Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11).

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.     

牛源近平滑念珠菌对氟康唑耐药性研究

以牛源近平滑念珠菌（Candida parapsilosis）为试验菌株,采用微量稀释法进行药物敏感性试验,PCR扩增测序检测ERG11基因突变,Realtime PCR检测ERG11、CDR1、MDR1、MRR1基因的mRNA表达量,探讨耐药相关基因在牛源近平滑念珠菌耐唑类药物中的作用,为牛源近平滑念珠菌的耐药研究提供参考。结果表明,近平滑念珠菌对5-氟胞嘧啶、两性霉素Ｂ的敏感率均高于75%,对唑类药物的耐药率均高于50%,其中对氟康唑的耐药率最高,达58.3%;所有菌株的ERG11基因中均检测出错义突变A395T,耐氟康唑和剂量依赖菌株的ERG11基因中检测出同义突变T591C;氟康唑耐药组ERG11、CDR1、 MDR1、MRR1基因表达水平均显著高于敏感组（P<0.05）。牛源近平滑念珠菌对唑类抗真菌药物的耐药率较高且具有多重耐药性。牛源近平滑念珠菌ERG11基因中的T591C突变以及ERG11、CDR1、MDR1、MRR1基因的高表达都可能在其对氟康唑耐药性的产生中起到一定的作用。     

额外拷贝ERG6基因对烟曲霉的影响

通过构建烟曲霉ERG6基因额外拷贝株．研究该基因对烟曲霉生长速度、抗药物敏感性的影响。在烟曲霉基因组找出烟曲霉可能的ERG6基因的开放读码框(ORF),PCR扩增ERG6的ORF连同其上下游各约1 kb的DNA片段,利用DNA重组的方法将该片段克隆到载体pRG-AMA1-NotI。用重组后的质粒转化烟曲霉尿嘧啶营养缺陷株AF293．1。在MM和YAG培养基上观察转化子的生长速度。采用纸片扩散法和微量液基稀释法测定转化子对抗真菌药物敏感性。烟曲霉基因组中存在一个拷贝的ERG6基因,ORF大小为1,256 bp。其编码的蛋白与白念珠菌、酿酒酵母固醇甲基转移酶(Ers6p)的氨基酸相同率分别为57%和50%,相似率分别为70%和63%。烟曲霉中ERG6基因被成功克隆到了pRG-AMA1-Not I,产生了质粒pERG6。用pERG6和空载体pRG-AMA1-Not I转化AF293．1后,分别得到转化子AF-pERG6和AF-empty。AF-pERG6在MM和YAG培养基上的生长速度均比AF-empty慢。AF-pERG6和AF-empty对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性没有差异。ERG6基因额外拷贝不影响烟曲霉对伊曲康唑、伏力康唑、特比萘芬、两性霉素B、卡泊芬净、灰黄霉素的敏感性,但是能使烟曲霉的生长速度减慢。     

近平滑念珠菌ERG 11基因编码区的克隆及生物信息学分析

目的克隆、测序近平滑念珠菌ERG11基因的编码区序列并进行生物信息学分析。方法运用生物信息学的方法 ,通过与白念珠菌ERG11基因碱基序列同源性比对,在近平滑念珠菌基因组(www.sanger.ac.uk/sequencing/Candida/pa-rapsilosis/)中寻找可能的ERG11基因序列(CpERG11),并据此序列设计引物,经PCR扩增近平滑念珠菌标准株(ATCC22019)的ERG11基因片段,产物经电泳、纯化、克隆到质粒prG-AMAI-NotI中,转染DH10B大肠杆菌细胞,并酶切鉴定筛选阳性克隆测序分析。结果近平滑念珠菌ERG11编码区由1569个碱基组成,编码一段含522个氨基酸的多肽。近平滑念珠菌ERG11的编码区序列与白念珠菌、热带念珠菌、光滑念珠菌、酿酒酵母菌ERG11基因的同源性分别为74%、75%、65%、64%。该近平滑念珠菌ERG11的编码区为唑类药物作用靶酶基因。结论成功克隆、测序、并生物信息学分析近平滑念珠菌ERG11基因的编码区序列,为进一步的功能研究奠定基础。     

