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。     

Adhesion of Candida albicans mutant strains to host tissue

Adhesion of Candida albicans to host cells is believed to represent a fungal virulence factor and a significant step in the development of candidiasis. As C. albicans strains may differ in their in vitro adhesion ability we initiated a study to investigate whether mutant strains differ in this respect from their parent wild-type. We assessed the in vitro adhesion of C. albicans CBS562 and two mutants obtained by mutagenesis with N′-nitrosoguanidine: a histidine auxotroph, SAG5, derived from CBS562, and a respiratory-deficient strain (a petite mutant), SAR1, derived from SAG5. The adhesion was tested in vitro using two target cell systems: (1) exfoliated human buccal epithelial cells (BEC); and (2) human keratinocyte tissue line cells (HaCaT cells). Adhesion to BEC was evaluated microscopically and that to HaCaT cells by a direct ELISA technique. The results indicated a 54% reduction in adhesion to BEC for SAG5 and 30% for SAR1 as compared to the wild-type, and a 25% reduction in adhesion to HaCaT cells for SAG5 and 20% for SAR1. To verify whether the prototrophy restores the adhesion ability, we complemented the his-negative auxotroph by transforming the strain with the HIS4 gene. Then we assayed the adhesion to BEC of the complemented his-negative mutant in comparison to that of the wild-type, the his-negative mutant (SAG5) and the plasmid-cured transformant. The adhesion values of the complemented his-negative strain were similar to those of the wild-type, whereas the values of the plasmid-cured strain were similar to those of SAG5.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

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。     

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。     

家族传播的口腔白色念珠菌基因型多态性分析

目的检测家族传播的口腔白色念珠菌基因多态性。方法采集35个家庭（119个样本）的口腔牙菌斑,采用PCR ITS1-ITS2基因分型方法,检测、分析家族传播的口腔白色念珠菌基因多态性。结果 18个家庭（18/35,61%）,34个样本（34/119,28.6%）有白色念珠菌感染,11个家庭存在家族传播（11/18,61%）。在5个母子（父子）垂直传播的家庭成员中,白色念珠菌基因型均不一致。在3个呈水平传播的家庭成员中,两家基因型一致,1家不一致。在3个垂直-水平传播的家庭成员中,两家基因型一致,1家不一致。白色念珠菌家族传播基因型差异有显著统计学意义（χ2=26.571,P〈0.01）。白色念珠菌感染与年龄、性别、学历、吸烟、饮酒、义齿和龋病均无显著相关。结论白色念珠菌在口腔定植,受宿主自身遗传背景影响较大,在家族垂直传播中呈明显的基因多态性。呈水平传播的白色念珠菌菌种具有较高的传染性,基因型可保持不变。     

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.     

Azole sensitivity in Candida albicans

Abstract In the present study, we assessed the influence of three culture media on the susceptibility 'in vitro' of twenty four clinical strains belonging to Candida albicans against three imidazole-derivatives and also, we investigated the situation of azole sensitivity in three of these strains.     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Avirulence of Candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis

Abstract The virulence of Candida albicans strain SC5413 and two isogenic derivatives have been investigated in a rat model of oropharyngeal candidiasis. The results demonstrate that both mutant strains are avirulent in this animal model while the parental strain readily initiates infection. Avirulence is not related to altered growth characteristics or the inability of the strains to undergo yeast-to-hyphal morphogenesis. The potential importance of nutritional sufficiency as a virulence factor as well as the possibility of utilizing such strains in the development of an in vitro expression technology system for Candida albicans is discussed.     

Role of Candida albicans mating in genetic variability and adaptation to the host

Since its discovery at the end of the XIX century, Candida albicans has emerged as one of the most important human pathogenic fungi. This yeast efficiently colonizes the gastrointestinal cavity of humans, which is an important source for gastrointestinal-mediated dissemination of the fungus to internal organs under immune suppression. Controlling colonization may therefore lead to the eradication of C. albicans which may, in turn, be a useful strategy in the prevention of candidiasis. Recent studies indicate that colonization is influenced by -and related to-the white opaque (wo) transition, an epigenetic transition that has been shown to mediate several aspects of the biology of this fungus. Efficient mating in C. albicans occurs by a two-step process which involves the conversion to a homozygous mating type cell followed by a transition to the opaque state. The discovery of the opaque cell as the mating competent phase of this fungus provided an interesting evolutionary example of the role of mating in the adaptation to a mammalian host in a pathogenic fungus. A full sexual cycle has not been observed; rather, after mating, return to a diploid state is achieved by concerted chromosome loss, being this an important source of genetic variability for this opportunistic pathogen.     

