ERG11 in Drug-resistant of C. Krusei

ERG11 in Drug- loathly of C. Kr commitiErg11 sportsmans and up-regulation in clinical itraconazole loathly isolates of Candida kruseiSome C. krusei isolates were repelling the antifungous do do drugsss.Mutations including T939C, T642C and A756T were put together in ERG11 of C. krusei.ERG11 was amplyly expressed in broad C. krusei strains.. mouseObjective We aimed to provide with light for better understanding of ERG11 constituent in drug- metro mechanisms in Candida krusei (C. krusei).Methods C. krusei strains were isolated from patients in the Dermatology from Jan 2010 to May 2013. Susceptibility assays, including 5-fluorocytosine (5-FC), amphotericin B (AMB), voriconazole (VCR), fluconazole (FLC) and itraconazole (ITR), was performed by broth microdilution method according to the subject field Committee for clinical Laboratory Standards M27-A2. Isolates were split up into sensitised strains and resistant strains based on their energy to ITR. Mutations in the ERG11 cistro n period were detected utilise PCR amplification and gene sequencing. Expression levels of ERG11 were measured by real-time PCR. Differences of ERG11 expression levels between susceptible strains and resistant strains were comp argond by two-tailed scholarly person t test.Results A total of 15 C. krusei strains were obtained, among which 8.0, 6.0 and 3.0% were resistant to FCA, ITR and 5-FC, individually, whereas all isolates were found to be susceptible to AMB and VRC. Three similar codon substitutions were found in ERG11among all the C. krusei strains, including T939C, T642C and A756T. Expression level of ERG11 was significantly higher(prenominal) in resistant C. krusei strains (1.34 0.08) than that in susceptible C. krusei strains (0.94 0.14) (t = 3.74, P Conclusions Our study demonstrates that hitch mutations accompanied with the overexpression of ERG11 might be involved in the molecular mechanisms of drug resistance in C. krusei.Keywords ERG11 drug resistance Candida kr usei mutation overexpression.IntroductionCandida species are pathogenic microorganisms for systemic and local opportunistic infections and the fourth leading experience of nosocomial bloodstream infections worldwide 1. In recent years, an increasing numeral of infections due to Candida krusei has been witnessed 2. C. krusei generally causes infections among immunocompromised patients, eespecially those suffering from Human immunodeficiency computer virus (HIV)-acquired immune deficiency syndrome and hematological malignancies 3. Mortality rates among such patients with C. krusei fungemia are account to be as high as 60-80% 4. Moreover, the increasing use of immunosuppressive drugs has inevitably increased the risk of C. krusei infection, and C. krusei has ranked in one-fifth place among all the species of Candida 5, 6.C. krusei has been regarded as a multidrug-resistant (MDR) fungal pathogen by the former of its intrinsic resistance to fluconazole (FLC) as well as its considera ble diminution in competency to flucytosine and amphotericin B (AMB) 2. Resistance to these antifungal drugs is a major problem among patients with acute myelogenous leukemia, neutropenia and/or critically ill, since these drugs are frequently apply for prophylaxis of C. krusei infection 7. Therefore, a better understanding of resistance mechanisms in C. krusei was urgently needed to effectively prevent and control infections ca utilize by C. krusei.It has been reported that double mechanisms are involved in drug resistance in C. krusei , including overexpression of some(prenominal) genes such as multidrug transporters (encoded by CDR1, CDR2, and MDR1), which lead to decreased intracellular assembly of FLC. Nowadays, an increasing evidence proposed that adjustments and overexpression of the ERG11 gene, which codes the target enzyme cytochrome P450 lanosterol 14-demethylase, are major resistance mechanism in C. krusei 8, 9. However, the scarce information ready(prenominal) r egarding ERG11 gene mutation and gene expression in the drug-resistant strains makes C. krusei resistance to azoles poorly understood 10.In the present study, we evaluated the susceptibility profiles of 15 C. krusei isolates, investigated the potential alterations in the ERG11 gene chronological sequence of C. krusei strains, and further detected the differentially expressed levels of ERG11 between susceptible and resistant isolates of C. krusei. We aimed to provide with light for better understanding of molecular mechanisms in drug resistance of C. krusei.Methods2.1 Fungal strains and mediaThe strains used in this study were isolated from patients in the Dermatology of the Second Hospital of Shanxi Medical University from Jan 2010 to May 2013. The standard strain, Candida krusei ATCC 6258, was purchased from fungi and