Functional expression of the Candida albicans beta-tubulin gene in Saccharomyces cerevisiae

Expression of the beta-tubulin-encoding gene (TUB2) of Candida albicans has been examined in Saccharomyces cerevisiae. Overexpression of the TUB2 gene of C. albicans, as well as that of S. cerevisiae, was found to be lethal. Chromosomal integration of the C. albicans TUB2 gene into a strain in which the native TUB2 gene had been deleted led to functional complementation. The results demonstrate that correct splicing of the two introns present in the C. albicans TUB2 gene occurs in the heterologous host strain containing this gene. Such strains are supersensitive to the tubulin-binding agent benomyl, indicating that the natural resistance of C. albicans to benomyl is not related to the structure of its beta-tubulin.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).     

Functional expression of the Candida albicans alpha-factor receptor in Saccharomyces cerevisiae

Candida albicans genes involved in mating have been identified previously by homology to Saccharomyces cerevisiae mating pathway components. The C. albicans genome encodes CaSte2p, a homolog of the S. cerevisiae alpha-mating pheromone receptor Ste2p, and two potential pheromones, alpha-F13 (GFRLTNFGYFEPG) and alpha-F14 (GFRLTNFGYFEPGK). The response of several C. albicans strains to the synthesized peptides was determined. The alpha-F13 was degraded by a C. albicans MTLa strain but not by S. cerevisiae MATa cells. The CaSTE2 gene was cloned and expressed in a ste2-deleted strain of S. cerevisiae. Growth arrest and beta-galactosidase activity induced from a FUS1-lacZ reporter construct increased in a dose-dependent manner upon exposure of transgenic S. cerevisiae to alpha-F13. Mating between the strain expressing CaSTE2 and an opposite mating type was mediated by alpha-F13 and not by the S. cerevisiae alpha-factor. The results indicated that CaSte2p effectively coupled to the S. cerevisiae signal transduction pathway. Functional expression of CaSte2p in S. cerevisiae provides a well-defined system for studying the biochemistry and molecular biology of the C. albicans pheromone and its receptor.     

Degradation of Candida albicans Can1 permease expressed in Saccharomyces cerevisiae

The Candida albicans amino-acid Can1 permease expressed in Saccharomyces cerevisiae is degraded in the vacuole after internalisation by endocytosis. The CaCan1 inactivation and degradation is slow and not inducible by ammonium ions or 'stress' conditions. Using Saccharomyces cerevisiae mutants defective in ubiquitin-protein ligase and ubiquitin-protein hydrolase we have shown that the degradation of heterologous CaCan1 permease is ubiquitin dependent.     

白色念珠菌CaBEM1基因的克隆及其在酿酒酵母丝状生长中的作用

白色念珠菌在不同的生长条件下能发生显著的形态变化 ,这种变化由多种调控因子与信号转导途径所调控。酿酒酵母的G1期细胞周期蛋白Cln1和Cln2参与其形态发生 ,cln1/cln1、cln2 /cln2双缺失株不能形成菌丝。把白色念珠菌基因组文库导入cln1/cln1、cln2 /cln2缺失株 ,筛选能校正菌丝形成缺陷的基因 ,分离得到白色念珠菌中的CaBEM 1基因。从核苷酸序列推导 ,CaBEM1编码一种 6 32个氨基酸的蛋白质 ,氨基酸序列分析表明在其N端有 2个SH3结构域 ,中部有 1个PX结构域 ,C端有 1个PB1结构域 ;CaBem1的氨基酸序列与酿酒酵母的Bem1同源性达 38% ,与裂殖酵母的Scd2同源性达 32 %。在酿酒酵母的缺失株中异源表达CaBEM1,能够部分校正它们在氮源缺乏条件下的菌丝形成缺陷。这种菌丝形成的校正作用绕过MAPK途径和cAMP/PKA途径 ,表明CaBem1在菌丝形成中的作用可能位于这两条信号转导途径的下游     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Organization of the human interleukin-1 receptor antagonist gene IL1HY1

 The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7600 nucleotides and found four coding exons ranging in size from 55 to 2288 nucleotides. The 5′ untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene. Received: 15 October 1999 / Revised: 30 December 1999     

Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.     