Cloning of the lanosterol 14-α-demethylase ( ERG11 ) gene in Trichosporon asahii: a possible association between G453R amino acid substitution and azole resistance in T. asahii

Lanosterol 14-α-demethylase ( Erg11 protein; Erg11p ), encoded by the ERG11 gene, is the primary target of azoles. Recently, a change in affinity of this enzyme for azoles has been reported as a resistance mechanism in several fungal species. Trichosporon asahii ( T.?asahii) is susceptible to fluconazole (FLC). This report identified the ERG11 gene of T.?asahii (NCBI accession; HQ176415). The Erg11p of T.?asahii, presumed from the DNA sequence, was closely related to the Erg11p of Cryptococcus neoformans. Furthermore, a FLC-susceptible strain was cultured in medium containing FLC at concentrations from 4.0 to 16?μg?mL(-1) in order to analyze the development of FLC resistance in T.?asahii. The degree of resistance was related to the FLC concentration of the growth medium. One highly resistant strain that was cultured in the medium containing 16?μg?mL(-1) FLC contained 1 point mutation (G1357C) that caused a single amino acid substitution at G453R. This amino acid is highly conserved among major fungal pathogens, and it is in a region very close to the heme-binding domain, which is characteristic of the cytochrome P450 superfamily, the primary target for the azole class of antifungal agents. This amino acid substitution may have caused the high resistance to azoles in T.?asahii.     

In Candida albicans, resistance to flucytosine and terbinafine is linked to MAT locus homozygosity and multilocus sequence typing clade 1

A panel of 637 isolates of Candida albicans that had been typed by multilocus sequence typing (MLST) and tested for susceptibility to amphotericin B, caspofungin, fluconazole, flucytosine, itraconazole, ketoconazole, miconazole, terbinafine and voriconazole was the material for a statistical analysis of possible associations between antifungal susceptibility and other properties. For terbinafine and flucytosine, the greatest proportion of low-susceptibility isolates, judged by two resistance breakpoints, was found in MLST clade 1 and among isolates homozygous at the MAT locus, although only three isolates showed cross-resistance to the two agents. Most instances of low susceptibility to azoles, flucytosine and terbinafine were among oropharyngeal isolates from HIV-positive individuals. Statistically significant correlations were found between terbinafine and azole minimal inhibitory concentrations (MICs), while correlations between flucytosine MICs and azole MICs were less strong. It is concluded that a common regulatory mechanism may operate to generate resistance to the two classes of agent that inhibit ergosterol biosynthesis, terbinafine and the azoles, but that flucytosine resistance, although still commonly associated with MAT homozygosity, is differently regulated.     

Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes

Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida . The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 μg mL⁻¹); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1 , CDR2 , MDR1 , encoding for efflux pumps, and ERG11 , encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly ( P >0.05), probably acting as a Cdrp blocker.     

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.     

The gene encoding squalene epoxidase from Saccharomyces cerevisiae: cloning and characterization.

The gene (ERG1) encoding squalene epoxidase (ERG) from Saccharomyces cerevisiae was cloned. It was isolated from a gene library, prepared from an allylamine-resistant (AlR) S. cerevisiae mutant, by screening transformants in a sensitive strain for AlR colonies. The ERG tested in a cell-free extract from one of these transformants proved to be resistant to the Al derivative, terbinafine. From this result, we concluded that the recombinant plasmid in the transformant carried an allelic form of the ERG1 gene. The nucleotide sequence showed the presence of one open reading frame coding for a 55,190-Da peptide of 496 amino acids. Southern hybridization experiments allowed us to localize the ERG1 gene on yeast chromosome 15.