Study On The Correlation Between Resistance Genes Expression In Candida Albicans With G487T And T916C Mutations And Fluconazole Resistance

The genus Candida (especially Candida albicans) is the important cause of illness that leads to opportunistic mycoses and one of the main pathogens of nosocomial fungal infections. Following the widespread application of immunodepressants, glucocorticoid, broad-spectrum antibiotics, therapeusis of intervention, organ transplantation and the expanding of population with immunity inhibition, the infection rate of fungi is adscendent tendency in the lately years. The infection caused by pathomycetes candida albicans was the most common.The azole agents is the most extensively used in the treatment of fungal infections. Especially fluconazole, as the first line azole derivatives, because of broad antibacterial spectrum, strong antibacterial activity, few side effect, safetyly for intravenous injection and so on, is commonly used to treat various candidiasis in clinic since the advent of fluconazole in the late 1980s. However, With the mass use of antifungal agents, resistant strains emerge more frequently under the drug selection pressure and prolonged administration of broad-spectrum antibacterial agents or prophylactic treatment. In 1980, Rosenblatt et al. had reported azole drugs resistance in treating chronic mucocutaneous candidids with ketoconazole for the first time. After that, the phenomenon of drugs resistance are increasing gradually.Ergosterol is typical lipidd in cellular membrane of fungi. It can maintain the integrity and function of Candida albicans (C. albicans) membrane.14 alpha-demethylase (Erg11p) is a key enzyme in the ergosterol synthesis pathway of C. albicans. The coding gene is ERG11. Mutations in ERG11 can result in changes in the Ergllp spatial configuration, affect the affinity between the azole drugs and the enzyme, and makes isolates resistant to azole finally. The other is that increased expression of the ERG11 gene associated with antifungal drug resistance. The next possible mechanism for drug resistance is over-expression of the CDR1 and CDR2 genes, encoding transporters of the ABC family, and the MDR1 gene, coding for a major facilitator transporter. The homology of CDR1 and CDR2 is greater than 80%, they can have a synergistic effect in azole drug resistance. Another major facilitator gene, FUU1, has also been identified in C. albicans. The FLU1 gene, similar to MDR1, is related with azole resistance. It is reported that this gene can increase azole susceptibility when it is deleted in the study about fluconazole-resistant candida albicans. Besides that, biofilms may be a new mechanism that correlated with resistance development in C. albicans. Biofilms may be associated with poor prognosis in untreated systemic infections. And finally, vesicular vacuoles from resistant isolates may act as a novel fluconazole resistance mechanism.Occurrence of drug resistance is a complex process involving many different factors. In an analysis about the prevalence of mechanisms of resistance in C. albicans clinical isolates showed that multiple mechanisms of resistance were combined in 75% of resistant isolates, overexpression of genes encoding efflux pumps were detected in 85% of isolates, amino acid substitutions in ERG11 were found in 65% of isolates,35% of the isolates showed overexpression of ERG11. The studies on azole-resistance mechanisms of Candida spp are becoming a very active subject in the world. But there is a few works about Candida drug-resistant in our national.In a previous investigation, we found G487T and T916C existed simultaneously in a collection of fluconazole-resistant C. albicans by sequencing in ERG11 gene. The two mutations coexisted in fluconazole-resistant isolates without any other mutations in the ERG11 gene. In clinical isolates with the two mutations remain unreported at the present time. In the process of drug resistance in C. albicans, multiple mechanisms can induce resistance in varying degrees. But it is not clear in the gene expression (such as MDR1, CDR1/2 and FLU1) of these clinical isolates. There is no report about this at present. Are there any other similar points associated with fluconazole resistance in these isolates? And what about the expression of resistance genes? The aim of this study was to study expression of resistant genes and efflux pumps in every fluconazole-resistant C. albicans with G487T and T916C for the first time, and further to explore the role of gene expression in fluconazole-resistant isolates, thus provide potent experimental datas for determining mechanisms of drug resistance in these isolates, and provide essential data analysis and experiment background for drug resistance research in future.Objectives1. To investigate biological features of fluconazole-resistant C. albicans with mutations G487T and T916C, and to determine the efflux of Rhodamine 6G in these isolates.2. To detect the mRNA expression of multidrug resistant genes in fluconazole-resistant C. albicans with mutations G487T and T916C by RT-PCR.3. To detect Cdrlp and Cdr2p expression in fluconazole-resistant C. albicans with mutations G487T and T916C by Western blot.Methods1.Indentification of Candida spp. in the hospital Clinical isolates of candida species were collected, including sputum, urine, blood, pus, etc. inoculating specimen on blood plate, choosing dubious specimen and yeast-like strains were seperating by Garm's stain. Then separating and purificating on modified SDA slants, identifing C. albicans by CHROMagar medium. Fluconazole susceptibility was tested in vitro using microdilution and disc diffusion assays. The sensitive isolates (S), dose-dependent sensitive isolates (S-DD) and resistant isolates (R) were decided. M27-A2 broth dilution method were recommended by CLSI. Strains ATCC 6258 (C. krusei) and ATCC22019 (C. parapsilosis) were used as controls. Repeated three times for every test.2. The isolates growth and Rhodamine 6G experiment Experiment samples:14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C and the control isolate ATCC10231. After activating twice, adjusting concentration of culture, yeast cells were transfered to RPMI1640 culture