【作者】 赵杰；

【导师】 李春阳；

【作者基本信息】 山东大学 ， 皮肤病与性病学， 2012， 博士

【摘要】 背景随着广谱抗生素和糖皮质激素等免疫抑制剂的广泛应用,介入治疗和器官移植的普遍开展,艾滋病、肿瘤和糖尿病病人的增加,念珠菌感染呈逐年上升的趋势。在临床治疗的过程中,由于抗真菌药物的广泛和长期应用导致了耐药现象的出现,影响临床治疗效果,因此研究真菌耐药的分子机制不仅有利于发现新的药物治疗靶点还能更好的指导临床用药。氟康唑是1978年发现的氟代三唑类药物,具有抗菌谱广、半衰期长(27-34h)、生物利用度高、不良反应发生率低、可通过血脑屏障等优点,是目前治疗念珠菌感染的首选药物。但长期反复的应用导致了氟康唑耐药菌株的出现。白念珠菌是念珠菌感染的首要致病菌。从基因水平研究白念珠菌对氟康唑耐药机制的报道主要集中于欧美国家,国内研究相对较少。目前研究认为,白念珠菌耐药主要有以下三种机制：(1)药物靶酶基因ERG11突变或过度表达；(2)外排泵基因(CDR1、CDR2、MDR1)的过度表达；(3)生物膜的形成。麦角固醇是真菌细胞膜的特有脂质。它维持着细胞膜的完整性和正常功能。细胞色素P450依赖的14α-去甲基化酶(Ergllp)是麦角固醇合成途径中的限速酶,其编码基因为ERG11基因。氟康唑可以与14a-去甲基化酶结合从而影响该酶的活性,进而阻止麦角固醇的合成。ERG11基因的点突变可导致编码蛋白的改变从而影响Erg11p的空间构象,引起Ergllp与药物的亲和力降低或底物/药物进出通道发生改变。迄今报道的ERG11基因错义突变已逾160种。在我们的前期研究中发现14株氟康唑耐药的白念珠菌仅含有G487T和T916C突变,且并不伴随其它突变。最近有文章报道在人工诱导的氟康唑耐药株中也同样发现这两个突变。到目前为止,G487T和T916C两突变仅在氟康唑耐药菌株中出现,但其与氟康唑耐药的关系并未得到实验室证实。目的运用表达质粒过表达和基因敲除的方法,研究ERG11基因G487T和T916C突变与白念珠菌氟康唑耐药的关系。方法1.白念珠菌GZ16和SC5314MIC值的测定和ERG11基因突变检测应用微量稀释法对前期分离的氟康唑耐药株GZ16和标准株SC5314(由Richard Calderone教授惠赠)进行氟康唑MIC的测定。分别提取两株菌的基因组DNA,参照白念珠菌ERG11标准序列(GeneBank X13296)设计引物,扩增GZ16和SC5314的ERG11基因并测序。2. ERG11基因突变片段质粒的构建和酿酒酵母突变表达载体的构建以GZ16的ERG11基因为模板,通过重叠PCR得到无突变、含G487T突变、含T916C突变、含G487T和T916C两突变的ERG11基因片段,胶回收纯化后分别与T载体pGM-T连接得到质粒pGM-N、pGM-487T、pGM-916C、 pGM-TWO。以GZ16、pFA-CaURA3、pFA-SAT1为模板,通过PCR得到ERG11基因下游片段、URA3选择标记片段、SAT1选择标记片段,经胶回收纯化分别与T载体pGM-T连接得到质粒pGM-DOWN、pGM-URA3、pGM-SAT1。用相应的限制性内切酶分别双酶切质粒pGM-916C、pGM-DOWN、pGM-URA3、和pcDNA3.1,得到具有粘性末端的4片段：含T916C突变的ERG11片段、URA3选择标记片段、ERG11基因下游片段和pcDNA3.1片段。4片段经连接酶连接后得到以URA3为选择标记含突变片段的质粒pcDNA3.1-916C-URA3,该质粒骨架为pcDNA3.1。重复以上步骤得到以URA3为选择标记的无突变的、含G487T突变、含G487T和T916C两突变的突变片段质粒pcDNA3.1-N-URA3、 pcDNA3.1-487T-URA3、pcDNA3.1-TWO-URA3,以及以SAT1为选择标记的无突变的、含G487T突变的、含T916C突变的、含G487T和T916C两突变的突变片段质粒pcDNA3.1-N-SAT1、pcDNA3.1-487T-SAT1、pcDNA3.1-916C-SAT1、 pcDNA3.1-TWO-SAT1。以pGM-N、pGM-487T、pGM-916C、pGM-TWO为模板,PCR扩增,得到无突变、含G487T突变、含T916C突变、含G487T和T916C两突变的ERG11基因片段,片段双酶切后分别与酿酒酵母表达载体YEP351G连接得到酿酒酵母表达质粒YEP351G-N、YEP351G-487T、YEP351G-916C和YEP351G-TWO。3.含不同突变的YEP351G表达载体在酿酒酵母中的表达与氟康唑药敏测定采用醋酸锂转化的方法,分别用质粒YEP351G-N、YEP351G-916C、 YEP351G-487T、YEP351G-TWO转化酿酒酵母INVSc1,用亮氨酸缺陷的培养基筛选阳性克隆,并测定阳性克隆对氟康唑的MIC值。4.白念珠菌T916C突变菌株的构建与氟康唑敏感性测定限制性内切酶Hind Ⅲ和NheI双酶切质粒pcDNA3.1-916C-URA3得到相应的长度为3.7kb的敲除片段,醋酸锂转化白念珠菌CAI4,尿嘧啶缺陷的培养基选择阳性克隆,并测定阳性克隆对氟康唑的MIC值。结果1.白念珠菌GZ16和SC5314MIC值的测定和ERG11基因突变检测白念株菌GZ16为我们以前实验验证的氟康唑耐药菌株,经5次传代后其氟康唑MIC值仍为64μ g/m1。标准株SC5314的氟康唑MIC值为2μ g/ml,为氟康唑敏感菌株。GZ16仅含有G487T和T916C突变。SC5314含有T462A、T495A、A504G、A530C、C558T、C805T、A1167G、A1587G突变,其中氨基酸错义突变为F105L(T462A)、D116E(T495A)、K128T(A530C).2.ERG11基因突变片段质粒的构建和酿酒酵母突变表达载体的构建构建了以URA3为选择标记的FRG11基因突变片段质粒pcDNA3.1-N-URA3、 pcDNA3.1-487T-URA3、pcDNA3.1-916C-URA、pcDNA3.1-TWO-URA3,以及以SAT1为选择标记的ERG11基因突变片段质粒pcDNA3.1-N-SAT1、 pcDNA3.1-487T-SAT、pcDNA3.1-916C-SAT1、pcDNA3.1-TWO-SAT1;并成功构建了酿酒酵母突变表达载体YEP351G-N、YEP351G-487T、YEP351G-916C和YEP351G-TWO。3. YEP351G表达载体在酿酒酵母中的表达与氟康唑药敏测定用质粒YEP351G-N、YEP351G-916C、YEP351G-487T、YEP351G-TWO转化酿酒酵母INVSc1后,用亮氨酸缺陷的培养基成功筛选出相应的阳性克隆株INVScl+N、INVScl+487T INVScl+916C、INVScl+TWO,其对氟康唑的MIC值分别为32μ g/ml、32μ g/ml、128μg/ml、128μg/ml,酿酒酵母INVScl对氟康唑的MIC值为8μg/ml。4.白念珠菌T916C突变菌株的构建与氟康唑敏感性测定在T916C突变菌株的构建中用尿苷缺陷的培养基成功得到5株阳性克隆株(T1-T5),其中有3株存在T916C突变(T1、T3、T5),而两株虽然发生了同源重组但无T916C突变(T2、T4)。CIA-4、T1、T2、T3、T4、T5对氟康唑的MIC值分别为2μ g/ml、4u g/ml、2u g/ml、4u g/ml、2μg/ml、4u g/ml。结论1.成功构建了含白念珠菌ERG11基因G487T和T916C突变的突变片段质粒和能表达白念珠菌ERG11基因G487T和T916C突变的酿酒酵母突变表达载体。2.首次证实了白念珠菌ERG11基因T916C突变与氟康唑耐药有关3.初步证实白念珠菌ERG11基因G487T突变与氟康唑耐药无关,并证实白念珠菌ERG11基因G487T和T916C突变在氟康唑耐药中不存在协同关系。更多还原

【Abstract】 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.更多还原