inhibitor,ammonia sink,chemical chaperone and histone deacetylase inhibitor(HDACI)HDACIs have great potential for treating cancers and immunological diseases.PBA also has beneficial effects against salmonella enterica during infection.An existing research study showed that the inhibition of HDACs by trichostatin A(TSA)could reduce the growth of C.albicans.If PBA has antifungal activity is not clear.In this paper,we explore the possible mechanism from three aspects.1 Antifungal effects of FLC in combination with PBA against biofilms of C.albicansThe anti-biofilm effect of different drugs was evaluated by hyphae observation.The control group,which was untreated with any drug presented large amounts of biofilm consisting of abundant filamentous and yeast forms.C.albicans(CA10)treated with FLC or PBA alone produced biofilms composed of yeast cells and hyphae.Almost no difference was observed for C.albicans(CA10)treated with PBA or PBA alone compared to the control.C.albicans(CA10)treated with FLC and PBA,had biofilms with no observed microcolonies of yeasts and hyphae.The results of RT-PCR assays showed that FLC alone caused down-regulation in the expression levels of ALS1,ALS3,HWP1 and up-regulation in the expression levels of EFG1compared with those of the control group.The combination of FLC and PBA significantly down-regulated the expression levels of ALS1,ALS3 and EFG1 compared with those for the FLC alone by a greater than 2-fold,6-fold,and 3-fold,respectively.The combined group down-regulated the expression level of HWP1 compared with that for the FLC-alone groups,but there was no significant difference.The expression level of RAS1 was down-regulated by the FLC or PBA challenge compared with that of the control group.The combined group down-regulated the expression level of RAS1 compared with that for the FLC-alone groups,but there was no significant difference.The expression level of CYR1 was increased with the FLC challenge by 3-fold compared with that of the control group.The combination of FLC and PBA significantly down-regulated the expression level of CYR1 compared with that of the FLC alone by greater than 3-fold,respectively.The expression level of TPK2 was slightly increased with the FLC challenge compared with that of the control group.The combination of FLC and PBA significantly down-regulated the expression level of TPK2 compared with that of the FLC alone by greater than 100-fold.The penetration of FLC through C.albicans biofilm was evaluated using“Sandwich”model.The combined concentration results were 512?g/mL for FLC and 32?g/mL for PBA.The diameter of inhibition zone(16mm)for the group treated with the combination of PBA and FLC was larger than the diameter of inhibition zone(10mm),of those treated with FLC alone.The results showed that PBA can enhance the effect of FLC penetrating the C.albicans biofilm.2 The effect of PBA cominbined with FLC against C.albicans virulenceOur studies have been carried out on the two virulence factors:phospholipase activity and the expression of aspartyl proteinase(SAP)genes.The combined treatment could decrease the phospholipase activity and significantly down-regulate the expression levels of SAP genes.The phospholipase activity was determined by measuring the size of the precipitation zone(Pz)after C.albicans growth treated different drugs on egg yolk agar treated with different drugs.Next,10-?l aliquots of a suspension(10~7 CFU/mL)were inoculated onto the egg yolk agar medium plates.The plates were incubated at 37°C for 72h,then,the Pz value was measured.Pz value was expressed as the ratio of the diameter of the colony to the diameter of the precipitation zone.The value of Pz was 0.90±0.03,which meant C.albicans(CA10)treated FLC(1?g/mL)with PBA(32?g/mL)means had very low phospholipase activity.And,the Pz values,of C.albicans(CA10)without drugs and treated with only FLC(1?g/mL),meant the phospholipase activity was very high.In addition,the value of Pz was0.70±0.02,which the group treated with PBA(32?g/mL)alone means the high phospholipase activity.The results of RT-PCR assays showed that the expression levels of SAP1 to SAP4 were decreased with the FLC challenge by 2-fold compared with those of the control group.The combination of FLC and PBA significantly down-regulated the expression levels of SAP1,SAP2,and SAP3 compared with those of the FLC challenge alone by 2-fold,respectively.The combined group down-regulated the expression level of SAP4 compared with that of the FLC-alone groups,but there was no significant difference.3.Rhodamine 6G efflux assays and efflux gene expression levelsFor the purpose of testing whether the presence of PBA could inhibit drug efflux,we investigated the activity of efflux pumps by rhodamine 6G assay and determined the expression of drug-resistant genes CDR1,CDR2 and MDR1 via RT-PCR.The results of the rhodamine 6G efflux assay indicated that the addition of PBA did not inhibited the efflux of rhodamine 6G in 120 minutes.No difference was observed within 120 minutes.The results of RT-PCR assays showed that FLC alone caused up-regulation of CDR1,CDR2 and MDR1expression compared with that of the control group.And,there is no significant differences of control group and drugs alone group,as well as drugs alone group and drugs combined group were observed.In conclusion,PBA has an obvious antifungal activity and has synergistic activity combined with azoles against resistant C.albicans.The synergistic mechanism may be related to enhancing the effect of FLC penetrating C.albicans biofilm,inhibiting biofilms formation regulating genes and attenuating C.albicans virulence.The surprising discovery from this study provides reference for the PBA widely used in the field of anti-fungal infection and encourages us to consider the future use of the combination of azoles and PBA against fungi.Moreover,PBA has been demonstrated to have wide effects with different mechanisms,and a more in-depth study of the mechanisms are highly warranted.