solution, YEPD culture solution and YEPD culture solution containing 64ug/ml fluconazole. Being cultured at 37℃, then observed growth of isolates by inverted phase contrast microscope.Rhodamine 6G was used as tracer agent and the fluorescence densities of samples were measured at an excitation wavelength of 529 nm and an emission wavelength of 553 nm. The efflux of Rhodamine 6G was determined in the strains with mutations G487T and T916C.3. Real-Time PCR experimentExperiment samples:14 fluconazole-susceptible C. albicans isolates (from the first part),14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C, the fluconazole-susceptible isolate ATCC 10231. The PCR primers to amplify of C. albicans were designed by referring C. albicans ERG11 sequence of GenBank X13296, CDR1(X77589), CDR2(U63812), MDR1(Y14703) and FLU1(AF188621). The housekeeping gene 18SrRNA was used as control. Total cellular RNA was extracted using the E.Z.N.A.TM Yeast RNA kit by manufacturer's description. The cDNA of each strain were synthesized in a two-step SYBR RT-PCR Kit. The reaction were performed at LightCyclerReal Time PCR machine. ATCC 10231 was used as a control isolate. The amplification products were separated using 2% agarose gel electrophoresis, visualized and photographed with an ImageMaster Video Documentation Systemto further verify their specificity. Using the 2-△△Ct method, one popular calculation formula to evaluate the expression levels of ERG11, CDR1, CDR2, FLU1 and MDR1 of C. albicans.4. Western Blot experimentExperiment samples:14 fluconazole-resistant C. albicans isolates with mutations G487T and T916C, Saccharomyces cerevisiae AD/CDR1 and AD/CDR2 were used as controls. Extracting plasma membrane proteins of samples, The concentration of protein were determined by BCA kit. The extracted proteins were electrophoresed on a 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The blots were visualized with enhanced chemiluminescene (ECL). The blots were incubated with a 1:500 dilution of anti-Cdrlp antibody, and a 1:1000 dilution of anti-Cdrlp antibody. For anti-Cdrlp antibody, immunoreactivity was detected with horseradish peroxidase-labeled goat anti-rabbit IgG antibodies. The Saccharomyces cerevisiae AD/CDR1 and AD/CDR2 were used as controls. Densitometric analysis of the band intensity was carried out using Quantity One software.Results1. Totally,161 clinical isolates of candida species were collected. Among the 161 strains of yeasts, Candida albicans was the most frequently species (69.57 %), followed by C.tropicalis (19.88%), C.parapsilosis (4.97%), C.krusei (2.48%), C.glabrata (1.86%)and others (1.24%).101 isolates from sputum specimen(62.73%),20 isolates from urine (12.42%),18 isolates from Vaginal secretions (11.18%),7 isolates from blood (4.35%) and 15 isolates from other sites (9.32%). The samples from department of respiratory tract were the most, the second is department of gynecology, the third is ICU and department of hematology. Among 112 candida albicans,108 sensitive isolates were detected (97.32%) by microdilution assay, and 109 sensitive isolates were detected (97.32%) by disc diffusion assay. The chi-square criterion displayed that there was no significant difference between them (P>0.05).2. In all the fluconazole-resistant C.albicans with mutations, mycelial growth had notably increase in RPMI1640 culture, decreased in YEPD. And they were still quite lively in YEPD containing 64μg/ml fluconazole.The experiment about Rhodamine 6G efflux showed that the efflux of Rhodamine 6G in ATCC10231 was weakest. All the fluconazole-resistant C. albicans isolates with mutations G487T and T916C can active efflux Rhodamine 6G, the efflux ability of Rhodamine 6G was enhanced as time prolonging. The efflux ability will weaken after 60 minutes.3. Expression of the efflux gene CDR1 and CDR2 in 14 fluconazole-resistant isolates was upregulated 1.6-to 8-fold. Likewise, a 3.7-to 52-fold increase in CDR2 transcript levels was observed relative to the susceptible control strain ATCC 10231. Isolate J4266 had the highest CDR1 expression level, the lowest CDR1 expression level is isolate J4263, and 1.7 times as the control strain. The isolate GZ18 had the highest expression level and J4263 had the lowest expression level in CDR2. The expression of MDR1, FLU1 and ERG11 varied in the different C. albicans isolates compared with ATCC 10231. MDR1 overexpression was observed in four isolates, and isolate GZ23 had the same MDR1 transcript level. Upregulation of the ERG11 gene were observed in six isolates, respectively were GZ03, J4266, GZ17, J592, GZ23 and GZ51. Overexpression of the FLU1 gene were observed in six isolates, respectively were J4266, J592, GZ17, GZ03, GZ23 and GZ34.4. The results of Western blot showed that expressed bands can be detected in the control strain Saccharomyces cerevisiae AD/CDR1 and AD/CDR2. Cdrlp were all detected in 14 fluconzazole-resistant C. albicans with G487T and T916C mutations, The analysis of the band intensity showed expression of J4266 was maxlmum, corresponding to the result of RT-PCR. But Cdr2p could not be detected in some isolates:GZ16, GZ34 and GZ51. The analysis of the band intensity showed expression of J592 was maximum, but the mRNA expression level of this isolate was inferior to GZ18 by RT-PCR. These results may suggest that stability of protein translation in CDR2 gene is uncertain.Conclusions1. The efflux pumps exist in all the fluconazole-resistant C. albicans with mutations G487T and T916C, and efflux of Rhodamine 6G will enhance as time prolonging, but the efflux ability will weaken after 60 minutes.2. The efflux pumps also exist in fluconazole-susceptible C. albicans, but the efflux ability is far inferior to fluconazole-resistant C. albicans. 3. The expression of efflux gene CDR1 and CDR2 can be detected in fluconazole-resistant C. albicans with mutations G487T and T916C. The efflux gene CDR1 and CDR2 may play an important role in fluconazole resistance, whereas MDR1 play a minor role in fluconazole resistance in these isolates.4. It may suggest that stability of protein translation in CDR2 gene is uncertain and decreased Cdr2p protein stability compared to Cdrlp in fluconazole-resistant C. albicans with mutations G487T and T916C.