白念珠菌新型耐药基因MXR1的敲除与鉴定

目的 构建用于白念珠菌MXR1基因敲除的载体质粒,并通过Ura-Blaster策略敲除MXR1两条等位基因.方法 分别扩增白念珠菌MXR1基因ORF两侧上下游的片段,通过酶切与连接反应,将上下游片段分别插入到p5921质粒的hisG-URA 3-hisG盒两端,从而形成MXR1敲除载体质粒pUC-MXR1-URA3.通过Ura-Blaster策略将载体质粒转染到白念珠菌RM 1000内,并采用PCR和Southern-blot杂交方法鉴定各步转染、复筛所得的阳性克隆.结果 成功获得MXR1基因缺失的菌株.结论 MXR1基因缺失菌株的构建,有助于深入研究白念珠菌耐药机制.     

Candida albicans ABG1 gene is involved in endocytosis

The human fungal pathogen Candida albicans undergoes reversible morphogenetic transitions between yeast, hyphal and pseudohyphal forms. The fungal vacuole actively participates in differentiation processes and plays a key role supporting hyphal growth. The ABG1 gene of C. albicans encodes an essential protein located in the vacuolar membranes of both yeast and hyphae. Using fluorescence microscopy of a green fluorescent protein-tagged version of Abg1p, a fraction of the protein was detected in hyphal tips, not associated with vacuolar membranes. Live cell imaging of emerging germ tubes showed that Abg1p migrated to the polarized growth site and colocalized with endocytic vesicles. Phenotypic analysis of a methionine-regulated conditional mutant confirmed that Abg1p is involved in endocytosis.     

The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) is essential for growth

The Candida albicans ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) was cloned by complementing a Saccharomyces cerevisiae erg26 mutant with a C. albicans genomic library. Sequence analysis showed a 70% identity between the C. albicans and S. cerevisiae ERG26 genes at the amino acid level. Sequential disruption of both copies of the ERG26 gene in the presence of an integrated rescue cassette containing a third copy of the ERG26 gene under the control of the inducible pMAL2 promoter, resulted in cells capable of growing only in the presence of the inducer. The results establish that the ERG26 gene is essential for growth and that inhibitors of the Erg26p may represent a new and highly effective class of antifungal agents.     

Cloning and expression of the 3-isopropylmalate dehydrogenase gene from Candida albicans

Abstract A variety of Saccharomyces cerevisiae genes e.g. HIS3, LEU2, TRP1, URA3 , are expressed in Escherichia coli and have been isolated by complementation of mutations in the corresponding E. coli genes [1]. The LEU2 gene was one of the first S. cerevisiae genes to be isolated in this way [2], and its isolation led to the development of transformation systems for S. cerevisiae [3,4]. The leuB gene in E. coli [5] and the LEU2 gene in S. cerevisiae [6] both code for 3-isopropylmalate dehydrogenase (3-IMDH; EC 1.1.1.85) which is essential for the biosynthesis of leucine in both organisms. This paper describes the cloning of a fragment of C. albicans DNA carrying the gene for 3-IMDH which will be useful in the development of transformation methods in C. albicans .     

Directional ligation of long-flanking homology regions to selection cassettes for efficient targeted gene-disruption in Candida albicans

