【作者】 陈腊梅；

【导师】 李春阳；

【作者基本信息】 山东大学 ， 皮肤病与性病学， 2010， 博士

【摘要】 念珠菌属是引起机会性真菌感染的常见病原体,也是医院真菌感染的重要病原菌之一。近年来,由于糖皮质激素、免疫抑制剂、广谱抗生素的广泛应用,器官移植的普遍开展,导管等生物材料应用的增多,原发及继发性免疫功能低下的人群扩大,真菌感染率呈不断上升的趋势。尤以白念珠菌的感染最为常见。唑类药物是目前应用最广的抗真菌药物。特别是氟康唑,自从20世纪80年代后期问世以来,由于其抗菌谱广、安全和可静脉给药,吸收好,毒性小等优点,成为目前临床治疗真菌感染的首选药物。但是,随着抗真菌药物的广泛使用,加之病人长时间的药物治疗和使用较低剂量的抗真菌药物,为耐药菌株的产生创造了条件。1980年Rosenblatt等人首次报道在应用酮康唑治疗慢性皮肤念珠菌病人身上出现药物耐药现象。此后有关耐药的报道越来越多。麦角固醇是真菌细胞膜中特有的脂质。它维持着细胞膜的正常结构和功能。14α-去甲基化酶(Ergllp)是念珠菌细胞膜麦角固醇合成途径中的关键酶,ERGll是14α-去甲基化酶的编码基因。大量研究认为这个基因的点突变或过度表达均与唑类耐药相关。ERG11的突变可以改变14α-去甲基化酶的氨基酸序列,从而影响唑类药物与该酶的亲和力,导致菌株耐药。过度表达的外排泵基因CDR1和CDR2以及MDR1基因是第二个与耐药相关的原因。CDR1和CDR2基因属于ABC转运蛋白超家族(ATP-binding cassette transporters, ABC transporters),二者的同源性超过80%,在耐药机制中可发挥协同作用。MDR1基因属于易化扩散载体超家族(major facilitator supeffamily, MFS),目前认为它的表达也与氟康唑耐药相关。近年来发现FLU1基因也与唑类耐药相关。该基因与MDR1基因同源性较高,在耐氟康唑白念珠菌的研究中发现这个基因被敲除后可增加菌株对唑类药物的敏感性。此外,生物膜形成是近年来与耐药相关的一个新发现。生物膜耐药也是导致临床上许多系统性感染难以根除的重要因素。最近的研究认为细胞内液泡封闭药物分子也可能引发菌株耐药。耐药的发生是一个多水平多因素参与的复杂过程。耐药白念珠菌的流行病学调查发现,85%的耐药株外排泵过度表达；35%的耐药株为药物靶酶的氨基酸改变或过度表达；75%的耐药株为多因素联合耐药。唑类药物对念珠菌耐药机制的研究已经成为当今医学研究的热点。但国内目前在念珠菌耐药性方面的研究仍然较少。前期研究中我们通过测序发现14株临床耐氟康唑白念珠菌的ERG11基因集中发生G487T和T916C突变,且不伴随有其他突变。目前尚未见过临床菌株存在该突变点的报道。在白念珠菌耐药的发生发展过程中,多种机制均可在不同程度上促进菌株耐药的形成。我们尚不清楚是否有其它耐药机制(如MDR1、CDR1/2和FLU1)介入了这一系列临床株的耐药形成过程。目前也未见该方面的研究报道。该系列菌株在耐氟康唑机制中还存在其它的共同点么?耐药相关基因的表达在这些白念珠菌中又起了什么作用呢?本课题首次对每一株携带G487T和T916C突变的耐氟康唑白念珠菌的耐药相关基因的表达及外排泵功能进行了研究,进一步探讨该系列菌株的基因表达在氟康唑耐药中的作用,从而为明确该系列菌株的耐氟康唑机制提供有力的实验数据,并为后续进一步研究菌株耐药现象提供必要的数据分析及实验支持。目的1.了解携带G487T和T916C突变的耐氟康唑白念珠菌在不同培养液的生长情况,使用若丹明6G作为示踪剂检测该系列菌株的外排能力。2.通过实时定量PCR方法检测携带G487T和T916C突变的耐氟康唑白念珠菌多个耐药相关基因mRNA表达水平。3.通过蛋白印迹方法检测携带G487T和T916C突变的耐氟康唑白念珠菌Cdrlp和Cdr2p蛋白的表达。方法1.院内念珠菌鉴定收集山东大学齐鲁医院检验科及皮肤科门诊的临床标本,包括痰液、尿液、血液及分泌物等。将标本接种于血平板,挑选可疑菌落涂片革兰染色确认为酵母样真菌后,转种于改良SDA平板分离纯化,通过科玛嘉念珠菌显色培养基鉴定,观察结果。应用微量稀释法和纸片扩散法测定氟康唑对试验菌株的MIC,确定敏感株(S)、剂量依赖敏感株(S-DD)和耐药株(R)。微量稀释法按照美国临床试验标准协会(CLSI)制定的M27-A2方案实施,ATCC 6258和ATCC22019被作为质控菌株。每次试验重复三次。2.菌株生长及若丹明6G实验实验菌株：携带G487T和T916C突变的耐氟康唑白念珠菌14株,敏感控制株ATCC102311株。将保存在SDA培养基的菌株经过两次活化,调整菌液浓度,将活化后的耐氟康唑白念珠菌分别转入lml RPMI1640培养液,lmlYEPD培养液和lml 64μg／ml氟康唑的YEPD培养液中,37℃静置培养,倒置相差显微镜下观察耐氟康唑白念珠菌的增殖情况。使用若丹明6G作为示踪剂,通过测定荧光强度(激发波长529nm,吸收波长553nm)来计算细胞外液若丹明的浓度,进而检测若丹明6G在携带G487T和T916C突变的耐氟康唑白念珠菌中的外排情况。3.实时定量PCR实验实验菌株：氟康唑敏感白念珠菌14株(来自第一部分的收集菌株),携带G487T和T916C突变的耐氟康唑白念珠菌14株,敏感株ATCC102311株。根据各耐药基因的基因序列：ERG11(GeneBank号：X13296,下同),CDR1(X77589), CDR2(U63812), MDR1(Y14703), FLU1(AF188621)设计相应的引物。内参照选用看家基因18SrRNA。标准株ATCC 10231被作为敏感控制株。采用酵母RNA试剂盒分别提取各菌株RNA,逆转录成cDNA后,在LightCycler Real Time PCR扩增仪上进行实时定量PCR反应。PCR产物进行凝胶电泳,在凝胶成像系统下观察是否为目的条带,有无杂带。将获得的域值循环数(Ct值)采用2-ΔΔCt方法转换为目的基因的相对表达量,检测多个耐药基因的表达情况,比较该系列菌株CDR1、CDR2、MDR1、ERG11和FLU1基因的表达差异。4.蛋白印迹实验实验菌株：携带G487T和T916C突变的耐氟康唑白念珠菌14株,内参照菌株Saccharomyces cerevisiae AD/CDR1和AD/CDR2。提取菌株的质膜蛋白,BCA蛋白浓度测定试剂盒测定蛋白含量,通过7.5%SDS-聚丙烯酰胺凝胶电泳,转膜,蛋白印记,ECL试剂盒显色等步骤,检测菌株CDR1和CDR2基因的表达情况。抗Cdr1p抗体(1：500),抗Cdr2p抗体(1：1000),二抗选用HRP标记的山羊抗兔二抗(抗Cdrlp抗体1：5000；抗Cdr2p抗体1：10000)。应用数字凝胶成像系统FluorChem 9900-50型进行扫描并记录图像,用Quantity One software软件进行半定量分析,计算各组目的蛋白相对于内参的相对灰度值作为各蛋白的相对含量。结果1.共收集酵母样菌161株,在161株菌中白念珠菌比例为69.57%,热带念珠菌为19.88%,近平滑念珠菌为4.97%,克柔念珠菌为2.48%,光滑念珠菌为1.86%,其他念珠菌为1.24%。痰标本最多,101份,占所有标本的62.73%。其次为尿液标本,20份(12.42%),来源于阴道分泌物的标本比例为11.18%,血液来源的标本占4.35%,其余(咽拭子、大便、导管分泌物、透析液、胆汁、脐分泌物、支气管肺泡冲洗液等)占9.32%。