间接免疫荧光法快速鉴别白念珠菌和都柏林念珠菌的初步研究

目的 用一种新制备的单克隆抗体MAb03．2Cl-C2鉴别生物学形态相近的白念珠菌和都柏林念珠菌。方法 用小鼠体内诱导法制备抗白念珠菌芽管胞壁外膜单克隆抗体MAb03．2Cl-C2。用不完全RPMI1640培养液、L—DMEM、H—DMEM、完全1640液、小牛血清诱导白念珠菌和都柏林念珠菌芽管及菌丝形成,间接免疫荧光（IIF）方法检测都柏林念珠菌芽管或菌丝表面有无可与该单抗相结合的成分。收集临床口腔念珠菌病标本涂片,直接做IIF试验。结果 用不完全RP-MI1640培养液37℃,6h可同时最高效率地诱导白念珠菌和都柏林念珠菌芽管或菌丝形成。单抗MAb03．2Cl-C2仅与白念珠菌芽管或菌丝特异性地结合,与都柏林念珠菌的孢子和菌丝不能结合。结论 单抗MAh03．2Cl-C2可用于白念珠菌和都柏林念珠菌实验室的速鉴别。     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

Promoter regulation in Candida albicans and related species

Regulation of gene expression has been studied extensively in Saccharomyces cerevisiae and Schizosaccharomyces pombe . Some, but by far not all, of the findings are also applicable to Candida albicans , an important ascomycete fungal pathogen of humans. Areas of research in C. albicans include the influence of key signal transduction cascades on morphology, and the response to host-generated influences, such as host immune effector cells, blood, pH or elevated carbon dioxide. The resistance to antifungal agents and response to stress are also well researched. Conditional gene expression and reporter genes adapted to the codon usage of C. albicans are now widely used in C. albicans . Here we present a comprehensive overview of the current techniques used to investigate regulation mechanisms for promoters in C. albicans and other Candida species. In addition, we discuss reporter genes used for the study of gene expression.     

Identification and immunochemical characterization of a germ tube specific antigen of Candida albicans

Abstract Germ tube specific fractions of the dimorphic pathogenic fungus Candida albicans were fractionated according to their ability to link fibrinogen. These fibrinogen binding factors were used as immunogens to prepare monoclonal antibodies (mAbs) with BALB/c mice. Among the resulting mAbs, one (mAb 3D9.3) was shown by indirect immunofluorescence to be specific to the surface of the mycelial phase of the C. albicans species. No labelling of the cell wall of any other Candida species was observed. This morphological shape specificity was confirmed by immunoblotting where a polydispersed high molecular mass component was identified. The molecular mass varied with the extraction procedure used; over 210 kDa with EDTA-2ME treatment, and ranging from 110 to 220 kDa after Zymolyase digestion. This phase-specific epitope was sensitive to proteolysis with pronase E, proteinase K and trypsin, but not to periodate treatment. Further purification of this material would allow further development of new serodiagnostic assays that might be more specific for invasive disease than currently available tests.     

白念珠菌MP65和SAP2蛋白原核表达质粒的构建与表达

目的构建以白念珠菌基因MP65和SAP2为目的基因的原核表达质粒,IPTG诱导其表达融合蛋白,并对其免疫原性进行分析。方法PCR法自白念珠菌标准菌株获取MP65和SAP2基因,分别插入至原核表达载体pGEx．4T之和pET32a中;将重组表达质粒转染感受态EcoliBL-21,经IPTG诱导表达、纯化后,SDS—PAGE和Western blot分析。结果PCR法克隆出全长为1140bp的MP65和1197bp的SAP2基因,构建的原核表达质粒pGEX-4T-2-MP65及pET32a-SAP2,可分别表达出66KD左右的GST融合蛋白和His融合蛋白。结论成功获取了白念珠菌基因^伊65和SAP2,所构建的原核表达质粒在BL-21中成功表达;两种蛋白均有免疫原性。     

白念珠菌生物被膜的基因表达及相关基因研究进展

白念珠菌是一种条件性致病菌,可在人体植入性器械表面形成生物被膜.与浮游和以个体形式存在的白念珠菌相比,生物被膜在结构及功能上有很大差异,这种差异本质上是由基因表达决定的.近年来,研究者们试图通过芯片和基因敲除等技术手段,探索与白念珠菌生物被膜形成及耐药相关的基因,揭示其分子机制,寻找药物作用的新靶点.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Infection of HEp2 epithelial cells with Candida albicans: adherence and postadherence events

We have previously observed that the infection of HEp2 epithelial cells with Candida albicans results in HEp2 cell actin rearrangement, and that a culture filtrate of C. albicans (Candida metabolite) caused the same changes and reduced membrane ruffling and motility. It was found that the Candida metabolite consisted of several proteins and nonproteinaceous components. In this study we report on the identity of three of the main proteins in the Candida metabolite, namely a secretory aspartate protease (Sap), an agglutinin-like adhesion sequence (Als) and a glucan 1,3-beta-glucosidase. The effect on HEp2 cells caused by the Candida metabolite, an inhibitor of the PKC MAP kinase signal pathway - bisindolylmaleimide (BIM), or the actin polymerization inhibitor - cytochalasin D (CyD) were studied alone and in combination. Exposure of HEp2 cells to the Candida metabolite, together with the BIM or CyD, had profound effects on HEp2 cell morphology, as compared to individually treated cells, and also reduced the adherence of the organisms to HEp2 cells. Our results show that the interaction of C. albicans with HEp2 cells is, not unexpectedly, complex, and involves changes in the host cell that may be related to the effect of Candida-secreted biomolecules.     

白念珠菌新型耐药基因MXR1的敲除与鉴定

目的 构建用于白念珠菌MXR1基因敲除的载体质粒,并通过Ura-Blaster策略敲除MXR1两条等位基因.方法 分别扩增白念珠菌MXR1基因ORF两侧上下游的片段,通过酶切与连接反应,将上下游片段分别插入到p5921质粒的hisG-URA 3-hisG盒两端,从而形成MXR1敲除载体质粒pUC-MXR1-URA3.通过Ura-Blaster策略将载体质粒转染到白念珠菌RM 1000内,并采用PCR和Southern-blot杂交方法鉴定各步转染、复筛所得的阳性克隆.结果 成功获得MXR1基因缺失的菌株.结论 MXR1基因缺失菌株的构建,有助于深入研究白念珠菌耐药机制.     

大鼠白念珠菌支气管肺感染时肺组织Toll样受体2的表达及意义

目的 探讨Toll样受体2(TLR2)在白念珠菌支气管肺感染大鼠肺组织模型中的表达及意义.方法 建立白念珠菌支气管肺感染大鼠模型,观察感染后肺组织病理形态学改变,采用免疫组织化学法观察感染后不同时间(第3天、第7天)白念珠菌支气管肺感染大鼠肺组织TLR2的表达,并与未感染组进行比较.结果 大鼠白念珠菌支气管肺感染后肺组织TLR2表达水平明显升高.结论 白念珠菌支气管肺感染肺组织TLR2表达增高可能参与感染的炎症反应.     

白色念珠菌ALS家族基因的克隆与功能分析

利用ＣＸ２ ０．５ｋｂ探针,研究在不同白色念珠菌菌株中ＣＸ２串联重复序列的分布,发现多种白色念珠菌中都含有这个重复序列。为证明ＣＸ２的表达与形态转变有关,选用了多种诱导和非诱导菌丝生长的条件培养白色念珠菌ＳＣ５３１４,然后用串联重复序列作控针进行Ｎｏｒｔｈｅｒｎ杂交分析,证明ＣＸ２ ｃＤＮＡ片段的表达与菌丝形态紧密相关。用它作为探针进行染色体定位和基因组Ｓｏｕｔｈｅｒｎ杂交分析,表明白色念珠菌中多     

都柏林念珠菌的鉴定及与白念珠菌3种表型鉴别方法的研究

目的 从临床分离的念珠菌中进一步鉴定都柏林念珠菌,并评价3种表型鉴别白念珠菌和都柏林念珠菌的方法.方法 对17株临床分离并初步鉴定的白念珠菌和1株ATCC白念珠菌标准株,采用PHR1同源序列PCR法检测,鉴定出其中的都柏林念珠菌;分别采用45℃生长试验、YEPD(1%酵母浸膏,2%蛋白胨,2%葡萄糖)液基39℃芽管生成试验、Staib琼脂(鸟食琼脂)厚壁孢子形成试验对两种菌的表型特点进行比较.结果 17株临床分离的白念珠菌中有3株鉴定为都柏林念珠菌;45℃时,两种菌在改良沙堡弱琼脂上均无明显生长,YEPD液基中仅有1株白念珠菌生长良好;YEPD液基39℃培养2种菌均无芽管生成;Staib琼脂培养72h,3株都柏林念珠菌中有2株可形成厚壁孢子,而白念珠菌则无,与PHR1同源序列检测结果基本一致.结论 PHR1同源序列检测是鉴别都柏林念珠菌与白念珠菌的可靠方法,Staib琼脂厚壁孢子形成试验有助于鉴别两菌,45℃生长试验和YEPD液基39℃芽管生成试验则不能有效鉴别两菌.     

Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans

Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of alpha-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.     

Purification and germination of Candida albicans and Candida dubliniensis chlamydospores cultured in liquid media

Candida albicans and Candida dubliniensis are the only Candida sp. that have been observed to produce chlamydospores. The function of these large, thick-walled cells is currently unknown. In this report, we describe the production and purification of chlamydospores from these species in defined liquid media. Staining with the fluorescent dye FUN-1 indicated that chlamydospores are metabolically active cells, but that metabolic activity is undetectable in chlamydospores that are >30 days old. However, 5–15-day-old chlamydospores could be induced to produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae depending on the incubation conditions used. Chlamydospores that were preinduced to germinate were also observed to escape from murine macrophages following phagocytosis, suggesting that these structures may be viable in vivo . Mycelium-attached and purified chlamydospores rapidly lost their viability in water and when subjected to dry stress, suggesting that they are unlikely to act as long-term storage structures. Instead, our data suggest that chlamydospores represent an alternative specialized form of growth by C. albicans and C. dubliniensis .     

白念珠菌对宿主的黏附机制

白念珠菌对宿主的黏附是白念珠菌感染过程的关键的第一步,因此阐明白念珠菌对宿主的黏附机制对探索新的方法预防和治疗白念珠菌感染至关重要。近年来,研究者们从白念珠菌的表面结构、黏附素以及黏附相关基因等方面对白念珠菌与宿主的黏附机制进行了大量研究。该文就白念珠菌对宿主的黏附机制进行综述。     

白念珠菌新型耐药基因IPF14744敲除质粒的构建

目的 构建用于白念珠菌IPF 14744基因敲除的载体质粒.方法 分别扩增白念珠菌IPF 14744基因ORF两侧上下游的片段,通过酶切与连接反应,将上下游片段分别插入到p5921质粒的hisG-URA3-hisG盒两端,从而形成IPF 14744敲除载体质粒pUC-14744- URA 3.结果 成功获得IPF 14744基因敲除载体质粒.结论 所获得的质粒pUC-14744- URA3可用于白念珠菌IPF 14744基因的敲除.     

HOG1基因对白念珠菌超微结构的影响

目的探讨HOG1基因对白念珠菌超微结构的影响。方法设置实验组、HOG21组(hog1/hog1双等位基因缺陷株);对照组、WT组(标准株),分别在扫描电镜及透射电镜下观察两组菌株细胞的超微结构。结果扫描电镜下观察两组细胞均呈圆形或椭圆形,呈多边出芽繁殖的生长方式。但HOG1基因缺陷株细胞表面粗糙、凹凸不平,出芽数目比标准株少;标准株细胞表面光滑,出芽数量较多,可见"花瓣样"结构的芽痕;透射电镜下HOG1基因缺陷株细胞壁结构不完整,电子透明层厚薄不一,部分细胞可见棉絮状电子致密外层局灶性缺失、细胞膜外凸、不连续以及细胞膜周围见囊泡聚集等现象。标准株细胞壁各层结构完整。结论HOG1基因对白念珠菌细胞壁结构具有一定影响。