Homology, disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.     

The Candida albicans gene HGT12 (orf19.7094) encodes a hexose transporter

Yeast cells of the human pathogen Candida albicans that enter the bloodstream can be engulfed by macrophage cells but survive in, and can escape from, the phagolysosome. The C. albicans gene HGT12, which is specifically expressed during macrophage infection, encodes a protein that transports fructose, glucose and mannose. Expression of this hexose transporter along with the shift from glycolysis to gluconeogenesis that occurs in these phagocytosed cells suggests a requirement for glucose that can be supplied in part by uptake from the lumen of the phagolysosome.     

The putative peroxisomal gene Pxt1 is exclusively expressed in the testis

Genes reported to be crucial for spermatogenesis are often exclusively expressed in the testis. We have identified a novel male germ cell-specific expressed gene named peroxisomal testis specific 1 (Pxt1) with expression starting at the spermatocyte stage during mouse spermatogenesis. The putative amino acid sequence encoded by the cDNA of the Pxt1 gene contains a conserved Asn-His-Leu (NHL)-motif at its C-terminal end, which is characteristic for peroxisomal proteins. Pxt1-EGFP fusion protein is co-localized with known peroxisomal marker proteins in transfected NIH3T3 cells. In addition, we could demonstrate that the peroxisomal targeting signal NHL is functional and responsible for the correct subcellular localization of the Pxt1-EGFP fusion protein. In male germ cells peroxisomes were reported only in spermatogonia. The Pxt1 gene is so far the first gene coding for a putative peroxisomal protein which is expressed in later steps of spermatogenesis, namely in pachytene spermatocytes.     

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.     

Homology,disruption and phenotypic analysis of CaGS Candida albicans gene induced during macrophage infection

During macrophage infection Candida albicans expresses differentially several genes whose functions are associated with its survival strategy. Among others, we have isolated CaGS gene, which is homologous to SNF3, a glucose sensor of Saccharomyces cerevisiae. To elucidate its potential role during infection, CaGS has been disrupted and the resulting phenotype analyzed on different solid media. The null mutant lost the ability to form hyphae on a medium with low glucose concentration and serum. Furthermore, this mutant does not disrupt macrophage in in vitro infections. We believe that this putative glucose sensor is involved in hyphal development during macrophage infection.     

Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid.

An IMP dehydrogenase gene was isolated from Candida albicans on a approximately 2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomyces cerevisiae and various other organisms. Like the S. cerevisiae IMH3 sequence characterized in the genome sequencing project, the open reading frame of the C. albicans IMH3 gene is interrupted by a small intron (248 bp) with typical exon-intron boundaries and a consensus S. cerevisiae branchpoint sequence. IMP dehydrogenase mRNAs are detected in both the yeast and hyphal forms of C. albicans as judged by Northern hybridization. Growth of wild-type (sensitive) C. albicans cells is inhibited at 1 microg of mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, per ml, whereas transformants hosting a plasmid with the IMH3 gene are resistant to MPA levels of up to at least 40 microg/ml. The resistance of cells to MPA is gene dosage dependent and suggests that IMH3 can be used as a dominant selection marker in C. albicans.     

Host-induced, stage-specific virulence gene activation in Candida albicans during infection

An understanding of the complex interactions between pathogenic microbes and their host must include the identification of gene expression patterns during infection. To detect the activation of virulence genes in the opportunistic fungal pathogen Candida albicans in vivo by host signals, we devised a reporter system that is based on FLP-mediated genetic recombination. The FLP gene, encoding the site-specific recombinase FLP, was genetically modified for expression in C. albicans and fused to the promoter of the SAP2 gene that codes for one of the secreted aspartic proteinases, which are putative virulence factors of C. albicans. The SAP2P-FLP fusion was integrated into one of the SAP2 alleles in a strain that contained a deletable marker that conferred resistance to mycophenolic acid and was flanked by direct repeats of the FLP recognition target (FRT). Using this reporter system, a transient gene induction could be monitored at the level of single cells by the mycophenolic acid-sensitive phenotype of the colonies generated from such cells after FLP-mediated marker excision. In two mouse models of disseminated candidiasis, SAP2 expression was not observed in the initial phase of infection, but the SAP2 gene was strongly induced after dissemination into deep organs. In contrast, in a mouse model of oesophageal candidiasis in which dissemination into internal organs did not occur, no SAP2 expression was detected at any time. Our results support a role of the SAP2 gene in the late stages of an infection, after fungal spread into deep tissue. This new in vivo expression technology (IVET) for a human fungal pathogen allows the detection of virulence gene induction at different stages of an infection, and therefore provides clues about the role of these genes in the disease process.     

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes.

The gene encoding Candida tropicalis peroxisomal trifunctional enzyme, hydratase-dehydrogenase-epimerase (HDE), was expressed in both Candida albicans and Saccharomyces cerevisiae. The cellular location of HDE was determined by subcellular fractionation followed by Western blot analysis of peroxisomal and cytosolic fractions using antiserum specific for HDE. HDE was found to be exclusively targeted to and imported into peroxisomes in both heterologous expression systems. Deletion and mutational analyses were used to determine the regions within HDE which are essential for its targeting to peroxisomes. Deletion of a carboxyl-terminal tripeptide Ala-Lys-Ile completely abolished targeting of HDE to peroxisomes, whereas large internal deletions of HDE (amino acids 38-353 or 395-731) had no effect on HDE targeting to peroxisomes in either yeast. This tripeptide is similar to, but distinct from, other tripeptide peroxisomal targeting sequences (PTSs) as identified in peroxisomal firefly luciferase and four mammalian peroxisomal proteins. Substitutions within the carboxyl-terminal tripeptide (Ala----Gly and Lys----Gln) supported targeting of HDE to peroxisomes of C. albicans but not of S. cerevisiae. This is the first detailed analysis of the peroxisomal targeting signal in a yeast peroxisomal protein.     

IL-13 attenuates gastrointestinal candidiasis in normal and immunodeficient RAG-2(-/-) mice via peroxisome proliferator-activated receptor-gamma activation

We recently demonstrated that in vitro peroxisome proliferator-activated receptor-gamma (PPARgamma) activation of mouse peritoneal macrophages by IL-13 or PPARgamma ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2(-/-)) mice with natural and synthetic PPARgamma-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARgamma antagonist, and are reduced in PPARgamma(+/-) mice. Overall, these data demonstrate that IL-13 or PPARgamma ligands attenuate C. albicans infection of the GI tract through PPARgamma activation and hence suggest that PPARgamma ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.     

Differential Candida albicans lipase gene expression during alimentary tract colonization and infection

The human pathogenic fungus Candida albicans, which can reside as a benign commensal of the gut, possesses a large family of lipase encoding genes whose extracellular activity may be important for colonization and subsequent infection. The expression of the C. albicans lipase gene family (LIP1-10) was investigated using a mouse model of mucosal candidiasis during alimentary tract colonization (cecum contents) and orogastric infection. LIPs4-8 were expressed in nearly every sample prepared from the cecum contents and infected mucosal tissues (stomach, hard palate, esophagus and tongue) suggesting a maintenance function for these gene products. In contrast, LIPs1, 3, and 9, which were detected consistently in infected gastric tissues, were essentially undetectable in infected oral tissues. In addition, LIP2 was expressed consistently in cecum contents but was undetectable in infected oral tissues suggesting LIP2 may be important for alimentary tract colonization, but not oral infection. The host responded to a C. albicans infection by significantly increasing expression of the chemokines MIP-2 and KC at the site of infection. Therefore, differential LIP gene expression was observed during colonization, infection and at different infected mucosal sites.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

Candida albicans secreted aspartyl proteinases in virulence and pathogenesis.

Candida albicans is the most common fungal pathogen of humans and has developed an extensive repertoire of putative virulence mechanisms that allows successful colonization and infection of the host under suitable predisposing conditions. Extracellular proteolytic activity plays a central role in Candida pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins). Although the consequences of proteinase secretion during human infections is not precisely known, in vitro, animal, and human studies have implicated the proteinases in C. albicans virulence in one of the following seven ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host. We have critically discussed the data relevant to each of these seven criteria, with specific emphasis on how this proteinase family could contribute to Candida virulence and pathogenesis.     

The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes

In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly. In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome. We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal. Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene. The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb. The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites. The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker. As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders. Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders. Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected. PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups.     

The effects on immune response of levamisole treatment following infection of U-937 macrophages with Candida albicans

The aim of this study was to investigate the effects on the immune response of levamisole alone and in conjunction with Candida albicans stimulation in human macrophage cell culture by determining the alterations in the levels of cytokine release. Levamisole treatment was performed before, during and after infecting U-937 human macrophage cells with C. albicans. In cell supernatants, interleukin (IL)-1b, IL-12, IL-18, tumour necrosis factor alpha (TNF-α) levels were measured by ELISA. In vitro levamisole treatment accompanied by C. albicans stimulation significantly increased IL-12, IL-1β and IL-18 production in macrophage cells (p < 0.05). It was observed that when administered before C. albicans infection, levamisole significantly increased IL-12 and IL-1β production in macrophage cells (p < 0.05). Another finding was that when applied to macrophage cells simultaneously with C. albicans infection, or before infection with C. albicans, levamisole suppressed the TNF-β production stimulating effect of C. albicans (p < 0.05). These results indicated that levamisole could be useful in treating patients infected with C. albicans or in protecting individuals under the risk of being infected with this pathogen. There is a need for further experimental and clinical studies on this hypothesis.     

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。