Association Of G487T And T916C Mutations In Candida Albicans ERG11with Fluconazole Resistance

BackgroundCandidiasis is becoming more common, because of the widespread application of broad-spectrum antibiotics glucocorticoid, therapeusis of intervention and organ transplantation and the expanding of population suffered from AIDS, cancer and diabetes mellitus. The utilization of antifungal agents promotes the process of resistance formation. Thus, understanding the molecular mechanisms involved in fluconazole resistance is needed to guide clinical therapy and discover new antifungals.Fluconazole, discovered in1978, is used for treating most infection with Candida albicans, because of broad-antibacterial spectrum, strong antibacterial activity, less adverse effect, safetyly for intravenous injection and so on. However repeated use leads to drug resistance.C. albicans is the most important pathomycete of candiasis. Overseas researches on molecular mechanisms of C. albicans to azoles have been carried out. Up to now, molecular mechanisms involved in fluconazole resistance in C. albicans are upregulation and mutation of ERG11, which encodes the target enzyme of fluconazole; decreased intracellular fluconazole concentration because of overexpression of drug efflux pumps encoded by MDR1, CDR1and CDR2; and formation of biofilms, which protect pathogens against the host immune system and antimicrobial therapies.Ergosterol is essential for maintaining the integrity and function of C. albicans membrane. Cytochrome P450-dependent14a-lanosterol demethylase (Erg11p), encoded by ERG11, is a key enzyme in the ergosterol synthesis pathway of C. albicans. Fluconazole can block the active centre of Ergllp, which influences the ergosterol synthesis. Point mutations in ERG11can lead to amino acid substitutions in Ergllp, which might result in changing the Ergllp spatial configuration and decreased affinity to fluconazole. To date, more than 160different amino acid substitutions of Erg11p have been described.In our previous study, T916C (Y257H) and G487T (A114S) mutations in ERG11were detected in fourteen fluconazole-resistant clinical isolates of C.albicans. However, the involvement of the two mutations in fluconazole resistance has not been studied by experimental proof.ObjectivesTo confirm the relationship between mutations of G487T and T916C in Candida albicans ERG11and fluconazole resistance.Methods1. Fluconazole susceptibility assay and detection of mutations of ERG11Fluconazole susceptibility for GZ16and SC5314was tested in vitro using microdilution to decide sensitive isolates(S), dose-dependent sensitive isolates (S-DD) and resistant isolates (R). Genomic DNA of the two strains was isolated, and then, ERG11was amplified by PCR. PCR products were purified with gel purification kit and sequenced.2. Construction of site-directed mutagenesis cassette and plasmid constructsERG11with G487T, T916C, G487T and T916C and without any mutations were generated by PCR overlapping. The PCR products verified by DNA sequencing were gel purified and ligated to pGM-T to yield pGM-487T pGM-916C, pGM-TWO and pGM-N. The downstream region of ERG11, auxotrophic marker URA3and anti-natamycin gene SAT1were amplified by PCR and ligated to pGM-T to yield pGM-DOWN、pGM-URA3and pGM-SAT1.Plasmids of pGM-916C, pGM-DOWN and pGM-URA3were digested with corresponding enzymes to obtain three fragments with cohesive ends, which were inserted into pcDNA3.1+to construct pcDNA3.1-916C-URA3. Using the same methods, plasmids of pcDNA3.1-N-URA3, pcDNA3.1-487T-URA3, pcDNA3.1-TWO-URA3, pcDNA3.1-N-SAT1, pcDNA3.1-487T-SAT1, pcDNA3.1-916C-SAT1and pcDNA3.1-TWO-SAT1were constructed.With pGM-N, pGM-487T, pGM-916C and pGM-TWO used as templates, the ERG11open reading frame was amplified by PCR. PCR products were cloned into YEP351G to obtain YEP351G-N, YEP351G-487T, YEP351G-916C and YEP351G-TWO.3. Overexpression of ERG11mutations in Saccharomyces cerevisiae INVScl and fluconazole susceptibilityPlasmids of YEP351G-N, YEP351G-487T, YEP351G-916C and YEP351G-TWO were transformed into S. cerevisiae INVScl by alithium acetate method. Yeast synthetic drop-out media without leucine was used to select Leu-positive transformants. Fluconazole susceptibility of transformants was measured by the M27-A2broth dilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI).4. Constructing C. albicans ERG11T916C mutant and fluconazole susceptibilityC. albicans CAI4cells were transformed with the3.7kb fragment from pcDNA3.1-916C-URA3by alithium acetate method. URA-positive colonies were selected by use of yeast synthetic drop-out media without uracil. Fluconazole susceptibility of transformants was measured by the M27-A2broth dilution method.Results1. Fluconazole susceptibility assay and detection of mutations of ERG11Fluconazole MIC was64μg/ml for GZ16and2μg/ml for SC5314. Only T916C and G487T mutations were founded in GZ16. Eight mutations (T462A, T495A, A504G, A530C, C558T, C805T, A1167G, A1587G) were detected in SC5314. Among these eight mutations, T462A (F105L), T495A (D116E) and A530C (K128T) caused amino acid substitutions.2. Construction of site-directed mutagenesis cassette and plasmid constructsPlasmids of pcDNA3.1-N-URA3, pcDNA3.1-487T-URA3, pcDNA3.1-916C-URA3, pcDNA3.1-TWO-URA3, pcDNA3.1-N-SAT1, pcDNA3.1-487T-SAT1, pcDNA3.1-916C-SAT1and pcDNA3.1-TWO-SAT1, which contained site-directed mutagenesis cassette, were successfully constructed. S. cerevisiae expression vectors of YEP351G-N, YEP351G-487T, YEP351G-916C and YEP351G-TWO were also constructed.3. Overexpression of ERG11mutations in Saccharomyces cerevisiae INVScl and fluconazole susceptibilityS. cerevisiae INVScl was transformed with plasmids of YEP351G-N, YEP351G-487T, YEP351G-916C and YEP351G-TWO, and Leu+transformants (INVScl+N, INVScl+487T, INVSc1+916C and INVScl+TWO) were obtained. The MIC vaules of fluconazole were8μg/ml,32μg/ml、32μg/ml、128μg/ml and128μg/ml for INVSc1, INVSc1+N, INVScl+487T, INVScl+916C and INVSc1+TWO.4. Constructing C. albicans ERG11T916C mutant and fluconazole susceptibilityIn the construction of CAI4transformant with T916C mutation, we assayed homologous recombination by identifying URA3+transformants and found five transformants (T1, T2, T3, T4and T5). These transformants were PCR-amplified and digested with Nde I which specifically digested the T916C mutant allele. Only transformants (T1, T3and T5) were founded to contain T916C mutation in ERG11. The MIC values of fluconazole were4μg/ml for Tl, T3and T5and2μg/ml for T2, T4, and the wild-type CAI4.Conclusions1. Plasmids containing site-directed mutagenesis cassette and expression vectors for S. cerevisiae were successfully constructed.2. We firstly confirmed T916C mutation in C. albicans ERG11was associated with fluconazole resisitance.3. Preliminary results showed G487T mutation was not associated with fluconazole resisitance and the two mutations did not play a synergistic role in fluconazole resistance.