PCR-product directed gene disruption with a marker cassette having short homology regions is often used in Candida albicans. However, it is quite inefficient due to the high frequency of non-homologous recombination at non-targeted loci, which necessitates extensive screening to identify the correct disruptants. Thus, many PCR-based methods to introduce long flanking homology regions have been developed to increase the frequency of integration at the targeted loci. However, these methods are not that amenable for use with the widely employed C. albicans marker cassettes having direct repeats, as these repeats tend to recombine during PCR, resulting in shorter amplified products without the selection marker. To circumvent this limitation, we have developed a dinucleotide-sticky-end-ligation strategy to add one flanking homology region to one side of the selection cassette, and the other flanking homology region to the other side of the selection cassette. This method involves release of the selection cassette from the plasmid by digestion with two different restriction enzymes, followed by partial fill-in, to provide a unique two base overhang at each end of the cassette. The flanking homology regions, corresponding to the gene to be disrupted, are individually PCR-amplified, and treated with T4-DNA Polymerase in the presence of appropriate dNTPs to yield two base-5' overhangs. The primers used for the PCR have additional bases at the 5' ends such that after T4 DNA Polymerase treatment, the two flanks will have distinct overhangs compatible with the overhangs of the partially filled-in selection cassette. The selection cassette and the flanks are then ligated together and directly used to transform C. albicans. We have successfully used this method for disruption of several C. albicans genes. We have also used this method to recreate insertion mutations obtained with transposons to reconfirm the mutant phenotypes. This approach can be extended to other organisms like Schizosaccharomyces pombe which also require long flanking regions of homology for targeted gene disruption.     

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.     

RTA2, a novel gene involved in azole resistance in Candida albicans

Widespread and repeated use of azoles, particularly fluconazole, has led to the rapid development of azole resistance in Candida albicans. Overexpression of CDR1, CDR2, and CaMDR1 has been reported contributing to azole resistance in C. albicans. In this study, hyper-resistant C. albicans mutant, with the above three genes deleted, was obtained by exposure to fluconazole and fluphenezine for 28 passages. Thirty-five differentially expressed genes were identified in the hyper-resistant mutant by microarray analysis; among the 13 up-regulated genes, we successfully constructed the rta2 and ipf14030 null mutants in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1. Using spot dilution assay, we demonstrated that the disruption of RTA2 increased the susceptibility of C. albicans to azoles while the disruption of IPF14030 did not influence the sensitivity of C. albicans to azoles. Meanwhile, we found that ectopic overexpression of RTA2 in C. albicans strain with deletions of CDR1, CDR2 and CaMDR1 conferred resistance to azoles. RTA2 expression was found elevated in clinical azole-resistant isolates of C. albicans. In conclusion, our findings suggest that RTA2 is involved in the development of azole resistance in C. albicans.     

Phosphatidylinositol 3-kinase VPS34 of Candida albicans is involved in filamentous growth, secretion of aspartic proteases, and intracellular detoxification

The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans influences vesicular intracellular transport, filamentous growth and virulence. To get a clearer understanding how these phenomena are connected, we analysed hyphal growth in a matrix under microaerophilic conditions at low temperature, the detoxification of metal ions and antifungal drugs, the secretion of aspartic proteinases (Saps), as well as expression of adhesion-associated proteins of the C. albicans vps34 null mutant strain. The hyphal growth in a matrix, which is repressed in the wild-type strain by Efg1p, was derepressed in the mutant. CZF1, which encodes an activator of hyphal growth in a matrix, was up-regulated in the mutant. In addition, CZF1 expression was pH-dependent in the wild-type. Expression of EFG1 was not changed. Examination of Saps secretion showed a reduction in the vps34 null mutant. Determination of sensitivity against metal ions and antimycotic drugs revealed defects in detoxification. Expression studies indicated that the vps34 mutant reacts to the phenotypical defects with an up-regulation of genes involved in these processes, including the aspartyl proteinases SAP2 and SAP9, adhesion proteins ALS1 and HWP1, and the ABC transporters CDR1 and HST6. We also found an increased expression of the PI 4-kinase LSB6 indicating a complex feed-back mechanism for the compensation of the multiple defects arising from the lack of the PI3-kinase VPS34.     

