Isolation of genes from Candida albicans by complementation in Saccharomyces cerevisiae

Summary A genomic library of the asexual pathogenic yeast Candida albicans was constructed in the S. cerevisiae vector YEp13. The library contains a representation of the entire genome with a probability of 99%. The expression of the genes of C. albicans in S. cerevisiae was examined and two mutations his3-1 and trp1-289 of S. cerevisiae were complemented by the cloned genes of C. albicans. The hybridization data indicates that the plasmids complementing the mutations of S. cerevisiae contain sequences from C. albicans.     

Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae

Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 micrograms of Hg (as HgCl2) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28 degrees C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows. (i) C. albicans was the more mercury-resistant species, but both yeast species failed to grow in the media containing 0.75 micrograms of Hg per ml. (ii) The amounts of organomercury produced by the two species were proportional to the amount of HgCl2 added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae. (iii) The amounts of elemental Hg produced were inversely proportional to the HgCl2 level added in the case of S. cerevisiae but were all similar in the case of C. albicans. (iv) Neither organomercury nor elemental Hg was produced in any of the control media.     

Transformations of inorganic mercury by Candida albicans and Saccharomyces cerevisiae.

Saccharomyces cerevisiae and Candida albicans were incubated with 0.25, 0.5, or 0.75 micrograms of Hg (as HgCl2) per ml of Nelson's medium in the presence of trace amounts of oxygen at 28 degrees C for 12 days. Two control media were used, one without added Hg and one without yeast inoculum. Yeast cell growth was estimated after 1, 2, 3, and 8 days of incubation. The contents of organomercury in the system and of elemental mercury released from the media and collected in traps were determined at the end of the experiments. The results were as follows. (i) C. albicans was the more mercury-resistant species, but both yeast species failed to grow in the media containing 0.75 micrograms of Hg per ml. (ii) The amounts of organomercury produced by the two species were proportional to the amount of HgCl2 added to the medium. In all cases C. albicans produced considerably larger amounts of methylmercury than S. cerevisiae. (iii) The amounts of elemental Hg produced were inversely proportional to the HgCl2 level added in the case of S. cerevisiae but were all similar in the case of C. albicans. (iv) Neither organomercury nor elemental Hg was produced in any of the control media.     

A functional analysis of the Candida albicans homolog of Saccharomyces cerevisiae VPS4

To investigate the role of the prevacuolar secretion pathway in the trafficking of vacuolar proteins in Candida albicans, the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS4 was cloned and analyzed. Candida albicans VPS4 encodes a deduced AAA-type ATPase that is 75.6% similar to S. cerevisiae Vps4p, and plasmids bearing C. albicans VPS4 complemented the abnormal vacuolar morphology and carboxypeptidase missorting in S. cerevisiae vps4 null mutants. Candida albicans vps4Delta null mutants displayed a characteristic class E vacuolar morphology and multilamellar structures consistent with an aberrant prevacuolar compartment. The C. albicans vps4Delta mutant degraded more extracellular bovine serum albumin than did wild-type strains, which implied that this mutant secreted more extracellular protease activity. These phenotypes were complemented when a wild-type copy of VPS4 was reintroduced into its proper locus. Using a series of protease inhibitors, the origin of this extracellular protease activity was identified as a serine protease, and genetic analyses using a C. albicans vps4Deltaprc1Delta mutant identified this missorted vacuolar protease as carboxypeptidase Y. Unexpectedly, C. albicans Sap2p was not detected in culture supernatants of the vps4Delta mutants. These results indicate that C. albicans VPS4 is required for vacuolar biogenesis and proper sorting of vacuolar proteins.     

Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae

In this study, we investigated methionine synthase from Candida albicans (CaMET 6p) and Saccharomyces cerevisiae (ScMET 6p). We describe the cloning of CaMet 6 and ScMet 6, and the expression of both the enzymes in S. cerevisiae. CaMET 6p is able to complement the disruption of met 6 in S. cerevisiae. Following the purification of ScMET 6p and CaMET 6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 108, 84, 95, and 13 microM, respectively. The Michaelis constants for CaMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 113, 129, 120, and 14 microM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) as a substrate. We conclude that ScMET 6p and CaMET 6p require a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to the bacterial metE homologs. The cloning, purification, and characterization of these enzymes lay the groundwork for inhibitor-design studies on the cobalamin-independent fungal methionine synthases.     