fungal disease research center of Peking University and included in from all(prenominal) one test run for quality control. C. Krusei were maintained on agar YPD i ntermediate (2% peptone, 1% yeast extract, 2% dextrose) and stored in our Fungi Laboratory. RPMI 1640 medium with L-glutamate (Sigma, St. Louis, Mo.) was used as recommended for susceptibility assays and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS).2.2 Susceptibility assaysThe susceptibility assays of the isolates was performed in 96-well polystyrene microtiter plates by broth microdilution method depict in the National Committee for Clinical Laboratory Standards M27-A2 (NCCLS) 11. The antifungal drugs including 5-fluorocytosine (5-FC), amphotericin B (AMB), voriconazole (VCR), fluconazole (FLC) and itraconazole (ITR), were obtained from their respective manufacturers and used for susceptibility assays. MIC was watchd as the intentness of the drug that lessen the fungus growth by 80% compared to that grown in the absence of the drug. The informative criteria for susceptibility to the above drugs were published by the NCCLS and listed in Table 1. Samples were divided into susceptible strains and resistant strains based on their susceptibility to ITR.2.3 PCR amplification and sequence alignment of ERG11 geneTo amplify ERG11 gene, genomic DNA was firstly isolated from C. krusei cells using UNIQ-10 Column Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China) according to the manufacturers instruction and used as a template for amplification of the ERG11 genes. precise primers of ERG11 (Table 2) were designed by Primer 3 12, based on the available sequence information of C. Krusei ERG11 gene (GI163311561) at the National Center for bioengineering Information (NCBI). The PCR amplification of ERG11 gene was conducted using 2 l of genomic DNA, 2 l specific forward and reverse primers (50 mol/L) and Taq PCR Master coalesce (TIANGEN, Beijing, China). The PCR condition was set as denaturation for 5 min at 94 C, followed by 35 cycles 94 C for 30 s, 55 C for 30 s and 72 C for 30 s, and a final examination trample of elongation (72 C for 8 min). PCR products were then separated and surface on a 1.5% agarose gel by electrophoresis, and visualized under UV light after staining with ethidium bromide. Successfully amplified PCR products were sent for sequencing (Invitrogen, Shanghai, China). To verify the request mutations, sequencing results were aligned with the character reference sequence of C. Krusei ERG11 gene (GI163311561) using BLAST (Basic local Alignment Search Tool) program in NCBI.2.4 real time PCR analysisFor vicenary real-time PCR analysis, total ribonucleic acid was extracted from C. krusei cultures with an Yeast RNAiso Kit (TaKaRa, Dalian, China) and reversely transcribed to cDNA with PrimeScript RT Master Mix (TaKaRa, Dalian, China) according to the instructions of the manufacturer. For the ERG11 target genes and GAPDH reference gene, a primer pair were designed with Primer 5.0 program (Table 2). Real-time PCR was processed with a 25-l volume containing the following reagents 12.5 l of SYBR mixing Ex TaqTM II (TaKaRa, Dalian, China), 2 l of total RNA sample, 1l of each primer pair at a concentration of 10 M and distilled water up to the final volume. Samples were subjected to an initial step at 95 C for 5 min, followed by 40 cycles each of which consisted of 10 s at 95 C and 30 s at 60 C. Melting curves were recorded every 5 s during the 65-95 C by PCR amplifier. Fluorescence data (Ct) in each reaction were collected and were analyzed with the Rotor-Gene Q Series Software 2.0.2 software. A 2--Ct algorithmic program was applied to analyze relative expression levels of ERG11 at susceptible strains and resistant strains.2.5 Statistical analysisStatistical analysis was performed using SPSS 17.0 software (SPSS inc lettuceUS). All data were presented as mean standard deviation (SD). The two-tailed Student t test was conducted to compare the differences of ERG11 expression levels between susceptible strains and resistant strains. A p-value ResultsAntifungal susceptibilities of C. krusei isolatesA total of 15 C. krusei strains were isolated from clinical samples during Jan 2010 to May 2013 in our laboratory, among which 14 isolates were from urine and 1 from hydrothorax (Table 3). Table 4 shows the rates of azole resistance for these C. krusei isolates. Among 15 C. krusei isolates, 8.0, 6.0 and 3.0% were resistant to FCA, ITR and 5-FC, respectively, whereas all isolates were found to be susceptible to AMB and VRC. Ultimately, by using an MIC 1 g/ml to define resistance to the investigational ITR, the 15 isolates included 6 that were resistant and 9 that were susceptible.mutational analysis in ERG11of C. krusei isolatesDNA fragment with consistent length was successfully amplified from C. krusei isolates (Figure 1). Sequence alignment showed ternion synonymous codon substitutions in ERG11among all the C. krusei strains, including T939C, T642C and A756T (Figure 2). Among the three synonymous mutations, T642C and A756T only presented in ITR-resistant st rains, while T939C in susceptible as well as resistant C. krusei strains. Nevertheless, no point mutation was notice in the standard strain.Different expression levels of ERG11gene in susceptible and resistant C. krusei strainsReal-time PCR was performed to verify the varied expression levels of ERG11 in ITR-susceptible and ITR-resistant C. krusei strains. Result showed that mRNA transcription level of ERG11 was significantly higher in resistant C. krusei strains (1.34 0.08) than that in susceptible C. krusei strains (0.94 0.14) (t = 3.74, P DiscussionWith the drug-resistance character, C. krusei has emerged as one of the leading agents create candidemia, especially in immunocompromised patients 13. Previous studies have shown the crucial power of ERG11 gene in FLC-resistant clinical isolates of Candida species, while the molecular mechanism specially in C. krusei remains unclear. Herein, we evaluated the susceptibility profiles of 15 C. krusei isolates, subsequently searched for mutations in the ERG11 gene sequence of C. krusei using PCR amplification and gene sequencing, and further detected the differentially expressed levels of ERG11 between susceptible and resistant isolates by real-time PCR. We found three synonymous codon substitutions in ERG11 of C. krusei which have not yet been described previously. Among the three point mutations, T642C and A756T only presented in ITR-resistant strains, while T939C were also presented in ITR-susceptible strains. Moreover, mRNA transcription level of ERG11 was significantly higher in ITR-resistant C. krusei strains than that in ITR-susceptible C. krusei strains.It is reported that point mutations in the ERG11 gene can lead to conformational changes which whitethorn affect the affinity of the drug with the target, but not influence the enzyme duty in ergosterol biosynthesis 14. Many studies have been focused on the ERG11 alterationz in Candida species . Ricardo et al. 9 found two different types of mutations by sequencing the C. krusei ERG11 gene, including a heterozygous alteration at 1,389 bp (TC) presented in all of the susceptible and resistant C. krusei strains in their study, and a missense mutation in two strains at position 418 bp (TC) which yields a TyrHis amino acid change. Tavakoli et al. 15 displayed a heterozygous polymorphism at position 939 (TC) in ERG11 coding region, and speculated that this polymorphism might play a key berth in the transcriptional regulation of genes and be involved in the processes of ergosterol biosynthesis. Sionov et al. 16 have indentified a single missense mutation at amino acid 145 in the ERG11 of C. neoformans strain isolated from an FLC-treated patient, and verified that this mutation was sufficient to lead high FLC resistance. In the present study, we discovered three novel synonymous codon substitutions in ERG11 of C. krusei, among which T642C and A756T only presented in ITR-resistant strains. These alteration in ERG11might be involved in th e resistance mechanism of C. krusei.Many studies have also been designed to research the exact molecular mechanism behind the ERG11 up-regulation in response to azoles and different antifungal drugs 14, 17, 18. A well-characterized matched pair of FLC-susceptible and FLC-resistant C. albicans isolates was analyzed and the resistant strains were found principally associated with up-regulation of ERG11 gene 14. Henry et al. 17 demonstrated that treating C. krusei with the triazole FLC at a concentration of 2 to 9 mg/ml could resulted in four- to five-fold increase in ERG11 RNA levels. Accompanied with previous reports, they hypothesized that the the upregulation of ERG11 gene contributed at least partly to the strength of C. krusei to tolerate azole 19-21. The overexpression of ERG11was also directly shown to confer FLC resistance in S. cerevisiae 18. In accordance with previous studies, our results reflected that mRNA transcription level of ERG11 was found to be significantly hig her in ITR-resistant C. krusei strains compared with that in ITR-susceptible C. krusei strains. The increased production of 14a-demethylase was reported to exceed the inhibitory capacity of the antifungal drugs. Therefore, we speculated that ERG11 gene overexpression might be relevant in the drug resistance in C. krusei.In conclusion, on that point synonymous codon substitutions were observed in ERG11 of C. krusei. These point mutations accompanied with the overexpression of ERG11 might be involved in the molecular mechanisms of drug resistance in C. krusei.