Biosynthesis of chemical compounds by Candida albicans and Candida glabrata

BackgroundIn the last three decades the species of Candida have been of great interest due to the high mortality rates that they cause in immunocompromised and hospitalized patients. These species are opportunistic pathogens and they have inhabited other environments long before colonizing human cells. Among these environments we find wastewater from mines, and water from aquifers and soils that contain high concentrations of precious metals as well as toxic and base metals.AimsThe aim of this study was to assess whether Candida albicans and Candida glabrata are able to maintain homeostasis in the presence of zinc, copper, cobalt or silver.MethodsTo achieve the objective, each of the Candida species was exposed to every single metal individually in a salt solution. Subsequently the treated cells were lysed to evaluate the compounds formed by means of Scanning Electron Microscopy-Energy Dispersive X-ray spectroscopy (SEM-EDS).ResultsWhen analyzing the compounds that both C. albicans and C. glabrata formed in the presence of each of the metals, we found that they had synthesized silver sulfide (Ag₂S), cobalt sulfate (CoSO₄), zinc phosphate (Zn₃(PO₄)₂), or copper oxide (CuO).ConclusionsOur results indicate that both C. albicans and C. glabrata have enzymatic and non-enzymatic mechanisms that allow them to achieve homeostasis in a different specific manner for each of the single metals to which they were exposed. To our knowledge, this is the first work reporting that C. albicans and C. glabrata can reduce different metals, with the subsequent formation of sulfides, sulfates, phosphates and oxides. This ability, developed over time by these Candida species, is probably a kind of biochemical mechanism in order to survive and colonize many different environments, from water or soil to humans. For this reason, C. albicans and C. glabrata make up an excellent model of study, both from a medical and biotechnical point of view.     

Evidence for recombination in Candida glabrata

Despite its clinical importance, little is known of the epidemiology and population structure of Candida glabrata. C. glabrata possesses a mating type system similar to that in Saccharomyces cerevisiae, however mating, meiosis and recombination have not been demonstrated. We performed multilocus sequence typing on a collection of 165 isolates to test for evidence of genetic recombination. A total of 3345 bp from six loci (FKS, LEU2, NMT1, TRP1, UGP1, and URA3) were sequenced for each isolate. The polymorphisms at these loci defined 34 sequence types. Significant evidence for a clonal population was revealed by the index of association and the number of phylogenetically compatible pairs of loci. However, 14 examples of phylogenetic incompatibility were also found. Thus we conclude that although C. glabrata has a predominantly clonal population structure, the multiple phylogenetic incompatibilities found strongly suggest that recombination occurred during the evolution of C. glabrata, and may infrequently still occur.     

Glutathione-coated cadmium-sulfide crystallites in Candida glabrata

Cadmium-sulfide crystallites form in the yeast Candida glabrata cultured in the presence of cadmium salts. The particles function to sequester and detoxify intracellular cadmium ions. The crystallites are peptide-coated, but the coating peptide varies with the nutrient conditions of the growth medium. When cultured in rich nutrient broth the yeast forms intracellular CdS particles coated with a mixture of glutathione and the gamma-glutamylcysteine dipeptide. In contrast, cultures in synthetic minimal medium yield particles coated with polymerized gamma EC peptides of general structure (gamma-Glu-Cys)n-Gly. Glutathione/gamma-glutamylcysteine particles exhibit properties analogous to quantum, semiconductor-type crystallites. The optical properties are dependent on particle size, and irradiation results in photoluminescence and photoreduction not observed in bulk CdS mineral. Aerobic irradiation leads to particle decomposition presumably via oxidation of the sulfide ions within the crystallite.     

A purine permease in Candida glabrata

Abstract Competition experiments revealed that adenine and guanine were transported by a purine permease in both Candida glabrata 4 and a C. glabrata 4 cytosine permease negative mutant. The C. glabrata 4 cytosine permease negative mutant was isolated using 5-fluorocytosine selection. This mutant no longer transported cytosine, but transported adenine and guanine. A transport system for hypoxanthine was not detected. Hence, in addition to the cytosine permease, a purine permease exists in C. glabrata . This differs from the purine cytosine permeases in Saccharomyces cereuisiae and Candida albicans which transport adenine, cytosine, guanine and hypoxanthine.     

