近平滑念珠菌形态多样性及毒力因子的分泌活性研究

目的比较近平滑念珠菌不同临床分离株的菌落和细胞形态及其分泌型天冬氨酸蛋白酶(SAP)酶活性,阐明该病原真菌的形态多样性及毒力差异。方法分别用Lee’s Glc NAc、Lee’s glucose、Spider 3种培养基于37℃培养7株近平滑念珠菌临床分离株,观察各菌株的菌落及细胞形态;用牛血清白蛋白培养基(BSA)于30℃培养7株近平滑念珠菌,通过观察形成晕圈能力的强弱确定其毒力因子(SAP)的分泌活性。结果 7株近平滑念珠菌临床分离株在不同条件下菌落表型有较明显的不同,菌落有光滑型、皱褶型及火山口样型,且染色程度不同,各分离株细胞在形成酵母相或菌丝相的能力方面呈现出不同程度的差异;SAP酶活性观察实验中检测到各菌株水解晕圈大小从2.55 cm到1.55 cm各不相等。结论近平滑念珠菌临床分离株具有形态多样性,各菌株SAP酶活性明显不同,提示菌株间的毒力有明显差异。     

白色念珠菌形态转换的调控

白色念珠菌 (Ｃａｎｄｉｄａａｌｂｉｃａｎｓ)是一种常见的致病真菌 ,属于半知菌亚门芽孢菌纲隐球酵母科念珠菌属。在健康人体粘膜表面 ,如肠道、口腔 ,白色念珠菌通常不会引起疾病。但在免疫系统受到损害或抑制的人体中 ,比如化疗病人、器官移植病人或是艾滋病人中 ,白色念珠菌能够引起严重的白色念珠菌病 ,危及生命。许多因素可能与白色念珠菌的致病性有关 ,如菌体对寄主细胞的粘附能力、白色念珠菌产生的分泌性蛋白酶、菌落形态的转换以及细胞形态的转换等[1] 。1 .影响白色念珠菌形态转换的因素白色念珠菌能够以三种形态生长 ,即菌体形…     

近平滑念珠菌引起真菌性膝关节炎：病例报告及文献回顾

报告1例由近平滑念珠菌引起的膝关节炎并进行相关骨关节真菌感染的文献回顾.患者,男,53岁,有糖尿病史及左侧腘窝皮肤浅表小肿瘤切除史,因左膝关节反复疼痛7 a,逐渐加重伴跛行6个月入院.临床表现为左膝关节明显红肿,影像学发现关节腔内滑膜增生,临近胫骨上端及股骨下端有灶性骨破坏、吸收,关节腔积液查见真菌,鉴定为近平滑念珠菌.经关节镜清理术及静脉用氟康唑治疗后症状改善,随访2 a无复发,已恢复日常活动.本文结合本例患者进行了骨关节真菌感染的相关文献复习,对骨关节感染的相关因素及治疗等内容进行了回顾,其结果对临床相关医生具有重要参考价值.     

牛源近平滑念珠菌对氟康唑耐药性研究

以牛源近平滑念珠菌（Candida parapsilosis）为试验菌株,采用微量稀释法进行药物敏感性试验,PCR扩增测序检测ERG11基因突变,Realtime PCR检测ERG11、CDR1、MDR1、MRR1基因的mRNA表达量,探讨耐药相关基因在牛源近平滑念珠菌耐唑类药物中的作用,为牛源近平滑念珠菌的耐药研究提供参考。结果表明,近平滑念珠菌对5-氟胞嘧啶、两性霉素Ｂ的敏感率均高于75%,对唑类药物的耐药率均高于50%,其中对氟康唑的耐药率最高,达58.3%;所有菌株的ERG11基因中均检测出错义突变A395T,耐氟康唑和剂量依赖菌株的ERG11基因中检测出同义突变T591C;氟康唑耐药组ERG11、CDR1、 MDR1、MRR1基因表达水平均显著高于敏感组（P<0.05）。牛源近平滑念珠菌对唑类抗真菌药物的耐药率较高且具有多重耐药性。牛源近平滑念珠菌ERG11基因中的T591C突变以及ERG11、CDR1、MDR1、MRR1基因的高表达都可能在其对氟康唑耐药性的产生中起到一定的作用。     

近平滑念珠菌ERG 11基因编码区的克隆及生物信息学分析

目的克隆、测序近平滑念珠菌ERG11基因的编码区序列并进行生物信息学分析。方法运用生物信息学的方法 ,通过与白念珠菌ERG11基因碱基序列同源性比对,在近平滑念珠菌基因组(www.sanger.ac.uk/sequencing/Candida/pa-rapsilosis/)中寻找可能的ERG11基因序列(CpERG11),并据此序列设计引物,经PCR扩增近平滑念珠菌标准株(ATCC22019)的ERG11基因片段,产物经电泳、纯化、克隆到质粒prG-AMAI-NotI中,转染DH10B大肠杆菌细胞,并酶切鉴定筛选阳性克隆测序分析。结果近平滑念珠菌ERG11编码区由1569个碱基组成,编码一段含522个氨基酸的多肽。近平滑念珠菌ERG11的编码区序列与白念珠菌、热带念珠菌、光滑念珠菌、酿酒酵母菌ERG11基因的同源性分别为74%、75%、65%、64%。该近平滑念珠菌ERG11的编码区为唑类药物作用靶酶基因。结论成功克隆、测序、并生物信息学分析近平滑念珠菌ERG11基因的编码区序列,为进一步的功能研究奠定基础。     

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。     

近平滑念珠菌性皮肤肉芽肿1例

<正>1临床资料患者男,59岁,"右手腕皮疹3a余,右上臂皮下结节3个月",于2014年4月就诊我院我科门诊。皮疹初起于右手腕伸侧,无明显外伤史,初起为一枚红色绿豆大丘疹,皮疹逐渐增多并融合,无明显痒痛,挤压可有黄色分泌物渗出。外院诊疗不详,无明显好转。就诊前3个月右上臂内侧出现皮下肿物,无压痛。既往体质可,高血压病史,血压控制可。查体:系统检查无特殊,右手腕伸侧见5cm×6cm大小红斑,上见多发大小不一红色斑块,表     

贵州地区临床分离近平滑念珠菌复合群分布及其生物膜形成相关基因研究

目的 研究贵州地区近平滑念珠菌复合群临床分布和药敏特点,检测近平滑念珠菌生物膜形成能力及其CDC28基因的表达水平。方法 收集贵州地区2所三级甲等医院送检标本中分离出的菌株,利用BACTEC^TM FX仪、Phoenix-100仪对菌种进行鉴定,通过基因测序对近平滑念珠菌复合群各分型菌株进行鉴定;采用ATB FUNGUS 3试剂盒检测菌株体外药物敏感情况;检测血源性近平滑念珠菌生物膜形成能力及利用qRT-PCR技术检测它们的CDC28基因表达水平。结果 共分离鉴定出163株近平滑念珠菌复合群,其中近平滑念珠菌Ⅰ型、Ⅱ型和Ⅲ型分别占73.0%、6.1%和20.9%;尿液和血液是分离菌株最多的标本(均为50株);血液途径感染的菌株中,Ⅰ型、Ⅱ型和Ⅲ型分别占54.0%、10.0%和36.0%;三种基因型菌株对常见抗真菌药物无明显耐药性;17株临床菌株中,16株(94.1%)具有生物膜形成能力,其中2株、4株和10株菌株生物膜形成能力强、一般、弱,CDC28在2株生物膜形成能力强以及3株生物膜形成能力一般的菌株中表达上调。结论 分离的近平滑念珠菌复合群以Ⅰ型感染为主,三种基...     

白念珠菌的形态发生与致病性

白念珠菌是一种广泛存在于人体内的共生真菌,也是人类最常见的机会性致病真菌,可引起浅表感染甚至威胁生命的系统性感染。白念珠菌具有很强的形态可塑性,而且这种形态可塑性与致病性密切相关。白念珠菌在侵染的过程中可以进行酵母、假菌丝和真菌丝之间的形态转换。除此之外,white形态、opaque形态、gray形态和GUT细胞在宿主不同的部位具有生长繁殖优势。本文总结了白念珠菌各种形态特征以及它们与致病性之间的联系,同时我们也简述了宿主环境因素调控这些形态的发生与转换的机理。     

白念珠菌的二型性转换：从共生菌到致病菌

白念珠菌Candida albicans是人体内的良性共生真菌,存在于宿主的口腔、表皮、胃肠道及阴道等处,在免疫能力低下的人群中可能引起严重的疾病。一般以二倍体的形式存在,且能在酵母、假菌丝和菌丝的状态之间转换。菌丝状态促进了白念珠菌的侵染能力,同时也可以使白念珠菌逃逸宿主的免疫攻击,在其对宿主的感染途径中起到了重要的作用。本综述将阐述白念珠菌菌丝形成的调控机制、菌丝的发育模式以及菌丝形态对宿主免疫系统的影响,并且简要介绍念珠菌属中热带念珠菌和耳念珠菌菌丝发育方面的相关研究。     

Experimental rat vaginal infection with Candida parapsilosis

The experimental vaginopathic potential of Candida parapsilosis was determined in ovariectomized rats maintained under pseudoestrus by estrogen administrations. Of the 3 strains of C. parapsilosis tested, that isolated from the vagina of a woman affected by vulvovaginal candidosis gave a prolonged and sustained experimental vaginitis, not different in extent and duration from that caused by a vaginal isolate of C. albicans from a vaginitis patient. The other two isolates of C. parapsilosis (one from the vagina of an asymptomatic subject and another from soil) were unable to infect rat vagina. Microscopic observations of PAS-stained vaginal smears from rats infected with the vaginopathic isolate of C. parapsilosis showed pronounced adherence of yeasts to exfoliated cells. In addition, this isolate of C. parapsilosis produced an elevated quantity of acid proteinase in vitro.     

Evaluation of xylitol production from corn cob hemicellulose hydrolysate by Candida parapsilosis

Candida parapsilosis was grown for 59 h in a medium containing corn cob hydrolysate consisting of 50 g xylose l^–1, 3.0 g glucose l^–1, 2.0 g arabinose l^–1, and 0.9 g acetic acid l^–1. A biomass of 9.1 g l^–1 was produced with 36 g xylitol l^–1 and 2.5 g ethanol l^–1. In a medium containing 50 g xylose l^–1 instead of corn cob hydrolysate, the concentrations of cells, xylitol, and ethanol were 8.6 g l^–1, 33 g l^–1, and 0.2 g l^–1, respectively. The differences between two cultures were due to the glucose and arabinose in the corn cob hydrolysate stimulating growth and the low concentration of acetic acid stimulating xylitol production.     

An ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells

This study describes the biochemical characterization of a phosphatase activity present on the cell surface of Candida parapsilosis, a common cause of candidemia. Intact yeasts hydrolyzed p-nitrophenylphosphate to p-nitrophenol at a rate of 24.30+/-2.63 nmol p-nitrophenol h(-1) 10(-7) cells. The cell wall distribution of the Ca. parapsilosis enzyme was demonstrated by transmission electron microscopy. The duration of incubation of the yeast cells with the substrate and cell density influenced enzyme activity linearly. Values of V(max) and apparent K(m) for p-nitrophenylphosphate hydrolysis were 26.80+/-1.13 nmol p-nitrophenol h(-1) 10(-7) cells and 0.47+/-0.05 mM p-nitrophenylphosphate, respectively. The ectophosphatase activity was strongly inhibited at high pH as well as by classical inhibitors of acid phosphatases, such as sodium orthovanadate, sodium molybdate, sodium fluoride, and inorganic phosphate, the final product of the reaction. Only the inhibition caused by sodium orthovanadate was irreversible. Different phophorylated amino acids were used as substrates for the Ca. parapsilosis ectoenzyme, and the highest rate of phosphate hydrolysis was achieved using phosphotyrosine. A direct relationship between ectophosphatase activity and adhesion to host cells was established. In these assays, irreversible inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells.     

Candida parapsilosis ATCC 7330 can also deracemise 1-arylethanols

Abstract

Candida parapsilosis ATCC 7330 grown using different culture conditions (inoculum size 4% (v/v), inoculum age 12 h, and harvest time 14 h) from those previously reported (inoculum size 2% (v/v), inoculum age 24 h, and harvest time 44 h) successfully deracemised racemic 1-arylethanols and 4-phenyl-2-butanol to the (R)-enantiomer (ee up to >99%). The deracemisation of racemic 1-aryl ethanol proceeds via (i) enantioselective oxidation of (S)-enantiomer followed by (ii) reduction of the ketone formed to give the racemic alcohol which gets kinetically resolved thus enriching for the (R)-enantiomer from the racemate. This is the first report on the deracemisation of 1-arylethanols using Candida parapsilosis ATTC 7330 via dynamic kinetic resolution.     

Dynamics of in vitro acquisition of resistance by Candida parapsilosis to different azoles

Candida parapsilosis is a common isolate from clinical fungal infectious episodes. Resistance of C. parapsilosis to azoles has been increasingly reported. To analyse the development of resistance in C. parapsilosis , four azole-susceptible clinical strains and one American Type Culture Collection type strain were cultured in the presence of fluconazole, voriconazole and posaconazole at different concentrations. The isolates developed variable degrees of azole resistance according to the antifungal used. Fluconazole was the fastest inducer while posaconazole was the slowest. Fluconazole and voriconazole induced resistance to themselves and each other, but not to posaconazole. Posaconazole induced resistance to all azoles. Developed resistance was stable; it could be confirmed after 30 days of subculture in drug-free medium. Azole-resistant isolates revealed a homogeneous population structure; the role of azole transporter efflux pumps was minor after evaluation by microdilution and cytometric assays with efflux pump blockers (verapamil, ibuprofen and carbonyl cyanide 3-chloro-phenylhydrazone). We conclude that the rapid development of azole resistance occurs by a mechanism that might involve mutation of genes responsible for ergosterol biosynthesis pathway, stressed by exposure to antifungals.     

Direct transformation of a clinical isolate of Candida parapsilosis using a dominant selection marker

Candida parapsilosis is a human pathogenic fungus with increasing importance, particularly in nosocomial infections. For detailed molecular genetic explorations of prototrophic clinical isolates of C. parapsilosis, we developed an efficient transformation system based on a dominant selectable marker. The gene encoding resistance to mycophenolic acid (MPA) was used for selection in yeast transformation. C. parapsilosis cells were transformed with a plasmid vector containing the Candida albicans inosine monophosphate dehydrogenase gene (IMH3) responsible for mycophenolic acid resistance. Transformation was carried out both by electroporation and by the lithium acetate (LiAc) method. The LiAc method resulted in very poor transformation efficiency, while the modified electroporation method yielded a high number of mitotically stable transformants exhibiting unambiguous MPA resistance. Two hundred transformants were analysed for the presence of the C. albicans IMH3(r) gene by polymerase chain reaction. Integration of single or multiple plasmid copies into the genomic DNA of C. parapsilosis was determined by Southern hybridization. To our knowledge, the present study is the first report about a method based on a dominant selectable marker for the transformation of a prototrophic, clinical isolate of C. parapsilosis. The described technique may prove to be an efficient tool for the examination of the biology and virulence of this pathogenic yeast.     

Cloning and characterization of Sapp2p, the second aspartic proteinase isoenzyme from Candida parapsilosis

The human fungal pathogen Candida parapsilosis possesses at least three genes encoding secreted aspartic proteinases. Whereas the Sapp1p isoenzyme has already been biochemically characterized, the SAPP2 and SAPP3 gene products have not. The Sapp2p precursor, pro-Sapp2p, was therefore expressed in Escherichia coli and purified. Autoactivation of pro-Sapp2p in acidic conditions was inefficient and resulted in a protein extended by eight amino acids at the N-terminus (Sapp2p(+8)). The correct promature junction KR/SSPSS was cleaved by trypsin or by a membrane-bound Kex2-like proteinase from Candida parapsilosis. The mature Sapp2p obtained by the assisted activation was proteolytically active. Its activity was more than twofold higher than that of the self-processed protein species Sapp2p(+8), as measured by the hemoglobin cleavage test. The substrate specificity of Sapp2p differs from that of Sapp1p. Peptides containing aromatic residues in the P1 and P1' positions are cleaved poorly by Sapp2p. A fluorogenic substrate was synthesized to facilitate further studies.     

Catabolism of 4-hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-hydroxybenzoate 1-hydroxylase

Abstract The first two steps in the catabolism of 4-hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. ¹⁹F-NMR experiments with cell extracts and 2-fluoro-4-hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.     

Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.