白念珠菌V-ATPase研究进展

白念珠菌是临床最常见的条件致病真菌,V-ATPase是真核生物中高度保守的质子泵转运复合物,可以维持液泡和细胞质pH稳态.近年来,V-ATPase作为一个抗白念珠菌感染的潜在靶点为人们所关注,本文就V-ATPase抑制剂、相关的其他靶点及抑制剂筛选等方面的研究进展作一综述.     

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.     

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 .     

Cleavage of human big endothelin-1 by Candida albicans aspartic proteinase

Abstract A Candida albicans aspartic proteinase (CAP), one of the secretory proteinases of Candida albicans , is thought to be a possible virulence factor in Candida albicans infection. Whereas endothelin-1 is found as an endothelium-derived strong vasoconstrictive peptide, it is known to have a role in the maintenance of vascular homeostasis and tissue survival. Endothelin-1 is generated from a precursor form of endothelin-1, the so-called big endothelin-1. It has recently been reported that cathepsin D, E and pepsin, which are aspartic proteinases, convert big endothelin-1 to endothelin-1. In this study, the relationship between CAP and big endothelin-1 was studied. High performance liquid chromatography analysis revealed that big endothelin-1 was cleaved into several amino acid sites by CAP, but endothelin-1 was not converted from big endothelin-1. CAP cleaved big endothelin-1 at different sites when compared with that of other known aspartic proteinases, and it suppressed endothelin-1 production through the degradation of big endothelin-1. CAP may break homeostatic mechanism of endothelin-1 in Candida albicans infectious lesion.     

Gluconeogenesis in Candida albicans

According to different metabolic situations in various stages of Candida albicans pathogenesis the regulation of carbohydrate metabolism was investigated. We report the genetic characterization of all major C. albicans gluconeogenic and glyoxylate cycle genes (fructose-1,6-bisphosphatase, PEP carboxykinase, malate synthase and isocitrate lyase) which were isolated after functional complementation of the corresponding Saccharomyces cerevisiae deletion mutants. Remarkably, the regulation of the heterologously expressed C. albicans gluconeogenic and glyoxylate cycle genes was similar to that of the homologous S. cerevisiae genes. A C. albicans DeltaCafbp1 deletion strain failed to utilize non-fermentable carbon sources but hyphal growth was not affected. Our results show that regulation of gluconeogenesis in C. albicans is similar to that of S. cerevisiae and that the current knowledge on how gluconeogenesis is regulated will facilitate the physiological understanding of C. albicans.     

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。     

白念珠菌凋亡诱导研究进展

近年来,白念珠菌为主的真菌感染病例日渐增多,新型抗真菌药物研发成为热点。在原有抗真菌机制的基础上,通过诱导凋亡抗真菌成为目前探寻抗真菌药物作用机制的一个新趋向。本文对目前国内外关于药物诱导白念珠菌凋亡研究做一综述。     

紫外线对白念珠菌的影响

紫外线作为重要的环境因子之一,能显著影响包括白念珠菌在内的多种生物的生长及生理过程.研究发现白念珠菌受紫外线照射后菌丝形成被抑制,孢子形成增多且没有向光性;脉冲紫外线辐射可通过多靶点程序使白念珠菌失活;rad 51缺陷株比rad 52缺陷株更易受紫外线损害,同时紫外线可导致白念珠菌杂合性丢失.研究还发现UVC治疗可明显减少烧伤后真菌微生物感染,核黄素/UVA治疗可明显抑制白念珠菌生长.因此紫外线对白念珠菌有一定的抑制作用.