Two different NO-dependent mechanisms account for the low virulence of a non-mycelial morphological mutant of Candida albicans

We have previously described the low virulence of a Candida albicans morphological mutant: 92′. We have now used this strain to examine the role of phagocytes in its pathogenesis. Our results show that C. albicans 92′ cannot evade innate host macrophage defence mechanisms as efficiently as the parental strain. In addition to the high susceptibility to phagocytosis by peritoneal macrophages, the NO produced by macrophages is a very important element in the low virulence of this agerminative mutant, a thesis supported by in vivo and in vitro experiments. Whereas the parental strain was able to inhibit macrophage NO production, the mutant was quite inefficient at reducing NO production by macrophages. In addition, the mutant showed high sensitivity to a NO generator. Treatment of mice with aminoguanidine (a preferred inducible NO synthase inhibitor) caused 90% mortality in 92′ systemic infection, thus supporting a role for NO in the low virulence of this strain. Our data show that both the low inhibitory effect of 92′ on macrophage NO production and the higher sensitivity to NO underlie the low virulence of this strain. Received: 2 January 2001     

Detection of phospholipase activity of Candida albicans and non albicans isolated from women of reproductive age with vulvovaginal candidiasis in rural area

Background: Vulvovaginal candidiasis (VVC) is most common accounting for 17 to 39% of symptomatic women. Both Candida albicans and non albicans Candida species are involved in VVC. Amongst various virulence factors proposed for Candida, extracellular phospholipases is one of the virulence factor implicated in its pathogenicity. With this background the present study was carried out to find the prevalence of different Candida species and to detect phospholipase producing strains isolated from symptomatic women with VVC. Materials and Methods: At least two vaginal swabs from 156 women of reproductive age with abnormal vaginal discharge were collected. Direct microscopy and Gram’s stained smear examined for presence of budding yeast and pseudo mycelia followed by isolation and identification of Candida species. Extracellular phospholipase activity was studied by inoculating all isolates on Sabouraud’s dextrose egg yolk agar (SDA) medium. Results: Of the 156 women with curdy white discharge alone or in combination with other signs, 59 (37.82%) women showed laboratory evidence of VVC. A total of 31 (52.54%) women had curdy white discharge followed by 12 (20.33%) with other signs and symptoms. C. albicans (62.59%) and non albicans Candida (37.28%) in a ratio of 1.68:1 were isolated. Of the 37 strains of C. albians 30 (81.08%) showed the enzyme activity. Seventeen (56.66%) strains showed higher Pz value of < 0.70 (++++). Conclusion: Although there may be typical clinical presentation of Candidiasis. all the patients did not show laboratory evidence of infection. Pregnancy was found to be major risk factor for development of VVC. C. albicans was prevalent species but non albicans species were also frequently isolated. Extracellular phospholipase activity was seen in C. albicans and not in non albicans Candida isolates.     

抗白念珠菌芽管单克隆抗体的制备及特性鉴定

目的制备抗白念珠菌芽管的单克隆抗体mAb03.2C1-C2,并对其特性进行分析,以探讨其应用于实验室检测的可行性。方法利用杂交瘤技术制备分泌抗白念珠菌芽管胞壁外膜抗原mAb的杂交瘤细胞株,对mAb的Ig亚类进行鉴定。用间接免疫荧光(IIF)法对mAb的抗体活性及特异性进行测定。在白念珠菌芽管形成条件下,加入mAb,观察其对白念珠菌芽管诱导的抑制及白念珠菌对上皮细胞、内皮细胞黏附的抑制。复苏冻存的杂交瘤细胞株,分析其持续分泌mAb的能力。结果获得1株mAb03.2C1-C2,其Ig亚类为IgG1。该mAb仅与白念珠菌芽管特异性结合,并能显著降低白念珠菌芽管的生成率以及在芽管形成条件下对上皮细胞、内皮细胞的黏附,其抑制作用与该mAb的浓度呈正比。结论制备的mAb03.2C1-C2抗体效价高,在体外能抑制白念珠菌芽管的形成,降低白念珠菌的侵袭力。而且其对白念珠菌芽管具有高度的特异性,可用于白念珠菌和都柏林念珠菌的快速鉴别。     

Comparison of adherence of Candida albicans and Candida parapsilosis to silicone catheters in vitro and in vivo

Objective  Although Candida parapsilosis has been associated with device-related infections in the clinical settings, factors that contribute to this association have not been previously examined. The objectives of this study were to compare in vitro and in vivo the adherence to silicone catheters of: (1) Candida albicans vs. C. parapsilosis , and (2) invasive vs. colonizing isolates of C. albicans and C. parapsilosis .
Methods  The records of 840 patients who had had Candida species isolated at three teaching hospitals during a three-month period were reviewed. A total of 20 clinical isolates of each of C. parapsilosis and C. albicans were examined for their adherence to silicone catheters in vitro and in a rabbit model of percutaneously placed catheters. For each Candida species, ten invasive isolates that had caused clinical device-related infection and 10 colonizing isolates that had caused only device colonization were studied.
Results  Candida parapsilosis accounted for <5% of yeast isolates from all sites, while three-quarters were C. albicans . Candida parapsilosis was isolated proportionately more often from blood and/or devices than C. albicans (34.3% vs. 8.5%, respectively, P  < 0.0001). There were no significant differences in the degrees of adherence in vitro and in vivo between C. albicans and C. parapsilosis or between invasive and colonizing Candida .
Conclusion  Although C. parapsilosis was isolated proportionately more often from blood and/or devices than C. albicans in our studied population, there was no significant difference in the adherence of the two Candida species to silicone, nor between invasive and colonizing Candida in our in vitro and in vivo models. Factors other than microbial adherence may help explain the observed association of C. parapsilosis with device-related infections.     

Intracellular killing of Candida albicans by human polymorphonuclear leucocytes: comparison of three methods of assessment

Three different methods, [3H]uridine uptake, viable count and 51Cr-release were used to assess the intracellular survival of a strain of Candida albicans, 19321, which was lethal for mice injected intravenously. Intracellular survival 1 h after ingestion ranged from 50 to 80% depending on the method employed and the detergent used to lyse the phagocytes. Inhibition of uridine uptake by detergents used to lyse the phagocytes led to difficulty in assessment of intracellular killing by this method.     

Molecular heterogeneity of fluconazole-resistant and -susceptible oral Candida albicans isolates within a single geographic locale

The emergence of drug-resistant Candida albicans in immunocompromised patients is common. A disconcerting aspect of this phenomenon is the rapid emergence of C. albicans strains that are resistant to a widely used azole drug, fluconazole (FLZ). To understand the origin of FLZ-resistant yeast isolates, we investigated molecular profiles of 20 geographically related oral C. albicans isolates using three genotyping methods: randomly amplified polymorphic DNA-PCR, with six different primers (OBU1, OBU2, OBU3 RSD6, RSD11 and RSD12); electrophoretic karyotyping by pulsed-field gel electrophoresis; and HinfI restriction fragment analysis. Of the 20 isolates studied, 10 were FLZ- resistant and originated from patients with oral candidosis with a history of FLZ therapy, and the remainder were FLZ susceptible from individuals with oral candidosis, but without a history of FLZ therapy. A composite genotype was generated for each strain by combining molecular types derived from the three independent molecular methods. The composite profiles indicated genetic diversity amongst both the FLZ-resistant as well as -sensitive isolates, and no specific features emerged distinguishing the drug-resistant and -sensitive groups. These observations cast doubt on the theory of a clonal origin of FLZ-resistant C. albicans isolates.     

Genotypic profiles and virulence attributes of Candida albicans isolates from patients with oral lichen planus

Candida albicans carriage has been found to be increased in patients with oral lichen planus. In the present work we have investigated the genotypic profiles of 112 C. albicans strains isolated from patients with erosive or nonerosive OLP, and from healthy controls. The virulence attributes of the isolated strains were compared to elucidate the pathogenetic mechanisms through which C. albicans may cause OLP. We have characterized the genotypic profiles of these isolated strains using a randomly amplified polymorphic DNA assay. In addition, we used assays to measure adhesion to buccal epithelial cells and phospholipase activity to evaluate the virulence attributes of these isolates. Our RAPD analyses revealed four different genotypes, named type A to D, among all isolates, and identified statistically significant associations with disease conditions. Specifically, type A (58.1%) and C (29.0%) were primarily found in erosive OLP, while type A (33.3%) and D (58.3%) were identified in nonerosive OLP. However, the healthy group seemed to have type B (38.5%) and D (61.5%). Using adhesion to BEC assay, we demonstrated that the isolates with genotype A had the strongest adherence among the four genotypes (P=0.000<0.05). The phospholipase activity of the isolates with genotype A and C was higher than for those with genotype B and D (P=0.000<0.05). In conclusion, some C. albicans isolates with special genotypic profiles and virulence attributes may contribute to the pathogenesis of OLP.     

Starvation survival of Candida albicans in various water microcosms

Candida is a major Human pathogen causing a variety of infections and can survive for extended period of time in aquatic environment including marine and fresh water. In this study we compared a colorimetric XTT assay to colony forming units (CFU) count to evaluate the survival potential of Candida albicans incubated in water microcosms. Our results showed that cells maintain cultivability within a long period followed by a decline in cultivability and a drop of plate counts to less than 20 cell ml(-1) after 150 days in tap water, 190 days in rain water and 200 days in seawater. In addition we noted that 10% of cells viability was reached after 150 days in seawater, 180 days in rain water and 210 days in tap water. Molecular method confirms the persistence of C. albicans cells in water during long time starvation period.     

老年人口腔白色念珠菌的分布与基因分型研究

目的研究健康老年人口腔白色念珠菌的分布、基因型特点及基因型特点与白色念珠菌分布的相关性。方法来自成都地区212例老年人分为4组：A1（男性，有义齿）；A2（男性，无义齿）；B1（女性，有义齿）和B2（女性，无义齿）。CHROMagar Candida^TM鉴定培养基、碳水化合物同化反应鉴定体系（API 20C AUX）和随机扩增片段多态性分析（randomly amplified polymorphic DNA assays，RAPD）鉴定白色念珠菌。RAPD分析健康老年人口腔白色念珠菌分离株的基因型特征。统计学分析比较不同分组健康老年人口腔白色念珠菌基因型之间的差异。结果212例受检个体中检出89株白色念珠菌（42％），A1、A2、B1、B2白色念珠菌检出率分别为56．2％、21．3％、56．6％、38％。A1组与A2组白色念珠菌检出率差异有统计学意义，P〈0．01。以JWFP和NA为随机引物的RAPD分析将白色念珠菌分为5型。RAPD分析基因型构成在A1组与A2组之间比较差异有统计学意义，P〈0．05，A1组主要以A型为主。结论健康老年人口腔白色念珠菌的检出率与义齿修复相关。义齿修复可能促进具有特定基因型特点的白色念珠菌检出率增高。     

桔梗皂苷D防御口腔黏膜上皮细胞感染白色念珠菌的作用

目的:探讨桔梗皂苷D对白色念珠菌黏附口腔黏膜上皮细胞的影响。方法:MTT法检测不同桔梗皂苷D对口腔上皮KB细胞存活率的影响;将白色念珠菌、KB细胞以及不同浓度的桔梗皂苷D共同培养,革兰染色检测白色念珠菌黏附数,台盼蓝排斥实验检测念珠菌活力和菌丝转换率;ELISA法检测上清液中IL-18与人β-防御素2(HBD-2)的蛋白含量;实时荧光定量PCR和Western blot法分别检测KB细胞中HBD-2 mRNA与蛋白表达的变化。结果:桔梗皂苷D对KB细胞存活率无影响;随着桔梗皂苷D浓度的增加,白色念珠菌的黏附数、菌活力和菌丝转换率逐渐下降,上清液中IL-18与HBD-2的蛋白含量以及KB细胞中HBD-2 mRNA表达与蛋白水平逐渐降低。结论:桔梗皂苷D具有抑菌作用,并参与了口腔黏膜上皮细胞防御白色念珠菌感染的免疫反应。     

Novel role of the Candida albicans ferric reductase gene CFL1 in iron acquisition,oxidative stress tolerance,morphogenesis and virulence

Ferric reductase catalyzes the reduction of ferric iron into ferrous iron and plays an essential role in high-affinity iron acquisition. In this study, we found that the cfl1Δ/Δ (orf19.1263) mutant was not defective in iron acquisition. However, deletion of CFL1 increased cellular iron accumulation by elevating surface ferric reductase activity in Candida albicans, revealing that there existed functional redundancy and/or a compensatory upregulation mechanism among ferric reductase genes. The absence of CFL1 resulted in increased expression levels of other alternative ferric reductase genes, including FRP1, CFL2 and FRE10. In addition, CFL1 played an important role in the response to different oxidative stresses. Further research revealed that the cfl1Δ/Δ mutant exhibited higher levels of both ROS production and SOD activity under oxidative conditions. Moreover, deletion of CFL1 led to a profound defect in filamentous development in an iron-independent manner at both 30 and 37 °C. The cfl1Δ/Δ mutant exhibited highly attenuated virulence and reduced fungal burdens in the mouse systemic infection model, indicating that CFL1 might be a potential target for antifungal drug development. In summary, our results provide new insights into the roles of ferric reductase gene in C. albicans.     

免疫功能低下Wistar大鼠白念珠菌肺炎模型的建立

目的建立用于免疫干预研究的免疫功能低下白念珠菌肺炎Wistar大鼠模型.方法醋酸考的松皮下注射制成大鼠免疫功能低下模型,并于第14天气管内灌注白念珠菌,制成免疫功能低下合并白念珠菌肺炎模型.检测免疫功能低下组,免疫功能低下+白念珠菌1、3、7?d组,正常组,正常+白念珠菌1、3、7?d组大鼠肺泡巨噬细胞的吞噬及杀菌功能;抗原提呈功能;培养上清中TNF-α活性和IL-1β、IL-6水平;血淋巴细胞杀伤功能;血清IFN-γ活性、IL-1β、IL-6水平;免疫功能低下+白念珠菌1、7?d组和正常+白念珠菌1、7?d组肺组织IFN-γ、TNF-α、IL-1β、IL-6表达;并于第7天进行左肺白念珠菌培养计数.结果免疫功能低下的大鼠肺泡巨噬细胞的吞噬和杀菌功能,抗原提呈功能,以及分泌TNF-α、IL-1β、IL-6的功能均明显低于免疫功能正常的大鼠;免疫功能低下的大鼠血中淋巴细胞的细胞毒作用明显低于免疫功能正常的大鼠;免疫功能低下的大鼠血清IFN-γ活性和IL-1β、IL-6水平明显低于免疫功能正常的大鼠.免疫功能低下合并白念珠菌肺炎大鼠肺组织IFN-γ、TNF-α、IL-1β、IL-6表达灌菌后第1天均低于白念珠菌肺炎大鼠,第7天均高于白念珠菌肺炎大鼠.免疫功能低下的大鼠左肺白念珠菌培养计数6.50×108CFU,免疫功能正常的大鼠左肺白念珠菌培养未见白念珠菌生长.结论该模型是免疫功能低下宿主肺部感染研究的适宜模型.     

正常儿童口腔中白色念珠菌的分布及基因分组

目的 研究以白色念珠菌为代表的念珠菌，以及白色念珠菌基因分组在不同年龄段儿童口腔中的分布情况。方法 4组不同年龄儿童和1组成人作为研究对象，粘膜拭子法取样，CHROMagar Candida鉴定培养基分离鉴定，PCR方法一组引物确认白色念珠菌，一组引物基因分组。结果 不同年龄组儿童口腔中念珠菌的检出率有所不同，除健康青年外，白色念珠菌在检出的念珠菌中均占多数。白色念珠菌3种基因分组的分布也存在差异，以A组为主。结论 不同年龄阶段健康儿童口腔中，A组白色念珠菌组占主导地位。     

Differential role of NK cells against Candida albicans infection in immunocompetent or immunocompromised mice

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK‐cell‐depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK‐cell‐deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B‐cell‐deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti‐C. albicans host defense in immunosuppressed hosts with defective T/B‐lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK‐cell function.     

Immunoblotting analysis of concanavalin A -isolated allergens of Candida albicans

The carbohydrate-containing fraction of Candida albicans was isolated from the crude extract with ConA Sepharose affinity chromatography and studied by IgE-immunoblotting with individual and pooled sera from C. albicans-allergic subjects. In the ConA-bound fraction there was a diffuse IgE binding in the high molecular weight area which also gave a carbohydrate stain (PAS). A distinct band corresponding to a molecular weight of 70 kD bound specific IgE antibodies. This glycoprotein, presumably a mannoprotein, gave a weak carbohydrate staining and a strong protein staining. Further biochemical studies are needed to reveal the exact nature of the epitopes in the ConA-bound mannose-containing fraction of C. albicans.     

以LongSAGE方法寻找不同菌相生长白念珠菌细胞差异表达基因

目的 以长片段基因表达系列分析技术（LongSAGE）寻找酵母相和菌丝相生长的白念珠菌细胞差异表达基因。方法 采用LongSAGE方法，分别构建白念珠菌酵母相和菌丝相细胞Long—SAGE标签文库，比较文库获得不同菌相生长白念珠菌细胞差异表达基因。结果 成功构建了白念珠菌酵母相和菌丝相细胞的LongSAGE标签文库，比较文库获得了白念珠菌酵母相和菌丝相细胞间差异表达的单基因匹配标签。结论 用LongSAGE方法较完整地获得了白念珠菌酵母相和菌丝相细胞基因表达谱及其丰度的量化信息，文库间比较可获得不同菌相生长白念珠菌差异表达基因。     

白念珠菌在肠腔中的增殖与局部IgA抗体分泌的关系

目的 观察白念珠菌（白念菌）在肠腔内增殖与局部粘膜免疫反应的关系。方法 采用无特殊原菌动物经口喂入白念菌,在不同时相处死后,观察肠内白念菌总数及肠粘膜表面白念菌粘附数量;取肠系膜淋巴结做白念菌选择培养,观察移位体内发生率;采用Ｂｒｄｕ体内掺入显示局部粘膜淋巴细胞的增殖情况;流式细胞计数潘伊尔结细胞表面ＩｇＡ（ＳｍＩｇＡ）阳性率;免疫组化染色后计数固有层中ＩｇＡ浆细胞数量变化;ＥＬＩＳＡ法测定肠粘液