白色念珠菌ERG11基因突变与其耐三唑类抗真菌药物的关系

发布时间：2017-12-28 06:41

本文关键词：白色念珠菌ERG11基因突变与其耐三唑类抗真菌药物的关系　出处：《郑州大学》2015年硕士论文　论文类型：学位论文

【摘要】：背景近些年来,在真菌感染患者的临床治疗中免疫抑制剂、广谱抗生素以及糖皮质激素等的使用愈来愈多见,此外,诸如气管插管、呼吸机等侵入性治疗操作的使用频率愈来愈高,这些都可能导致患者中肺部真菌感染几率的增大。最多见的造成深部感染的真菌主要为念珠菌、青霉菌、曲霉菌、隐球菌、孢子丝菌、和毛霉菌等几种。深部真菌感染的愈后差,病死率高,药物应用相对局限。因此,对深部真菌感染的致病菌的鉴定对临床诊断、治疗、愈后等有着很重要的影响,但深部真菌感染的菌种鉴定是临床微生物实验室的一项难题。在传统的真菌的分类鉴定中,常采用的方法均是基于真菌菌种的形态学、生理生化等特点,并参考菌株的生理生化指标进行综合鉴定和分析。采用这种分析鉴定方法的弊端在于真菌菌株的检出及菌株的培养需过程需要保证特殊的培养环境和培养步骤;另外,由于实际培养中大部分的真菌菌株的生长相对缓慢,这给真菌菌种的鉴定工作造成了相当的困难。最终也就降低了针对深部真菌感染的致病菌进行鉴定获得的鉴定结果对于临床治疗的意义。近年来,得益于分子生物学的快速发展和进步,利用分子生物学的方法能够快速、准确地对真菌菌种进行鉴定,且该方法的应用范围正变得愈来愈广泛,现已逐渐发展为真菌菌种鉴定的重要辅助手段之一。念珠菌(Candida)在自然界中的分布即为广发,通常可以对人体的皮下组织、内脏和黏膜等造成侵袭,并且是能够引起目标对象全身性感染的条件致病真菌。在患者的临床诊治过程中,发现的致病真菌基本包括了白色念珠菌(Canida albicans)、光滑念珠菌(Canida glabrata)、热带念珠菌(Canida tropicalis)、近平滑念珠菌(Canida parapsilosis)以及克柔念珠菌(Canida krusel)等5大类。这里面在真菌的临床诊治上的致病菌以白色念珠菌出现概率最高。白色念珠菌的Secreted Aspartyl Proteinases(SAP)多基因家族至少有九名成员(SAP1到SAP9),通过编码分泌产出型天冬氨酸蛋白酶(Saps),而该蛋白酶能够参与到白色念珠菌对组织的侵袭性;白色念珠菌的组织侵袭性被公认为是白色念珠菌作为致病菌感染人体的重要致病因素。实验证实,当SAP6基因被敲除时,白色念珠菌的生存能力明显下降,对宿主细胞的侵袭力明显降低,对组织的损伤显著减少;因此,白色念珠菌所独有的功能性基因SAP基因可用于白色念珠菌菌种的鉴定工作。唑类抗真菌药物因其疗效好、抗菌谱广、生物利用吸收好、安全性比较高等优点,广泛应用于真菌感染的预防和治疗。但是,鉴于唑类抗真菌药物已长期、广泛应用于真菌制冷中,使得耐药菌株日渐多现,这使得肺部真菌病的防治难度愈来愈高。白色念珠菌获得对唑类抗真菌药物的耐药性的具体机制不一而足,其中较为多见的一种机制如下,即编码唑类抗真菌药物靶酶的ERG11基因发生突变或过度表达,并最终引起白色念珠菌菌株的耐药性的产生,最为多见。唑类抗真菌药物的靶酶为由ERG11基因通过编码产生的14α-去甲基化酶(CYP51)。若ERG11基因发生突变则会给Ergllp的氨基酸序列带来改变,从而使的14α-去甲基化酶的空间结构有了变化,致使酶分子与药物分子之间不能结合或结合力变弱,致使真菌获得耐药性。有研究发现,白色念珠菌菌株获得耐药性是通过唑类药物的靶酶的过量表达实现的,实验通过将耐药菌株和敏感菌株比较,发现大部分靶酶的m RNA含量升高的菌株,它们的MIC值也有较明显的增高。比较基因组学(comparative genomics)技术是将基因测序技术和基因组图谱二者相结合,通过将实际检测到的基因序列同目前已知的基因和基因组结构进行比较,从而对解基因的功能、基因的表达机制,乃至物种的进化过程等作出科学推断的技术学科。本研究通过对耐药白色念珠菌ERG11基因测序,与已知的标准白色念珠菌ERG11基因的进行比较,通过对比发现两者的基因序列中存在基因多态性差异,力图发掘出可能的与耐药性相关的基因;比较基因组学的方法对于念珠菌耐药机制研究的发展和进步有着极其重要的应用。此次研究以白色念珠菌的ERG11基因为对象展开,通过比较基因组学的技术方法,对ERG11基因突变同念珠菌耐药性产生之间的相关性进行探讨。材料与方法菌株的收集时间范围:2013年3月至2014年3月;菌株采集来源:郑州大学第一附属医院呼吸内二科住院部收治的肺部真菌感染患者中所采集到的符合标准的临床菌株共57株。菌株的培养:将临床上收集到的真菌菌株标本依时间先后顺序在沙保弱培养基上接种,温度条件维持在35℃,培养时间为24~48h;持续观察,当念珠菌生长显现出酵母样菌落时,从中挑取单个菌落,并在科玛嘉显色培养基上接种,培养温度35℃看,培养时间为24h;菌落鉴定的判据为培养菌产生颜色,若认为有必要则可使用由法国生物梅里埃公司生产的VITEK-32型全自动细菌分析系统中的YBC卡对念珠菌菌种进行进一步鉴定。(若鉴定发现为某菌株分离自同一患者相同部位,则将其排除)。应用ATBFugus3试条对32株白色念珠菌进行药敏实验,实验步骤严格按照标准操作规程进行,在35℃的有氧环境中培养24个小时,最后判读药敏结果并进行记录。氟康唑、伏立康唑、伊曲康唑分别是所采用的3种抗真菌药。选取白色念珠菌保守基因序列SAP6作为目的基因进行检测,并根据Gen Bank公布的白色念珠菌SAP6基因序列,应用Primer5.进行0软件设计白色念珠菌SAP6基因的PCR引物,对目的基因扩增,将PCR产物纯化并进行测序,将测序结果与SAP6基因对比,序列相同者认为是白色念珠菌。用以对照的标准的白色念珠菌ERG11基因序列以Gen Bank的公布为准,通过Primer5.0软件对白色念珠菌ERG11基因的PCR引物开展设计,经目的基因的扩增后,此后可对PCR产物进行纯化操作,此后进行基因的测序工作。基因测序的结果在Blast分析软件中进行,该软件导出的基因序列与标准得到白色念珠菌ERG11基因标准序列X13296进行比对和分析,最终确定是否有基因突变位点的产生。结果1.收集到符合标准的念珠菌有57株,鉴定菌株种类以白色念珠菌为主,共32株,占56.1%;光滑念珠菌紧随其后,为12株,占到了21.1%;热带念珠菌8株,占到了14.0%;克柔念珠菌则只有3株,占到了5.3%;其他念珠菌仅为2株,只占到3.5%。2.通过32株白色念珠菌进行体外药敏实验可以发现:白色念珠菌共32株,其中有4株对氟康唑有耐药性,共有7株对伊曲康唑有耐药性,而仅有2株对伏立康唑有耐药性。3.对32株白色念珠菌进行SAP6基因扩增并测序,所有白色念珠菌都检测出了SAP6基因。4.经过实验分析32株白色念珠菌的ERG11基因测序结果,并将其与标准的X13296序列进行比对分析,结果发现:碱基突变位点共有37处,其中错义突变则共9处有发现。敏感菌株有3株出现错义突变,9株耐药菌株中存在1~5处错义突变。此次试验发现了白色念珠菌ERG11基因错义突变及氨基酸改变,其中敏感菌株的错义突变有T945A、G1309A、A530C,与此相对应的氨基酸突变分别为D116E、V437I、K128T,耐药菌株则分别T495A、A530C、T541C、G622A、G979A、A945C、G1309A、G1496A、G1728T,对应的氨基酸突变分别为D116E、K128T、Y132H、V159I、E226D、D278N、V437I、G450E、M527I。结论通过对白色念珠菌ERG11基因测序,发现耐三唑类抗真菌药物的白色念珠菌多有ERG11基因突变,白色念珠菌ERG11基因突变导致D116E、V437I、K128T氨基酸改变,与白色念珠菌对三唑类抗真菌药物耐药性无关;D226E、G450E、Y132H氨基酸改变与白色念珠菌对三唑类抗真菌药物耐药性有关;V159I、D278N、M527I氨基酸改变与白色念珠菌对三唑类抗真菌药物耐药性关系需要进一步研究。多位点错义突变可产生协同作用,使得耐药性增加或交叉耐药。一些耐三唑类抗真菌药物的白色念珠菌ERG11基因未发现有意义的氨基酸改变,考虑有其他机制参与其耐药。