A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis

Morphogenesis in Saccharomyces cerevisiae and the pathogenic yeast Candida albicans is governed in part by the same molecular circuits. In S. cerevisiae, FLO11/MUC1 expression has been shown to be modulated by multiple signalling pathways required for pseudohyphal development. We have established a screen in S. cerevisiae to identify regulators of fungal development in C. albicans based on FLO11::lacZ expression as a reporter. This screen identified both known components of the mitogen-activated protein kinase (MAPK) cascade and the cAMP cascade that are important for hyphal development in C. albicans, as well as genes not yet known to be involved in morphogenesis. The Candida homologue of MCM1 is one of the novel factors identified in this screen as being important for morphogenesis. CaMcm1p levels do not vary significantly in different cell types and respond to an autoregulatory feedback mechanism, arguing that CaMcm1p activity is regulated by post-translational modifications. Both overexpression and repression of this essential gene led to the induction of hyphae. Moreover, we found that the expression of HWP1, a hyphae-specific gene, was induced by repression of CaMCM1. The changes in morphology and HWP1 expression were not the result of a change in expression levels of NRG1 or TUP1, known repressors of hyphal development. Thus, CaMcm1p is a component of a hitherto unknown regulatory mechanism of hyphal growth.     

Identification and characterization of a functional Candida albicans homolog of the Saccharomyces cerevisiae TCO89 gene

As one of the components of target of rapamycin complex 1 (TORC1), ScTco89p is involved in rapamycin sensitivity and cellular integrity in Saccharomyces cerevisiae. Here we provide evidence showing that deletion of ScTCO89 causes yeast cells to be hypersensitive to salt stress in a high osmolarity glycerol pathway-independent fashion. In addition, we have identified and characterized a functional Candida albicans homolog (CaTCO89) of ScTCO89, which encodes a protein of 708 amino acids that shows overall 15% identity with ScTco89p at the amino acid level. However, CaTCO89 could complement the functions of ScTCO89 in rapamycin sensitivity, salt tolerance, and cellular integrity. Candida albicans cells disrupted for CaTCO89 are also sensitive to rapamycin and lithium salt, but not susceptible to challenges to cellular integrity.     

CaSRB9基因的克隆及其在酿酒酵母形态发生中的作用

白色念珠菌是一种重要的人体致病真菌 ,致病机制与其形态发生紧密相关。酿酒酵母Flo8因子在其形态发生中起重要作用 ,我们把白色念珠菌基因组DNA导入酿酒酵母flo8基因缺失株中 ,筛选能够互补 flo8侵入生长缺陷的基因 ,分离到了一个与酿酒酵母SRB9同源的新基因 ,命名为CaSRB9。该基因全长 4998bp ,编码一种16 6 5个氨基酸的蛋白质。在双倍体酿酒酵母中CaSRB9可以部分互补MAPK途径基因缺失株以及 flo8缺失株的菌丝生长缺陷 ;在单倍体酿酒酵母中表达能够互补 flo8缺失株的侵入生长缺陷 ,但在MAPK途径基因缺失株中不能形成侵入生长     

白念珠菌高铁还原酶FRP1基因的功能

白念珠菌((Candida albicans)获得铁的能力影响细胞的生长和毒力,高铁还原酶是白念珠菌高亲和铁吸收系统的重要组成部分.[目的]构建高铁还原酶FRP1(Ferric reductase protein)基因缺失突变株,对FRP1基因功能进行初步研究.[方法]使用Northem杂交的方法分析FRP1基因在缺铁和富铁条件下的表达.利用PCR介导的基因敲除技术构建frp1缺失突变株,并且对野生型和缺失突变株在细胞高铁还原酶活性以及缺铁条件下的生长情况进行比较分析.[结果]缺铁条件可以诱导FRP1基因的表达.frp1缺失突变株不能在铁缺陷的固体培养基上生长.[结论]FRP1蛋白可能是白念珠菌在缺铁条件下起主要作用的高铁还原酶.     

氟康唑体外诱导白假丝酵母菌耐药基因表达的研究

目的 对白假丝酵母菌耐药机制进行研究.方法 将临床分离对氟康唑敏感的白假丝酵母菌种,经体外诱导产生耐药.半定量PCR检测敏感株、耐药株、回复敏感株多药耐药基因CDR1、CDR2、MDR1和转录调控因子TAC1编码基因表达水平的变化,并对TAC1编码基因进行测序.结果 与敏感株和回复敏感株比较,耐药株CDR1、CDR2相对表达量增高,发现1株TAC1 N977D氨基酸置换.结论 氟康唑体外诱导白假丝酵母菌产生耐药的机制与CDR1、CDR2表达相关.TAC1基因突变在诱导耐药中的机制有待进一步研究.     

The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.

Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.     

Candida albicans INT1-induced filamentation in Saccharomyces cerevisiae depends on Sla2p

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans.