白念珠菌高铁还原酶FRP1基因的功能

白念珠菌((Candida albicans)获得铁的能力影响细胞的生长和毒力,高铁还原酶是白念珠菌高亲和铁吸收系统的重要组成部分.[目的]构建高铁还原酶FRP1(Ferric reductase protein)基因缺失突变株,对FRP1基因功能进行初步研究.[方法]使用Northem杂交的方法分析FRP1基因在缺铁和富铁条件下的表达.利用PCR介导的基因敲除技术构建frp1缺失突变株,并且对野生型和缺失突变株在细胞高铁还原酶活性以及缺铁条件下的生长情况进行比较分析.[结果]缺铁条件可以诱导FRP1基因的表达.frp1缺失突变株不能在铁缺陷的固体培养基上生长.[结论]FRP1蛋白可能是白念珠菌在缺铁条件下起主要作用的高铁还原酶.     

白念珠菌高铁还原酶职FRP1基因的功能

白念珠菌((Candida albicans)获得铁的能力影响细胞的生长和毒力,高铁还原酶是白念珠菌高亲和铁吸收系统的重要组成部分.[目的]构建高铁还原酶FRP1(Ferric reductase protein)基因缺失突变株,对FRP1基因功能进行初步研究.[方法]使用Northem杂交的方法分析FRP1基因在缺铁和富铁条件下的表达.利用PCR介导的基因敲除技术构建frp1缺失突变株,并且对野生型和缺失突变株在细胞高铁还原酶活性以及缺铁条件下的生长情况进行比较分析.[结果]缺铁条件可以诱导FRP1基因的表达.frp1缺失突变株不能在铁缺陷的固体培养基上生长.[结论]FRP1蛋白可能是白念珠菌在缺铁条件下起主要作用的高铁还原酶.     

利用定点突变分析白念珠菌依赖铁基因FRP1启动子元件

通过对白念珠菌高铁还原酶基因FRP1启动子进行突变分析, 确认启动子中特殊调控元件。我们通过分析FRP1起始密码子上游1000 bp序列发现在-160 和-650处有2个推测的Rim101p结合位点, 对其分别进行定点突变, 然后构建启动子与报告基因LacZ融合质粒, 转化整合到白念珠菌rim101-/-株和野生株中, 检测不同缺铁条件下b-半乳糖苷酶活性。结果发现碱性条件, Rim101p能够正向调控FRP1的表达; 启动子-160处突变对启动子功能影响较弱, 而-650突变使启动子活性大大降低, 此结果和双突变的结果相同, 表明Rim101p主要通过与启动子-650处结合位点相互作用来调控FRP1的表达。     

白色念珠菌CRK1基因的敲除及其功能研究

在筛选白色念珠菌促分裂原活化蛋白激酶（ＭＡＰＫ）相关蛋白激酶基因的过程中,得到了ＣＲＫ１（ＣＤＣ２－ｒｅｌａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ １）基因。利用同源重组原理,敲除了ＣＲＫ１双拷贝基因片段,构建了ＣＲＫ１纯合缺失株,发现ＣＲＫ１基因的缺失影响到白色念珠菌的生长速度,絮凝生长和形态发生。     

白念珠菌黏附基因研究进展

白念珠菌具有很强的黏附和侵袭能力,其黏附特性是致病力的重要原因,与白念珠菌黏附相关的基因称为黏附基因。对黏附基因的研究不仅有助于更好地解读白念珠菌的致病机制,而且也为研制新的抗真菌药物和真菌疫苗提供了理论依据。     

白念珠菌基因分型的相关研究

白色念珠菌定植于大多数人群的口腔中,在一定条件下可成为优势菌种而导致感染。基因分型是近年来白念分子生物学研究中的一个热点,随着医学科学技术的发展,由于深部真菌感染比例不断增加,分子生物学方法已经越来越广泛的应用于临床真菌病的研究中,从而为控制白念感染及为早期诊断、治疗提供基础。本文综述了限制性片段长度多态性、随机扩增多态性DNA分析、ITS区域序列分析等分子生物学技术在白念珠菌基因分型方面的相关研究,比较了它们的优缺点,并且讨论了将基因分型研究应用于临床诊断、治疗及开发新型抗真菌药物的发展趋势和广阔前景。认为目前更倾向于多种分型方法联合应用,并借助计算机软件进行分析,但是仍需进一步探索。     

白色念珠菌氟康唑耐药相关基因研究进展

近 2 0年来由于临床上对癌症患者、器官移植患者大量使用化疗药物、广谱抗生素和免疫抑制剂 ,以及艾滋病 (AIDS)的流行 ,使深部真菌感染的发生率在免疫受损的病人群体中急剧升高。深部真菌感染已日益成为一种常见病、多发病 ,并已逐渐成为这类疾病患者死亡的主要原因之一。深部真菌感染中最常见菌属是念珠菌属 (Candidaspecies) ,其中以白色念珠菌 (Candidaalbicans)为最常见菌种。目前临床上常用的抗真菌药物有两大类 :干扰真菌细胞膜脂质合成的药物(二性霉素B ,唑类药物 )和干扰真菌核酸合成的药物 (5 氟胞嘧啶 )。由于唑类药物中的氟…     

白念珠菌生物膜相关基因研究进展

近年来，以白念珠菌为主的真菌感染发生率呈逐年上升趋势。白念珠菌病已经成为影响人类生活质量、威胁生命健康的重要疾病之一。目前，临床上常用的抗真菌药物很少有令人满意的疗效，其主要原因之一在于白念珠菌容易形成生物膜（biofilm）。生物膜是细菌或真菌附着于活体组织或非活体组织表面、     

白念珠菌临床分离调查及基因分型研究

目的 了解白念珠菌临床分离情况,并探讨其药敏结果与基因分型的相关性.方法 回顾性分析本院2011年3～11月间临床分离白念珠菌分布及耐药性;随机选取232株,采用PCR方法扩增白念珠菌25S rDNA基因内含子区进行基因分型研究;采用ATB真菌药敏试剂条进行药敏分析;统计分析药敏结果与基因分型的相关性.结果 期间共检出酵母样真菌973例,占病原菌阳性样本数比率为15.7％ (973/6196);其中分离白念珠菌562株,占58％ (562/973),主要分布科室为呼吸科(39.1％)、老年科(13.2％)、ICU(7.7％)、神经内科(7.5％)、免疫科(6.0％)以及其他科室(26.5％);标本类型以下呼吸道为主(81.7％),其次为尿路(9.4％)、血液(1.8％)等.对氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑及伏立康唑的耐药率分别为0.9％、0％、1.4％、1.6％和1.1％.随机选取的232株白念珠菌经PCR方法可分为3型:A型125株,B型96株,C型11株.各型在5种药物的耐药性上并无差异.结论 临床分离酵母样真菌以白念珠菌为主,感染部位以下呼吸道为主;临床分离株对5种抗真菌药物敏感度较高,主要基因型为A和B型,不同基因分型间药敏结果并无统计学差异.     

白念珠菌CaGDT1基因的功能研究

人类跨膜蛋白TMEM165与酿酒酵母Sc Gdt1均属于阳离子/钙离子交换器家族的成员,在本研究中,通过序列比对在白念珠菌中发现了Sc GDT1的同源基因Ca GDT1,表型互补实验显示Ca GDT1基因的表达能够抑制Sc GDT1基因缺失所造成的钙离子敏感性,证明Ca GDT1是Sc GDT1的同功基因。此外,通过同源重组原理敲除了Ca GDT1的2个等位基因。表型筛选结果表明gdt1/gdt1缺失株对钙离子、细胞壁和内质网3种胁迫均不敏感,而对酮康唑和特比萘芬2种抗真菌药物具有耐受性。     

Characterization of Candida albicans dihydrofolate reductase

Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes.     

白假丝酵母Vps74蛋白的鉴定及其功能

【背景】Vps74/GOLPH3是参与高尔基体蛋白糖基化修饰的关键蛋白,并且是重要的磷酸磷脂酰肌醇效应因子,在胞内参与多种信号通路。【目的】鉴定白假丝酵母Vps74蛋白,并探索其在该病原菌压力应答、蛋白分泌、形态发生及致病过程中的功能。【方法】采用在线序列比对方法,初步鉴定白假丝酵母Vps74蛋白;采用两步PCR介导的同源重组方法,构建白假丝酵母vps74基因缺失菌株vps74Δ/Δ及回补菌株VPS74c;采用反向遗传学方法,探究Vps74在白假丝酵母的压力应答、蛋白分泌、形态发生及致病过程中的功能。【结果】白假丝酵母中存在典型的Vps74/GOLPH3同源蛋白,Vps74参与蛋白糖基化修饰过程,vps74基因缺失导致白假丝酵母蛋白分泌能力、形态发生能力、黏附能力以及侵染宿主能力的显著降低。【结论】Vps74通过影响蛋白分泌、形态发生、黏附、嵌入式生长等过程,在白假丝酵母致病过程中发挥重要作用。     

白假丝酵母CFL1基因通过转录调控参与氧化压力应答

【背景】CFL1基因是白假丝酵母高铁还原酶基因,介导胞外铁离子的还原,在白假丝酵母胞内铁稳态的维持方面发挥着重要作用。【目的】研究CFL1基因调节氧化压力应答的分子机制。【方法】采用液体培养及巨噬细胞模型,测定CFL1缺失对氧化压力耐受性和杀伤巨噬细胞能力的影响;使用羟基自由基清除剂二甲基亚砜(DMSO)分析其对缓解氧化压力敏感性的影响;采用实时荧光定量PCR分析CFL1缺失对氧化压力应答基因表达的影响;采用过氧化氢酶(CAT)活性测定方法研究CFL1缺失对CAT1基因表达的影响;通过构建WT-CAT1-GFP和cfl1Δ/Δ-CAT1-GFP菌株分析过氧化氢酶基因过表达对cfl1Δ/Δ氧化压力敏感性的影响。【结果】白假丝酵母CFL1基因的缺失会造成杀伤巨噬细胞能力的减弱,氧化压力应答基因表达的下降。过氧化氢酶基因的过表达则能恢复与野生型几乎一致的氧化压力水平。【结论】CFL1基因通过转录调控参与白假丝酵母氧化压力应答过程。     

Characterization of Mucosal Candida albicans Biofilms

C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.