这些标本中,来自呼吸科的标本占的比例最大,妇科、ICU、血液科次之。112株白念珠菌中,通过微量稀释法筛选出108株敏感株,占96.43%,纸片扩散法试验筛选出109株敏感株(97.32%)。两种方法经卡方检验P>0.05,二者无明显统计学差异。2.在RPMI1640培养液中,该系列白念珠菌可形成大量菌丝；在YEPD培养液中白念珠菌的菌丝形成数目明显减少；在64μg／ml氟康唑的培养液中它们仍表现活跃。若丹明6G外排实验结果显示：敏感控制株ATCC10231主动外排若丹明6G能力在所有受试菌株中表现最弱。携带G487T和T916C突变的耐氟康唑白念珠菌在能量供给下可高效主动外排若丹明6G。随着时间的延长该外排能力增强,但60min之后转运能力减弱至平衡或下降。3.实时定量PCR方法显示携带G487T和T916C突变的耐药组CDR1和CDR2基因的表达明显高于敏感组；而两组的ERG11,MDR1和FLU1基因在mRNA表达水平上差异无统计学意义。与敏感控制株ATCC10231相比,14株携带G487T和T916C突变的耐氟康唑白念珠菌的主动外排泵CDR1和CDR2基因的mRNA表达水平都是明显增高的,分别达到1.6-8倍和3.7-52倍。其中菌株J4266的CDR1 mRNA表达水平最高,而菌株J4263的CDR1mRNA表达水平在14株中最低,但仍然是敏感株的1.7倍。菌株GZ18的CDR2基因mRNA表达是最高的。J4263的CDR2基因mRNA表达水平在14株中最低。在MDR1,FLU1和ERG1l基因中,各个菌株的表达水平是高低不一的。MDR1的表达在4株菌中表达是增高的,分别是菌株GZ17、GZ03、J4266和J592。菌株GZ23的MDR1基因的mRNA表达水平与敏感控制株相同。6株菌中发现ERG11基因过度表达,分别是GZ03、J4266、GZ17、J592、GZ23和GZ51。6株菌的FLU1基因过度表达,分别是J4266、J592、GZ17、GZ03、GZ23和GZ34。4.蛋白印迹实验显示：内参照Saccharomyces cerevisiae AD/CDR1可以稳定表达Cdr1p, Saccharomyces cerevisiae AD/CDR2菌株可以稳定表达Cdr2p。在这些携带G487T和T916C突变的耐氟康唑白念珠菌中,所有菌株的CDR1基因均有明显的表达,半定量分析结果示菌株J4266表达量最高。这与实时定量mRNA结果相符。但在菌株GZ16、GZ34和GZ51,无法检测到CDR2基因的完整表达。半定量分析结果示菌株J592的Cdr2p蛋白表达最高。而在实时定量PCR比较中它的mRNA表达水平要低于GZ18。这可能提示该系列菌株CDR2基因的蛋白翻译并不是十分稳定的。结论1.携带G487T和T916C突变的耐氟康唑白念珠菌均具有主动外排若丹明6G的能力,且该能力随着时间延长而增强,但在60min之后逐渐下降至平衡。2.氟康唑敏感白念珠菌也可具有主动外排若丹明6G的能力,但该能力远低于携带G487T和T916C突变的耐氟康唑菌株。3.主动外排泵基因CDR1和CDR2的过度表达存在于每一株携带G487T和T916C突变的白念珠菌。主动外排泵基因CDR1和CDR2在携带G487T和T916C突变的白念珠菌耐氟康唑机制中可能发挥重要作用,而易化扩散载体蛋白MDR1基因的作用可能并不明显。4.在携带G487T和T916C突变的耐氟康唑白念珠菌中,CDR2基因转录后的蛋白翻译并不十分稳定,Cdr2p蛋白翻译的稳定性可能弱于Cdr1p。更多还原

【Abstract】 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.更多还原