大蒜素对白念珠菌形态转换的影响研究

目的探讨大蒜素对白念珠菌形态转换的影响及其作用机制。方法倒置显微镜观察白念珠菌菌丝形成的体外动力学过程;采用CLSI-M27-A3微量液基稀释法检测大蒜素对白念珠菌的最小抑菌浓度(minimum inhibitory concentration,MIC);倒置显微镜观察不同浓度大蒜素对白念珠菌在Spider液体培养基中菌丝形成的影响;qRT-PCR法检测在不同浓度大蒜素作用下白念珠菌菌丝相关基因HWP1、ALS1、EFG1、PDE2表达水平的变化。结果白念珠菌在Spider液体培养基中6 h时出现较长菌丝,24 h后镜下可见大量念珠菌菌丝包裹酵母细胞,紧密交错;大蒜素对白念珠菌的MIC值为25μg/mL;倒置显微镜观察(25~100)μg/mL浓度的大蒜素能明显抑制Spider液体培养基中白念珠菌菌丝的生长;qRT-PCR结果显示,在(25~100)μg/mL浓度的大蒜素作用下,白念珠菌菌丝相关基因表达下调。结论大蒜素能有效抑制白念珠菌的形态转换,其作用机制可能与调节菌丝形成相关基因的表达水平有关。     

pH及氧气对白念珠菌菌丝形成的影响

目的探讨pH值和氧气对白念珠菌菌丝形成的影响。方法通过调节Muller—Hinton液体培养基的pH值和去除培养基中的氧气来观察白念珠菌的生长曲线、倍增时间和菌丝形成率的变化。结果在无氧气的液体培养基中,白念珠菌生长缓慢,不能产生菌丝结构,只有酵母细胞形成。生长曲线的延缓期内各组没有明显差异,而在生长的对数期pH3和pH4的条件下念珠菌生长速度明显慢于pH5、pH6、pH7、pH8和pH9。菌丝形成率在pH3、pH4和pH5条件下〈20％,而在pH6、pH7、pH8和pH9条件下可高达70％。结论厌氧条件抑制白念珠菌的菌丝形成,只形成酵母细胞。白念珠菌在pH3—9的范围内均能生长,偏酸性环境有利于白念珠菌酵母形成,偏碱性的环境有助于菌丝的形成。     

黄芩苷联合氟康唑通过降低cAMP抑制白念珠菌酵母-菌丝形态转化

目的探讨中药有效成分黄芩苷(Baicalin,BA)联合氟康唑(Fluconazole,FLC)对白念珠菌(Candida albicans)形态转化的影响及机制。方法在菌丝诱导培养基(液体和固体培养基)上观察两药联用对白念珠菌酵母-菌丝形态转化的影响;ELISA法检测两药联用对胞内cAMP含量的影响;回补实验验证两药联用对胞内cAMP的影响;qRT-PCR检测两药联用对白念珠菌cAMP相关基因RAS1,CDC35和PDE2表达的影响。结果黄芩苷联合氟康唑能显著抑制白念珠菌酵母-菌丝形态转化,降低胞内cAMP水平;外源性cAMP能逆转两药联用所造成的菌丝抑制;两药联用使RAS1和CDC35表达分别下调35%和27%,使PDE2上调1.21倍。结论黄芩苷协同氟康唑显著抑制白念珠菌形态转化,其作用机制可能与下调胞内cAMP有关。     

龙胆泻肝汤氯仿提取物对白念珠菌VVC临床株菌丝抑制作用研究

目的观察龙胆泻肝汤氯仿提取物(chloroform extracts of Longdan xiegan decoction,CELX)对白念珠菌菌丝形成的影响,探讨其可能的作用机制。方法采用微量稀释法测定龙胆泻肝汤不同溶剂提取物对15株VVC临床株的最低抑菌浓度(minimal inhibitory concentration,MIC);采用固体培养基和半固体培养基观测菌落形态及菌丝侵袭能力;XTT还原法评价白念珠菌菌丝活性;平板法计数6h内药物对白念珠菌CFU的影响;荧光显微镜下观察菌丝代谢活力;采用实时荧光定量PCR(qRT-PCR)检测其菌丝相关基因:ALS3、SSA1、SUN41、HWP1、UME6和ECE1的表达量。结果 CELX对15株VVC临床株MIC介于32~64mg/L之间,效果优于总提物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物和水提取物的MIC;256mg/L的CELX能显著抑制菌丝的形成;256mg/L的CELX在固体培养基中可抑制菌落褶皱,在半固体培养基中可抑制菌丝侵袭;qRT-PCR结果显示256 mg/L CELX可使菌丝相关基因ALS3、SSA1、SUN41、HWP1、UME6和ECE1分别下调81.6%、60.2%、69.8%、73.7%、49.5%、52.8%。结论龙胆泻肝汤氯仿提取物(CELX)可通过影响菌丝相关基因的表达来抑制菌丝的形成,从而降低白念珠菌对机体的侵袭力。     

白念珠菌对特比萘芬耐药性的体外梯级诱导和回复研究

目的以浓度梯级倍增的特比萘芬在体外诱导白念珠菌标准株获得耐药子代菌株,并观察其耐药稳定性,从细胞水平研究白念珠菌对特比萘芬耐药前后生物学特性的变化,为进一步采用基因芯片在基因表达水平上研究特比萘芬对白念珠菌的药理作用及其诱导耐药机制提供理想的实验模型。方法将白念珠菌ATCC90028株在特比萘芬浓度逐渐梯级倍增的YPD液体培养基中分别转种传代,直到最后转种至含1024μg／ml特比萘芬的YPD液体培养基中培养,分别测定诱导后形成的各子代菌株的MIC值;选用以1024μg／ml特比萘芬诱导形成的耐药菌株,在不含特比萘芬的YPD液体培养基中连续传代10次后,测定其MIC值,观察其耐药表型的稳定性;并分别用肉眼、光镜和电镜观察白念珠菌耐药性产生前后的形态学特征。结果特比萘芬MIC值为8μg／ml的白念珠菌母本菌株（白念珠菌ATCC90028）成功地被诱导成特比萘芬MIC值为≥512μg/ml的子代菌株,进一步的耐药稳定性实验说明诱导后形成的子代菌株的表型是相对稳定的,诱导后的子代耐药菌株与其母本相比,生长繁殖速度减慢,细胞形态不规则,部分细胞胞膜不完整。结论通过在药物浓度梯级倍增的培养基中连续传代培养的方法可成功建立相同基因型的对特比萘芬敏感的白念珠菌母本和对特比萘芬耐药的子代模型,为获取有亲本的耐药白念珠菌菌株提供了一个有效的实验方法,是在基因水平研究白念珠菌对特比萘芬耐药机制的理想实验模型。     

不同物质对白鬼笔菌丝生长的影响

本文研究了不同物质对白鬼笔菌丝生长的影响.采用平板培养法,以白鬼笔菌株Phallus impudicus-1为供试菌株,分析了 5种物质对白鬼笔菌丝生长的影响.供试碳源中,当以红糖为碳源时,白鬼笔菌丝生长速率最快,为1.445mm/d,蔗糖和葡萄糖次之;供试氮源中,当以酵母膏为氮源时,白鬼笔菌丝浓密,菌丝生长速率最快,...     

酵母相和菌丝相白念珠菌感染阴道上皮细胞免疫反应的比较

目的白念珠菌作为一种二相菌,存在酵母相和菌丝相二态,本实验研究这两种不同形态的白念珠菌刺激阴道上皮细胞,比较其引起的免疫应答差异。方法以阴道上皮细胞株VK2/E6E7为模型,分别给予不同浓度的活的白念珠菌和热灭活白念珠菌SC5314(感染指数MOI分别为10、50、100)进行刺激,以实时荧光定量PCR(RT-qPCR)和酶联免疫吸附法(ELISA)检测3 h、6 h、12 h感染组细胞的促炎因子TNF-α和趋化因子IL-8的分泌水平。结果活的白念珠菌在K-SFM细胞培养基中呈菌丝相,热灭活的白念珠菌在K-SFM培养基中呈酵母相。两相感染组分泌IL-8和TNF-α水平与未感染组相比具有显著差异。在各个MOI条件下,菌丝相感染组释放细胞因子mRNA水平均显著高于酵母相;在MOI=10时,菌丝相感染组释放细胞因子的蛋白水平强于酵母相感染组,而随着MOI的增大,结果相反。结论酵母相和菌丝相白念珠菌均能诱发阴道上皮细胞出现显著免疫反应。同时,在MOI=10时,菌丝相白念引起的免疫反应强于酵母相白念。两相感染组免疫反应强度的差异提示两者引起免疫应答的机制可能不同。     

白念珠菌酵母相与菌丝相对5种抗真菌药物的敏感性比较

目的比较白念珠菌酵母相与菌丝相对常用抗真菌药物的敏感性的差异,为病原真菌学研究及临床用药提供理论指导。方法应用法国生物-梅里埃ATB FUNGUS 3药敏试剂盒对临床分离的220株白念珠菌进行抗真菌药物的敏感性测定。结果 220株白念珠菌酵母相与菌丝相对两性霉素B、氟康唑、伊曲康唑、伏立康唑、5-氟胞嘧啶5种常用的抗真菌药物的敏感率分别为97.27%与97.73%,81.82%与85.00%,87.27%与90.46%,92.72%与95.00%,96.82%与97.27%。氟康唑、伊曲康唑和伏立康唑对白念珠菌酵母相MIC值明显高于菌丝相,差异有显著性(P0.05),两性霉素对白念珠菌酵母相和菌丝相MIC值差异不显著(P0.05)。受试菌株两相细胞对5种抗真菌药物敏感度总体一致性为85.46%(188/220),有32株菌株酵母相表现为耐药及中介,但菌丝相却为中介与敏感。结论白念珠菌酵母相和菌丝相对5种抗真菌药物的敏感性存在差异,抗真菌药物对菌丝相的抗菌活性强于酵母相。     

小G蛋白抑制剂CASIN的体外抗真菌活性研究CSCD

目的通过对小G蛋白抑制剂体外抗真菌活性的测定,筛选具有新结构类型的抗真菌活性化合物。方法采用微量液基稀释法考察不同小G蛋白抑制剂对于念珠菌的最低抑菌浓度(MIC),并从中筛选活性较强的化合物进一步进行抗真菌谱测定;采用时间-生长曲线和时间-杀菌曲线考察CASIN对于白念珠菌生长增殖的影响;采用菌丝诱导实验考察化合物CASIN对于白念珠菌菌丝形成的抑制效果;采用甲基四氮盐(XTT)法考察化合物CASIN对于白念珠菌生物被膜形成的抑制作用。结果在考察的小G蛋白抑制剂中,CASIN的体外抗真菌活性最强,对于近平滑念珠菌、克柔念珠菌、热带念珠菌、光滑念珠菌中均有较好的抑制作用,MIC为8~16μg/mL。16μg/mL CASIN可以有效抑制YPD培养基中白念珠菌的生长增殖,也在RPMI1640培养基中展现出对白念珠菌的杀菌活性;当培养基中CASIN浓度高于8μg/mL时,白念珠菌菌丝受到明显抑制;此外,8μg/mL的CASIN浓度对于白念珠菌的被膜生成具有80%以上的抑制率。结论小G蛋白抑制剂CASIN具有较强的体外抗念珠菌活性。     

尖顶羊肚菌液体培养基质与条件的研究

通过对尖顶羊肚菌液体培养基质与条件的研究,明确其菌丝生长的最适pH值、最适温度、适宜光照条件、适宜葡萄糖和蛋白胨浓度、适宜培养基,以便应用于尖顶羊肚菌液体菌种的生产和工业发酵。结果表明:菌丝的最适生长温度为2 5℃;最适生长pH值为6 ;葡萄糖和蛋白胨最适浓度分别为2 0 0g/L和10g/L ;菌丝在黑暗环境下生长良好,光照对菌丝生长具有抑制作用;用胡萝卜酵母膏培养基振荡培养形成的菌丝球多,菌丝生长量大;菌丝球在不同培养基中生长,可引起培养液pH值的上升或者下降;菌丝球可利用培养基内的氨基酸,使氨基酸降解,在胡萝卜酵母膏培养基中振荡培养8d的菌液总氨基酸含量较原液减少了36 71% ,亮氨酸、异亮氨酸和甲硫氨酸含量的下降幅度最大     

Identification of salivary components that induce transition of hyphae to yeast in Candida albicans

Candida albicans , the major human fungal pathogen, undergoes a reversible morphological transition from single yeast cells to pseudohyphae and hyphae filaments. The hyphae form is considered the most invasive form of the fungus. The purpose of this study is to investigate the effect of saliva on hyphae growth of C. albicans. Candida albicans hyphae were inoculated in Roswell Park Memorial Institute medium with whole saliva, parotid saliva or buffer mimicking the saliva ion composition, and cultured for 18 h at 37 °C under aerobic conditions with 5% CO₂. Whole saliva and parotid saliva induced transition to yeast growth, whereas the culture with buffer remained in the hyphae form. Parotid saliva was fractionated on a reverse-phase C8 column and each fraction was tested for inducing transition to yeast growth. By immunoblotting, the salivary component in the active fraction was identified as statherin, a phosphoprotein of 43 amino acids that has been implicated in remineralization of the teeth. Synthetically made statherin induced transition of hyphae to yeast. By deletion of five amino acids at the negatively charged N-terminal site (DpSpSEE), yeast-inducing activity and binding to C. albicans were increased. In conclusion, statherin induces transition to yeast of C. albicans hyphae and may thus contribute to the oral defense against candidiasis.     

复合碳源培养对酒糟厌氧消化液中丙酸互营氧化菌及群落结构的影响

丙酸累积是影响厌氧消化系统稳定性的主要因素,为了考察酒糟厌氧消化过程中间代谢产物的累积情况,以总固体含量(TS)（质量分数） 5%和7%的白酒糟为发酵原料进行了批次试验。结果表明,乙酸(最高浓度33~129 mmol/L)、丙酸(39~61 mmol/L)、丁酸(5~44 mmol/L)和15种氨基酸(0.01~0.3 mmol/L)为主要中间代谢产物。为了探究其中关键的丙酸降解菌群,以酒糟原始沼液JO为植种源,10 mmol/L丙酸和0.1 mmol/L混合氨基酸为复合碳源进行富集培养,获得中温厌氧丙酸-氨基酸培养系JO-AP。高通量测序分析表明,互营丙酸降解菌与厚壁菌门（Firmicutes）的丙酸厌氧降解菌（Pelotomaculum schinkii）近缘,16S rRNA基因相似性100%,占细菌总丰度的16.7%。对比酒糟原始沼液JO、丙酸培养系JO-P及丙酸-氨基酸培养系JO-AP中的主要功能菌群,发现采用单一碳源和复合碳源获得的优势互营丙酸降解菌不同;传统培养与分子生物学技术相结合可以更全面地掌握系统中的微生物群落组成。     

Evaluation of a new chromogenic medium (Candida ID) for the isolation and presumptive identification of Candida albicans and other medically important yeasts

Candidiasis is a frequent human infection caused mainly by Candida albicans. However, other species are emerging as important pathogens, as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei or Candida guilliermondii. Rapid identification of clinical isolates could facilitate diagnosis and treatment. Candida ID (bioMerieux, Spain) is a new medium for the isolation and presumptive identification of yeasts: C. albicans grows as blue colonies, and C. tropicalis, C. guilliermondii, Candida kefyr and Candida lusitaniae as pink ones. The utility of Candida ID was evaluated with more than 700 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Saccharomyces, and Rhodotorula. Presumptive identification was confirmed by germ tube test, microscopic morphology and chlamydoconidia production on corn meal agar and carbohydrate assimilation on API-ATB ID 32C or Vitek (bioMerieux). Growth on Candida ID was rapid (18-24 h) for most of the yeast strains tested. Sensitivity and specificity of identification of C. albicans was significantly high (>98%), since a very low number of isolates were found to be false negative or false positive. A better result was obtained for species growing as pink colonies (>99.5%). Detection of different species of medical important yeasts was easy with Candida ID, as perfectly distinct colors and textures of colonies were observed on this medium. Candida ID allowed the discrimination between C. glabrata (creamy and smooth) and C. krusei (rough and white) colonies. Other species showed different colony textures and colours, white being the predominant colour. Candida ID was very useful for the presumptive identification C. albicans isolates.     

Nutrition-dependent modulations of protein synthesis in Candida albicans during germ-tube formation or maintenance of the yeast form in N-acetyl glucosamine media

Abstract Stationary phase, yeast-form cells of Candida albicans grown in glucose-yeast extract medium were shifted to N -acetylglucosamine (GlcNAc) and/or glucose medium, and the pattern of protein synthesized under conditions of a progressive decrease in the rate of total protein synthesis was analyzed by SDS-PAGE and autoradiography.
Marked temporal modulations in the rate of synthesis of some cytoplasmic proteins were detected both in cells forming germ-tubes (at 37°C) and in yeast cells (at 28°C). The major modulated components showed molecular weights of 63, 53, 48 and 34 kDa. These products could not be qualified as heat-shock or heat-stroke proteins, because analogous modulations were observed on shifting cells from 28°C to 37°C or from 28°C to 28°C. However, no marked modulations in the synthesis of specific proteins were detected when amino acids were added to the medium fostering germ-tube formation under conditions of unimpaired overall rate of protein synthesis.
It is suggested that the modulations observed in cells incubated in GlcNAc-glucose medium could represent a response to a nutritional stress.     

Genetic analysis of Candida albicans morphological mutants

In contrast to some other strains, Candida albicans 1001 gave rise, upon UV irradiation, to mutants displaying a 'rough colony' morphology associated with a permanent alteration in morphogenesis which determined growth of the cells mostly as pseudohyphae. One of these mutants, C. albicans 1001FR, could form sectored (rough/smooth) colonies spontaneously, and with increasing frequency by treatment with mild UV doses (32-64 microJ mm-2). Rough sectors corresponded to stable 'rough-filamentous' strains which never segregated smooth strains. On the other hand, smooth sectors consisted mainly of yeast cells which could occasionally revert to a rough-filamentous phenotype. We suggest that C. albicans 1001 is heterozygous for some gene involved in the control of morphogenesis, and that the described mutants should be of help in the characterization of the genetic control of dimorphism in C. albicans.     

An ER packaging chaperone determines the amino acid uptake capacity and virulence of Candida albicans

The Candida albicans CSH3 gene encodes a functional and structural homologue of Shr3p, a yeast protein that is specifically required for proper uptake and sensing of extracellular amino acids in Saccharomyces cerevisiae. A Candida csh3delta/csh3delta null mutant has a reduced capacity to take up amino acids, and is unable to switch morphologies on solid and in liquid media in response to inducing amino acids. CSH3/csh3delta heterozygous strains display normal amino acid induced morphological switching. However, although heterozygous cells apparently sense and properly react to amino acid induced signals they cannot take up amino acids at wild-type rates. Strikingly, both CSH3/csh3delta heterozygous and csh3delta/csh3delta homozygous strains are unable to efficiently mount virulent infections in a mouse model. The haploinsufficiency phenotypes indicate that both CSH3 alleles contribute to maintain high-capacity amino acid uptake in wild-type strains. These results strongly suggest that C. albicans cells use amino acids, presumably as nitrogen sources, during growth in mammalian hosts.     

Ultraviolet microscopy of Candida albicans

Balish, Edward (Argonne National Laboratory, Argonne, Ill.), and George Svihla. Ultraviolet microscopy of Candida albicans. J. Bacteriol. 92:1812-1820. 1966.-Yeast and mycelial strains of Candida albicans were grown in medium supplemented with sulfur amino acids in an effort to determine factors that control the morphology and pathogenicity of the organism. Ultraviolet microscopy revealed a greater concentration of S-adenosylmethionine in the vacuoles of the mycelial phase than in those of yeast phases. Supplementation with amino acids greatly increased the concentration of S-adenosylmethionine in the mycelial phase, and made these cells more sensitive to the lytic action of snail gut enzymes than two yeast phase strains. This indicates a difference in cell wall structure that may be related to the pathogenicity of the mycelial phase.     

Transport of basic amino acids in Candida albicans

In Candida albicans, ATCC 46977, transport of basic amino acids is mediated by two systems (S1 and S2). Kinetic data and competitive inhibition studies of the different systems showed that transport of L-lysine, L-arginine and L-histidine have distinct specificities. System S1 of L-lysine and L-arginine was highly specific for the respective single basic amino acid. However, S2 of L-lysine and S1 of L-histidine were shown to be specific systems for most of basic amino acids. S2 of L-arginine was different from S2 of L-lysine and S1 of L-histidine. The effect of a thiol reagent, N-ethylmalemide, revealed that S2 of L-lysine and S1 of L-histidine were sensitive to this reagent, while all other systems were insensitive. The transport activity of different systems of L-lysine, L-arginine and L-histidine was followed during the growth of C. albicans. It was observed that different basic amino-acid systems have maximum activity during different stages of C. albicans growth.