[Abstract]:Background in recent years, the fungal infection in the clinical treatment of immunosuppressant, broad-spectrum antibiotics and glucocorticoid use more and more, in addition, such as endotracheal intubation, ventilator use and invasive treatment and operation frequency is higher, which may lead to an increased risk of pulmonary fungal infection in patients. The most common fungi causing deep infection are Candida, Penicillium, Aspergillus, Cryptococcus, spores, and mucormycosis. The healing of deep fungal infection is poor, the mortality rate is high, and the drug application is relatively limited. Therefore, the identification of pathogenic bacteria for deep fungal infection has a very important impact on clinical diagnosis, treatment, recovery and so on, but the identification of deep fungal infection is a difficult problem in clinical microbiology laboratory. In traditional classification and identification of fungi, the methods commonly used are based on the morphological, physiological and biochemical characteristics of fungi, and refer to the physiological and biochemical indicators of strain for comprehensive identification and analysis. The drawbacks of this method for the analysis and identification of the fungal strains and strain detection is to develop the process needs to ensure that the special culture environment and culture step; in addition, due to the actual training in most of the fungi growth is relatively slow, which caused considerable difficulties for the identification of fungal strains. In the end, the results of identification of the pathogenic bacteria for deep fungal infection have also been reduced for clinical treatment. In recent years, thanks to the rapid development of molecular biology and progress, using molecular biology method can quickly and accurately identify the fungal species, the application scope and the method is becoming more and more widely, has gradually developed into one of the important means of identification of fungi. The distribution of Candida (Candida) in nature is GFA, which can invade subcutaneous tissue, viscera and mucous membrane of human body, and is a conditional pathogenic fungus that can cause systemic infection of target subjects. In the process of clinical diagnosis and treatment of patients, including the basic pathogenic fungi found in Candida albicans (Canida albicans), Candida glabrata, Candida tropicalis (Canida glabrata) (Canida tropicalis), c.parapsilosis (Canida parapsilosis) and Candida krusei (Canida krusel) and other 5 categories. The highest probability of Candida albicans is the pathogenic bacteria in the clinical diagnosis and treatment of fungi. Secreted Aspartyl Proteinases of Candida albicans (SAP) gene family with at least nine members (SAP1 to SAP9), secreted aspartic proteinase production by encoding (Saps), and the protease can be involved in the invasion of Candida albicans to the organization; organization of invasive Candida albicans is considered as Candida albicans the important pathogenic factors of human infection. Experiments show that when the SAP6 gene was