Differential Virulence of Candida glabrata Glycosylation Mutants

The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.     

Three mating type-like loci in Candida glabrata

Candida glabrata, the second most prevalent Candida species colonizing humans, possesses three mating type-like (MTL) loci (MTL1, MTL2, and MTL3). These loci contain pairs of MTL genes with their respective coding regions on complementary Crick and Watson DNA strands. Each pair of genes is separated by a shared intergenic promoter region, the same configuration found at the mating type loci of Saccharomyces cerevisiae. Two of the MTL loci, MTL1 and MTL2, contain either the MTLa1/MTLa2 configuration or the MTLalpha1/MTLalpha2 configuration in different strains. All but one of the 38 tested C. glabrata strains were either aaalpha or aalphaalpha. One test strain was alphaalphaalpha. Based on the mating type genotype, the MTL genes at the MTL1 or MTL2 loci, and the size of the XbaI fragment harboring MTL1 or MTL2, four classes of C. glabrata strains (I, II, III, and IV) were distinguished. Northern analysis revealed that strains were either a-expressors or alpha-expressors and that expression always reflected the genotype of either the MTL1 or MTL2 locus, depending on the class. The expression pattern in each class, therefore, is similar to that observed in S. cerevisiae, which harbors two silent cassette loci, HMR and HML, and the expression locus MAT. High-frequency phenotypic switching between core phenotypes in an alpha-expressing, but not in an a-expressing, strain modulated the level of MTL expression, suggesting a possible relationship between core phenotypic switching and mating.     

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.     

Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland

Over the last decades, Candida spp have been responsible for anincreasing number of infections, especially in patients requiring intensive care.Knowledge of local epidemiology and analysis of the spread of these pathogens isimportant in understanding and controlling their transmission. The aim of this studywas to evaluate the genetic diversity of 31 Candida albicans and17 Candida glabrata isolates recovered from intensive care unitpatients from the tertiary hospital in Krakow between 2011-2012. The strains weretyped by random amplified polymorphic DNA (RAPD) polymerase chain reaction using fiveprimers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the presentinvestigation revealed a high degree of genetic diversity among the isolates. Noclonal relationship was found among the C. albicans strains, whereastwo C. glabrata isolates were identical. The source ofCandida infection appeared to be mostly endogenous; however, the presenceof two clonal C. glabrata strains suggested the possibility ofcross-transmission of these pathogens. Our study confirmed the high discriminatorypower of the RAPD technique in the molecular typing of Candidaclinical isolates. This method may be applied to the evaluation of transmissionroutes of pathogenic fungi on a local level.     

Assimilation of NAD(+) precursors in Candida glabrata

The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.     

Uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans

The uptake of pyrimidines and their derivatives into Candida glabrata and Candida albicans was measured using a novel technique in which the cells were rapidly separated from their suspending medium by centrifugation through a layer of an inert oil. The uptake of [14C]cytosine was linear for 30 s for all concentrations of pyrimidine tested. In C. glabrata but not C. albicans cytosine transport was mediated by both a high affinity (Km 0.8 +/- 0.1 microM), low capacity [V 40 +/- 4 pmol (microliters cell water)-1 s-1] and a low affinity [Km 240 +/- 35 microM], high capacity system [V 770 +/- 170 pmol (microliters cell water)-1 s-1]. The cytosine permease in C. glabrata was specific for cytosine and 5-fluorocytosine. In C. albicans there was only one cytosine transport system [Km 2.4 +/- 0.3 microM; V 50 +/- 4 pmol (microliters cell water)-1 s-1]; this system also transported adenine, guanine and hypoxanthine. Differences in nucleoside transport were also observed for C. glabrata and C. albicans, with the uridine permease in C. glabrata transporting only uridine and 5-fluorouridine whereas cytidine and adenosine were also transported by the uridine permease in C. albicans. Studies on the effect of nucleoside analogues on uridine transport in C. glabrata demonstrated the importance of the sugar moiety in determining the specificity of transport, with a hydroxyl residue on C-2 being apparently essential for transport.     