Functional equivalence of translation factor eIF5B from Candida albicans and Saccharomyces cerevisiae

Eukaryotic translation initiation factor 5B (eIF5B) plays a role in recognition of the AUG codon in conjunction with translation factor eIF2, and promotes joining of the 60S ribosomal subunit. To see whether the eIF5B proteins of other organisms function in Saccharomyces cerevisiae, we cloned the corresponding genes from Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and Candida albican and expressed them under the control of the galactose-inducible GAL promoter in the fun12Delta strain of Saccharomyces cerevisiae. Expression of Candida albicans eIF5B complemented the slow-growth phenotype of the fun12Delta strain, but that of Aspergillus nidulance did not, despite the fact that its protein was expressed better than that of Candida albicans. The Arabidopsis thaliana protein was also not functional in Saccharomyces. These results reveal that the eIF5B in Candida albicans has a close functional relationship with that of Sacharomyces cerevisiae, as also shown by a phylogenetic analysis based on the amino acid sequences of the eIF5Bs.     

Plasma membrane structures of Saccharomyces cerevisiae and Candida albicans revealed by freeze-fracturing before and after treatment with filipin

Abstract Plasma membrane structures of Saccharomyces cerevisiae and Candida albicans during growth were studied by means of freeze-fracturing before and after filipin treatment. Undifferentiated regions of the plasma membrane were severely deformed by filipin, indicating the existence of a high level of ergosterol. The plasma membrane of small buds was mildly deformed by filipin, which suggested the existence of a low level of ergosterol. The bottom part of invaginations and the plasma membrane of the neck between the mother cell and the bud usually lacked filipin-induced deformations. Constraints existed in these regions which might restrict the ability of filipin to deform the membrane.     

Uptake of 14C-chlorhexidine gluconate by Saccharomyces cerevisiae, Candida albicans and Candida glabrata

Uptake of radiolabelled chlorhexidine gluconate (¹⁴C-CHG) to Saccharomyces cerevisiae, Candida albicans and C. glabrata was very rapid and near maximal within 30 s. The organism, S. cerevisiae , most sensitive to the lethal action of chlorhexidine, took up significantly more biocide than the other organisms. Cells from cultures of different ages took up different amounts of ¹⁴C-CHG.     

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).     

Isolation of Helicobacter pylori from the intestinal mucosa of patients with Crohn's disease

BACKGROUND: Helicobacter species are associated with inflammatory bowel disease in rodents and in nonhuman primates. Therefore, we prospectively investigated the presence of Helicobacter species in the intestinal mucosa of patients with and without Crohn's disease by culture and polymerase chain reaction (PCR) assays. MATERIALS AND METHODS: Mucosal fragments were obtained from the ileum, different colon regions, and rectum of 43 patients with Crohn's disease and of 74 patients without inflammatory bowel disease. RESULTS: Helicobacter pylori strains, identified by 16S rRNA gene sequencing, were more frequently isolated and PCR-detected in the intestinal mucosa of patients with ulcerative colitis-like Crohn's disease than in intestinal mucosa of the control group. Otherwise, anti-H. pylori immunoglobulin G levels were significantly lower in fibrostenosing and fistulating Crohn's disease subgroups. No other Helicobacter species were found in the intestinal mucosa of the patients. CONCLUSIONS: Although our results suggest an association between the presence of H. pylori in the intestine and ulcerative colitis-like phenotype of Crohn's disease, H. pylori infection in the actual causality of Crohn's disease is still to be determined.     

Evidence for the occurrence of calmodulin in the yeasts Candida albicans and Saccharomyces cerevisiae

The lectin extracted from Vicia graminea seeds has been purified by conventional techniques but such procedures did not give a satisfactory yield. We describe a new purification which involves 3 steps after obtention of the crude extract. The first step is based on affinity chromatography on con A—Sepharose. Further purification steps were performed on DEAE-Sephacel chromatography and ultrogel AcA₄₄ gel filtration. The homogeneity of the lectin was demonstrated by polyacrylamide gel electrophoresis. Purification of the lectin by this new method was less time consuming, the yield was higher and the specific activity increased.     

幽门螺杆菌感染对冠心病患者血清生化水平及颈动脉硬化的影响

目的 探讨幽门螺杆菌感染对冠心病患者血清生化水平及颈动脉硬化的影响。方法 选取150例冠心病患者,所有患者均接受14C呼气试验检查,将呼气试验阳性患者分为观察组,阴性患者分为对照组。两组患者均检测甘油三酯（TG）、胆固醇（TC）、低密度脂蛋白（LDL-C）、血清同型半胱氨酸（HCY）和血清超敏C反应蛋白（hs-CRP）水平,并接受彩色多普勒超声检查颈动脉。观察并比较两组患者血清生化水平及颈动脉情况。结果 观察组患者血清TC、LDL-C、HCY和hs-CRP水平显著高于对照组（P0.05）。观察组患者内膜厚度显著大于对照组（P0.05）。结论 冠心病患者感染幽门螺杆菌会影响脂蛋白代谢,激活炎症反应,增加冠心病患者动脉硬化发生的风险,应早期积极干预治疗。     

Physicochemical modification of the excretion product of Saccharomyces cerevisiae killer strains results in fungicidal activity against Candida albicans and Tricophyton mentagrophytes

It is known that certain yeast strains, so called 'killers', can produce and excrete proteinaceous toxins that can induce death of other sensitive strains. We obtained a stable fungicidal factor (SKF) through concentration and stabilization of the excretion product of certain killer strains of Saccharomyces cerevisiae (K1 and K2). The isolated proteinaceous complex exhibited activity at broad ranges of pH (4-7.5) and temperatures (20-37.5 degrees C). It was significantly lethal against Candida albicans and Tricophyton mentagrophytes. SKF showed stability and activity after storage, with a mean half-life of 6 months at 4 degrees C or at -20 degrees C.     

Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae.

Naturally occurring strains of Candida albicans are opportunistic pathogens that lack a sexual cycle and that are usually diploids with eight pairs of chromosomes. C. albicans spontaneously gives rise to a high frequency of colonial morphology mutants with altered electrophoretic karyotypes, involving one or more of their chromosomes. However, the most frequent changes involve chromosome VIII, which contains the genes coding for ribosomal DNA (rDNA) units. We have used restriction fragment lengths to analyze the number and physical array of the rDNA units on chromosome VIII in four normal clinical strains and seven morphological mutants derived spontaneously from one of the clinical isolates. HindIII does not cleave the rDNA repeats and liberates the tandem rDNA cluster from each homolog of chromosome VIII as a single fragment, whereas the cleavage at a single site by NotI reveals the size of the single rDNA unit. All clinical strains and morphological mutants differed greatly in the number of rDNA units per cluster and per cell. The four clinical isolates differed additionally among themselves by the size of the single rDNA unit. For a total of 25 chromosome VIII homologs in a total of 11 strains considered, the variability of chromosome VIII was exclusively due to the length of rDNA clusters (or the number of rDNA units) in approximately 92% of the cases, whereas the others involved other rearrangements of chromosome VIII. Only slight variations in the number of rDNA units were observed among 10 random C. albicans subclones and 10 random Saccharomyces cerevisiae subclones grown for a prolonged time at 22 degrees C. However, when grown faster at optimal temperatures of 37 and 30 degrees C, respectively, both fungi accumulated higher numbers of rDNA units, suggesting that this condition is selected for in rapidly growing cells. The morphological mutants, in comparison with the C. albicans subclones, contained a markedly wider distribution of the number of rDNA units, suggesting that a distinct process may be involved in altering the number of rDNA units in these mutants.     

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI⁺], [URE3], and [RNQ/PIN⁺] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI⁺] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI⁺] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.     

肝癌中幽门螺杆菌CagA+亚型菌株的感染

目的:探讨肝癌与H.pylori感染的相关性.方法:采用金标免疫斑点法和酶免疫检测法(EIA)分别检测33例肝癌患者及169例普通成人Hp-Ab和CagA-Hp-IgG抗体.结果:肝癌组Hp-Ab和CagA -Hp-IgG检出率分别为78.8%和45.5%,而普通成人组分别为55.0%和27.2%,经x2检验,P＜0.05,差异均有显著性.结论:肝癌患者由于整体免疫力低下,易受H.pylori感染.     

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     

Fungal heme oxygenases: Functional expression and characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans

Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiae and Candida albicans express putative heme oxygenases that are required for the acquisition of iron from heme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 and CaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminal membrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncated enzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorption maxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1 are 0.34 +/- 0.09 microM and 1.0 +/- 0.2 microM, respectively. The resonance Raman spectra of Hmx1 reveal a heme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants, including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxin reductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin production by Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation by Hmx1 and CaHmx1 regiospecifically produces biliverdin IXalpha. Spectroscopic analysis of aerobic reactions with H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal heme oxygenases to be heterologously overexpressed and characterized. Their heme degradation activity is consistent with a role in iron acquisition.