knockout, Candida albicans viability decreased significantly, the host cell invasion significantly reduced tissue damage significantly reduced; therefore, the functional SAP genes unique to Candida albicans can be used for identification of Candida albicans strains. Azole antifungal drugs are widely used in the prevention and treatment of fungal infections due to their good efficacy, wide antimicrobial spectrum, good bioavailability and high safety. However, in view of the fact that azole antifungal drugs have been widely used in fungal refrigeration for a long time, the drug-resistant strains are becoming more and more frequent. This makes the prevention and treatment of pulmonary fungal diseases more and more difficult. This is not the only one specific mechanism of Candida albicans acquire resistance to azole antifungal drugs, one of the more common mechanisms are as follows, namely ERG11 gene encoding the target enzyme of azole antifungals mutated or overexpressed, and eventually lead to the resistance of Candida albicans strains, the most common. The target enzyme of the azolic antifungal agent is the 14 alpha demethylation enzyme (CYP51) produced by the encoding of the ERG11 gene. If ERG11 gene mutation occurs, it will change the amino acid sequence of Ergllp, resulting in the change of the spatial structure of the 14 alpha demethylation enzyme, resulting in the lack of binding or binding force between enzyme molecules and drug molecules, resulting in the drug resistance of fungi. It has been found that the resistance of Candida albicans isolates is achieved by over expression of target enzyme of azole drugs. Through comparing the resistant strains and sensitive strains, it is found that most of the m RNA strains with higher target enzymes have higher MIC values. Comparative genomics (comparative genomics) technology is the gene sequencing and genome combination of the two, compared with the known gene and genome structure through gene sequences will be detected, and the mechanism for the expression of the solution of gene function, gene technology, and evolution process to make scientific inference the. Through the study on drug resistance of Candida albicans ERG11 gene sequencing, compared with the standard of Candida albicans ERG11 gene known, by comparing the differences exist between genetic polymorphism of gene sequences, trying to explore the resistance associated gene; comparative genomics method has important applications for the study of mechanism of drug resistance of Candida albicans the development and progress of. In this study, the ERG11 basis of Candida albicans was launched, and the correlation between ERG11 gene mutation and Candida resistance was discussed through comparative genomics. The collection time and range of materials and methods were from March 2013 to March 2014. Strain collection sources: 57 strains of clinical isolates collected from two Department of respiratory medicine in the First Affiliated Hospital of Zhengzhou University were collected from the Department of pulmonary fungal infection in inpatient department. Culture of strains: the specimens collected from clinical fungi in chronological order
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R519

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