MYO2 is not essential for viability,but is required for polarized growth and dimorphic switches in Candida albicans

The human fungal pathogen Candida albicans changes from a budding yeast form to a polarized hyphal form in response to various external conditions. Dimorphic switching of C. albicans has been implicated in the development of pathogenicity. Morphogenic transformation requires polarized cell growth and rearrangement of the cytoskeleton. We previously showed that myosins play key roles in the conversion from the bud to the hyphal form of C. albicans by inhibiting myosin activities with 2,3-butanedione-2-monoxime (BDM), a general myosin ATPase inhibitor. In this study we investigated the function of MYO2 in C. albicans using deletion mutants. The amino acid sequence of CaMYO2 shows 60% identity and 77% homology with MYO2 and 54% identity and 70% homology with MYO4 of budding yeast Saccharomyces cerevisiae, suggesting that CaMYO2 is the only class V myosin in C. albicans. Cells in which both CaMYO2 alleles were deleted were viable, suggesting that MYO2 is nonessential in C. albicans. The proliferation of CaMYO2delta cells, however, was sharply decreased. In addition, CaMYO2delta cells showed defects in assembly and polarized localization of F-actin as well as an inability to induce germ tube formation and hyphal growth. The deletion of CaMYO2 also disrupted the shape and migration of the nucleus. These results strongly suggest that CaMYO2 is essential for polarized growth and hyphal transition in C. albicans.     

Purification of a biologically active recombinant glyceraldehyde 3-phosphate dehydrogenase from Candida albicans

We report here the purification of a functionally active recombinant glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Candida albicans. The GAPDH protein encoded by the TDH1 gene was obtained as a glutathione S-transferase fusion protein by expression in the vector pGEX-4T-3, and purified by affinity chromatography and thrombin digestion. The purified protein displays GAPDH enzymatic activity (42 micromol NADH min(-1) mg(-1)) as well as the laminin and fibronectin binding activities previously described. In addition, the recombinant GAPDH is covalently modified by NAD linkage; this modification is stimulated by nitric oxide and probably involves a sulfhydryl group (cysteine) residue since it is inhibited by Hg(2+) and cysteine.     

CaIPF7817 is involved in the regulation of redox homeostasis in Candida albicans

CaIPF7817, a functionally unknown gene in Candida albicans, was suggested to be involved in the redox system previously, but its exact role is unknown. In this study, ipf7817 null mutant was generated with the URA-blaster method. After the deletion of CaIPF7817, intracellular levels of reactive oxygen species were significantly increased; mitochondrial membrane potential, a direct indicator of mitochondrial function, was elevated; some important redox-related genes, including GLR1, SOD2, and TRR1, were up-regulated; and the GSH/GSSG ratio was raised. These changes indicated that CaIPF7817 played important roles in the regulation of redox homeostasis in C. albicans.     

Catalase gene disruptant of the human pathogenic yeast Candida albicans is defective in hyphal growth, and a catalase-specific inhibitor can suppress hyphal growth of wild-type cells

Although the catalase gene (CAT1) disruptant of the human pathogenic yeast Candida albicans was viable under ordinary growth conditions, we previously found that it could not grow on YPD (yeast extract/peptone/dextrose) containing SDS or at higher growth temperatures. To investigate the pleiotrophic nature of the disruptant, we examined the effect of the catalase inhibitor 3-AT on the growth of wild-type strains. Surprisingly, the addition of 3-AT and SDS caused the wild-type cells to be non-viable on YPD plates. We found an additional phenotype of the catalase gene disruptant: it did not produce normal hyphae on Spider medium. Hyphal growth was observed in a CAP1 (Candida AP-1-like protein gene) disruptant, a HOG1 (high-osmolarity glycerol signaling pathway gene) disruptant, and the double CAP1/HOG1 disruptant, suggesting that the defect in hyphal formation by the catalase disruptant was independent of these genes. Addition of 3-AT and SDS to hyphae-inducing media suppressed growth of normal hyphae in the wild-type strain. The potential necessity for catalase action upon exposure to hyphae-inducing conditions was confirmed by the immediate elevation of the catalase gene message. In spite of the requirement for catalase during hyphal growth, the catalase gene disruptant was capable of forming germ tubes in medium containing serum.     

Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans

A site-directed mutagenesis of the GFA1 gene encoding Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase afforded its GFA1S208A version. A product of the modified gene, lacking the putative phosphorylation site for protein kinase A (PKA), exhibited all the basic properties identical to those of the wild-type enzyme but was no longer a substrate for PKA. Comparison of the C. albicans Deltagfa1/GFA1 and Deltagfa1/GFA1S208A cells, grown under conditions stimulating yeast-to-mycelia transformation, revealed that the latter demonstrated lower GlcN-6-P synthase specific activity, decreased chitin content and formed much fewer mycelial forms. All these findings, as well as the observed effects of specific inhibitors of protein kinases, suggest that a loss of the possibility of GlcN-6-P synthase phosphorylation by PKA strongly reduces but not completely eliminates the germinative response of C. albicans cells.     

The Cek1 MAPK is a short-lived protein regulated by quorum sensing in the fungal pathogen Candida albicans

Mitogen activated protein kinase (MAPK) cascades are signal transduction mechanisms present in eukaryotic cells that allow adaptation to environmental changes. MAPK activity is mainly regulated by dual phosphorylation in a TXY motif present in the kinase subdomain VIII as well as dephosphorylation by specific phosphatases. The Cek1 MAPK is involved in filamentous growth in Candida albicans and is an important determinant of virulence in this microorganism; its activation is controlled by the Sho1 adaptor protein. Here we show that Cek1 phosphorylation is regulated by quorum sensing (QS). Cek1 phosphorylation is prevented by farnesol, a compound that also regulates the dimorphic transition in this fungus. Farnesol also induced the activation of Mkc1, the MAPK of the cell integrity pathway. The role of farnesol in Cek1 phosphorylation is independent of the Chk1 histidine kinase, a putative QS sensor, as revealed by genetic analysis. In addition, Cek1, not Hog1, is degraded by proteasome, as revealed by the use of a conditional lethal protein degradation mutant. Our data therefore describe two different mechanisms (QS and protein degradation) that control a MAPK pathway that regulates virulence in a fungal pathogen.     

白念珠菌ALS3、SSA1基因表达在念珠菌性阴道炎免疫机制中的作用

目的探讨白念珠菌ALS3、SSA1基因缺失对阴道上皮细胞激发免疫反应的作用。方法培养白念珠菌野生株及ALS3、SSA1基因敲除株(SC5314、Δals3、Δssa1),对其进行形态测定。按不同MOI感染人阴道上皮细胞系VK2/E6E7细胞,通过台盼蓝染色观察和乳酸脱氢酶(LDH)活性检测,评价不同MOI白念珠菌对上皮细胞的损伤作用;使用酶联免疫吸附试验(ELISA)评估感染过程中炎性细胞因子及趋化因子在共培养上清中的差异。结果 ALS3基因的缺失对白念珠菌芽管长度影响差异无统计学意义,而SSA1基因的缺失与其他两个菌株相比芽管长度减少约30%～40%(P<0.001)。台盼蓝染色观察及LDH测定发现,3株菌在感染上皮细胞时,其细胞损伤能力均与菌载量成正比;与野生型相比,Δssa1突变体在相同比率感染上皮细胞时,细胞损伤能力明显降低,且差异有统计学意义(P<0.05),Δals3突变株影响较小,甚至略微升高。检测炎性细胞因子及趋化因子发现,突变株在诱导上皮细胞产生促炎因子及趋化因子(GM-CSF、G-CSF、IL-1α、IL-8)的能力上明显减弱,差异均有统计学意义(P<0.05)。结论 ALS3和SSA1基因表达在阴道上皮细胞抗白念珠菌感染的局部免疫应答过程中可能起到重要作用,且SSA1基因表达意义更大。     

ABG1, a novel and essential Candida albicans gene encoding a vacuolar protein involved in cytokinesis and hyphal branching

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching.