Interplay between Humoral and CLA+ T Cell Response against Candida albicans in Psoriasis

Candida albicans (CA) infections have been associated with psoriasis onset or disease flares. However, the integrated immune response against this fungus is still poorly characterized in psoriasis. We studied specific immunoglobulins in plasma and the CA response in cocultures of circulating memory CD45RA⁻ cutaneous lymphocyte antigen (CLA)^+/− T cell with autologous epidermal cells from plaque and guttate psoriasis patients (cohort 1, n = 52), and also healthy individuals (n = 17). A complete proteomic profile was also evaluated in plaque psoriasis patients (cohort 2, n = 114) regarding their anti-CA IgA levels. Increased anti-CA IgA and IgG levels are present in the plasma from plaque but not guttate psoriasis compared to healthy controls. CA cellular response is confined to CLA⁺ T cells and is primarily Th17. The levels of anti-CA IgA are directly associated with CLA⁺ Th17 response in plaque psoriasis. Proteomic analysis revealed distinct profiles in psoriasis patients with high anti-CA IgA. C-C motif chemokine ligand 18, chitinase-3-like protein 1 and azurocidin were significantly elevated in the plasma from plaque psoriasis patients with high anti-CA levels and severe disease. Our results indicate a mechanism by which Candida albicans exposure can trigger a clinically relevant IL-17 response in psoriasis. Assessing anti-CA IgA levels may be useful in order to evaluate chronic psoriasis patients.     

Inhibition of yeast‐to‐filamentous growth transitions in Candida albicans by a small molecule inducer of mammalian apoptosis

The opportunistic fungal pathogen of humans Candida albicans is able to grow in different morphological forms such as round or oval yeasts and filamentous hyphae and pseudohyphae. Morphogenesis, the ability to switch between the yeast and filamentous growth forms, is important for adapting to new microenvironments in the human host and for pathogenesis. The molecular pathways governing morphogenesis are complex and incompletely understood. Previously, we identified several small organic molecules that specifically inhibit the initiation of hyphal growth in C. albicans without affecting cell viability or budded growth. One molecule from that screen is known to induce apoptosis in mammalian cells. In this study, we have screened additional inducers of mammalian apoptosis and identified BH3I‐1, as well as several structural derivatives of BH3I‐1, that act as specific inhibitors of morphogenesis under a variety of environmental conditions. Chemical epistasis experiments suggest that BH3I‐1 acts downstream of the hypha‐specific gene regulators Rfg1, Nrg1 and Ume6.     

Candida albicans homologue of GGP1/GAS1 gene is functional in Saccharomyces cerevisiae and contains the determinants for glycosylphosphatidylinositol attachment

The GGP1/GAS1/CWH52 gene of Saccharomyces cerevisiae encodes a major exocellular 115 kDa glycoprotein (gp115) anchored to the plasma membrane through a glycosylphosphatidylinositol (GPI). The function of gp115 is still unknown but the analysis of null mutants suggests a possible role in the control of morphogenesis. PHR1 gene isolated from Candida alibicans is homologous to the GGP1 gene. In this report we have analysed the ability of PHR1 to complement a ggp1Δ mutation in S. cerevisiae. The expression of PHR1 controlled by its natural promoter or by the GGP1 promoter has been studied. In both cases we have observed a complete complementation of the mutant phenotype. Moreover, immunological analysis has revealed that PHR1 in budding yeast gives rise to a 75–80 kDa protein anchored to the membrane through a GPI, indicating that the signal for GPI attachment present in the C. albicans gene product is functional in S. cerevisiae.     

Cloning of Candida albicans SEC14 gene homologue coding for a putative essential function

The yeast SEC14 gene product is required for the transport of proteins from the Golgi complex. We have cloned the homologous Candida albicans SEC14 gene (CaSEC14) by functional complementation of a Saccharomyces cerevisiae thermosensitive mutant, sec14. Some putative TATA boxes have been identified in CaSEC14 and, contrary to S. cerevisiae SEC14, no introns were found in the Candida homologue. Sequence analysis revealed that CaSec14p is a 301 amino acid protein, 67% identical to S. cerevisiae and Kluyveromyces lactis Sec14p, and 61% identical to the 300 amino-terminal residues of Yarrowia lipolytica Sec14p. Hydrophatic profile analysis of CaSec14p suggests a soluble protein without transmembrane domains, as has been described for the S. cerevisiae counterpart. While it was easy to disrupt one allele of SEC14 in C. albicans, repeated attempts to disrupt the second allele were unsuccessful, thus suggesting that the gene could be essential for vegetative growth in C. albicans. The sequence has been deposited in the EMBL data library under Accession Number X81937.     

Sterol glycosides and cerebrosides accumulate in Pichia pastoris, Rhynchosporium secalis and other fungi under normal conditions or under heat shock and ethanol stress

The occurrence of glycolipids such as sterol glycosides, acylated sterol glycosides, cerebrosides and glycosyldiacylglycerols was examined in the three yeast species Candida albicans, Pichia pastoris and Pichia anomala, as well as in the six fungal species Sordaria macrospora, Pyrenophora teres, Ustilago maydis, Acremonium chrysogenum, Penicillium olsonii and Rhynchosporium secalis. Cerebroside was found in all organisms tested, whereas acylated sterol glycosides and glycosyldiacylglycerols were not found in any organism. Sterol glycosides were detected in P. pastoris strain GS115, U. maydis, S. macrospora and R. secalis. This glycolipid occurred in both yeast and filamentous forms of U. maydis but in neither form of C. albicans. This suggests that sterol glycoside is not correlated with the separately grown dimorphic forms of these organisms. Cerebrosides and sterol glycosides from P. pastoris and R. secalis were purified and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. The cerebrosides are beta-glucosyl ceramides consisting of a saturated alpha-hydroxy or non-hydroxy fatty acid and a Delta4,8-diunsaturated, C9-methyl-branched sphingobase. Sterol glycoside from P. pastoris was identified as ergosterol-beta-D-glucopyranoside, whereas the sterol glucosides from R. secalis contain two derivatives of ergosterol. The biosynthesis of sterol glucoside in P. pastoris CBS7435 and GS115 depended on the culture conditions. The amount of sterol glucoside in cells grown in complete medium was much lower than in cells from minimal medium and a strong increase in the content of sterol glucoside was observed when cells were subjected to stress conditions such as heat shock or increased ethanol concentrations. From these data we suggest that, in addition to Saccharomyces cerevisiae, new yeast and fungal model organisms should be used to study the physiological functions of glycolipids in eukaryotic cells. This suggestion is based on the ubiquitous and frequent occurrence of cerebrosides and sterol glycosides, both of which are rarely detected in S. cerevisiae. We suggest P. pastoris and two plant pathogenic fungi to be selected for this approach.     

真菌群体感应分子法尼醇及其作用机制

群体感应(quorum sensing,QS)是取决于细胞密度的微生物通信机制,可调节细菌毒力因子分泌、生物膜形成、感受态和生物发光等行为。真菌群体感应系统10年前在法尼醇控制致病多态性真菌白色念珠菌中的细丝化研究中被发现。研究显示法尼醇作为群体感应分子(QSM)对宿主和其他微生物发挥多种作用;还发现芳香醇酪醇是控制白色念珠菌生长、形态发生和生物膜形成的另一群体感应分子。在酿酒酵母中,发现另两种芳香醇苯乙醇和色氨酸是在氮饥饿条件下调节形态发生的群体感应分子。此外,类似于群体感应的种群密度依赖性行为已在几种其他真菌中描述。本综述总结了目前发现群体感应效应的几种关键真菌物种,并重点阐述了研究较多的白色念珠菌和法尼醇作用的微观机制研究进展。     

香辛料提取物对白色念珠菌的抑制及工艺优化研究

研究24种香辛料的抑菌作用,并比较其醇提取液的抑菌效果。通过大量的实验测定白色念珠菌抑菌圈,其中肉桂提取液对白色念珠菌抑制作用最佳。然后运用响应面(RSM)分析优化肉桂乙醇提取工艺条件。在单因素实验基础上,以白色念珠菌抑菌圈为指标,研究提取温度、液固比、提取时间及乙醇浓度对白色念珠菌抑菌圈的影响。结果表明,肉桂醇提法最佳提取工艺为提取温度69.24℃,液固比10:1(mL/g),提取时间5h,乙醇浓度95%,在此条件下提取液对念珠菌的抑菌圈是39.46mm。     

抗白念珠菌人源性单链抗体库的构建及筛选

目的构建多样性良好的抗白念珠菌人源性单链抗体库,筛选抗白念珠菌特异性的噬菌体抗体。方法从20份人外周血淋巴细胞(包括正常成年人5份、新生儿5份和白念珠菌感染恢复期患者10份)中提取总RNA,反转录为cDNA,PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,以重叠延伸PCR法将VH和VL拼接成scFv基因,克隆入噬菌粒载体pCANTAB-5E中,电转化大肠杆菌XL1-Blue,构建人源抗白念珠菌天然噬菌体抗体库,并从中筛选阳性克隆抗体。结果构建的人源性抗白念珠菌天然噬菌体抗体库库容为2.8×109,多样性良好。共筛选出18个阳性克隆。结论已成功构建了1个多样性良好的抗白念珠菌人源性噬菌体抗体库,为筛选有效治疗白念珠菌和耐药性白念珠菌感染的药物提供了条件。     

Bax-induced cell death in Candida albicans

Bax is a pro-apoptotic member of the Bcl-2 family of proteins involved in the regulation of genetically programmed cell death in mammalian cells. It has been shown that heterologous expression of Bax in several yeast species, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris, also induces cell death. In this study we investigated the effects of Bax expression in the pathogenic yeast Candida albicans. Cell death inducing expression of Bax required a synthetic BAX gene that was codon-optimized for expression in Candida albicans. Expression of this BAX gene resulted in growth inhibition and cell death. By fusing Bax with the yeast enhanced green fluorescent protein of Aequoria victoria, the cell death-inducing effect of Bax was increased due to reduced proteolytic degradation of Bax. Using this fusion protein we showed that, upon expression in C. albicans, Bax co-localizes with the mitochondria. Furthermore, we showed for the first time that expression of Bax in yeast causes the mitochondria, which are normally distributed throughout the cell, to cluster in the perinuclear region.