住院患者光滑念珠菌检出的回顾性分析

目的调查住院患者光滑念珠菌检出的特征。方法回顾性调查分析白求恩国际和平医院2008年1月～2009年4月住院患者中光滑念珠菌检出阳性者的临床资料,以同期白念珠菌检出患者为对照。结果其间共有52例详细病史资料记录的光滑念珠菌检出患者,以60岁以上老年人为主,占65.4%;主要分离自痰标本,占76.9%。患者患有多种基础疾病,以肺部感染(28例,53.8%)、恶性肿瘤(20例,38.5%)、脑梗死(15例,28.8%)常见。使用抗生素(52例,100%)、留置导尿管(15例,28.8%)是光滑念珠菌检出者的主要实施医疗措施。氟康唑是临床最常用的治疗光滑念珠菌感染药物(23例,44.2%)。光滑念珠菌检出患者死亡率高(14例,26.9%),高于同期白念珠菌检出对照组(6.2%,P=0.004)。结论光滑念珠菌检出患者与白念珠菌检出具有相似的临床流行病学特征。     

Inner kinetochore of the pathogenic yeast Candida glabrata

The human pathogenic yeast Candida glabrata is the second most common Candida pathogen after Candida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA of C. glabrata (CgCEN), although structurally very similar to that of Saccharomyces cerevisiae, is not functional in S. cerevisiae. To further examine the structure of the C. glabrata inner kinetochore, we isolated several C. glabrata homologs of S. cerevisiae inner kinetochore protein genes, namely, genes for components of the CBF3 complex (Ndc10p, Cep3p, and Ctf13p) and genes for the proteins Mif2p and Cse4p. The amino acid sequence identities of these proteins were 32 to 49% relative to S. cerevisiae. CgNDC10, CgCEP3, and CgCTF13 are required for growth in C. glabrata and are specifically found at CgCEN, as demonstrated by chromatin immunoprecipitation experiments. Cross-complementation experiments revealed that the isolated genes, with the exception of CgCSE4, are species specific and cannot functionally substitute for the corresponding genes in S. cerevisiae deletion strains. Likewise, the S. cerevisiae CBF3 genes NDC10, CEP3, and CTF13 cannot functionally replace their homologs in C. glabrata CBF3 deletion strains. Two-hybrid analysis revealed several interactions between these proteins, all of which were previously reported for the inner kinetochore proteins of S. cerevisiae. Our findings indicate that although many of the inner kinetochore components have evolved considerably between the two closely related species, the organization of the C. glabrata inner kinetochore is similar to that in S. cerevisiae.     

Sir3 Polymorphisms in Candida glabrata Clinical Isolates

The opportunistic fungal pathogen Candida glabrata adheres tightly to epithelial cells in culture, mainly through the adhesin Epa1. EPA1 is the founding member of a family of up to 23 putative adhesin-encoding genes present in the C. glabrata genome. The majority of the EPA genes are localized close to the telomeres, where they are repressed by subtelomeric silencing that depends on the Sir, Ku, Rif1, and Rap1 proteins. EPA6 and EPA7 also encode functional adhesins that are repressed in vitro. EPA1 expression in vitro is tightly controlled both positively and negatively, and in addition, presents high cell-to-cell heterogeneity, which depends on Sir-mediated silencing. In this work, we characterized the ability to adhere to HeLa epithelial cells and the expression of several EPA genes in a collection of 79 C. glabrata clinical isolates from several hospitals in Mexico. We found 11 isolates that showed increased adherence to mammalian cells compared with our reference strain under conditions where EPA1 is not expressed. The majority of these isolates displayed over-expression of EPA1 and EPA6 or EPA7, but did not show increased biofilm formation. Sequencing of the SIR3 gene of several hyper-adherent isolates revealed that all of them contain several polymorphisms with respect to the reference strain. Interestingly, two isolates have polymorphisms in positions flanked by clusters of amino acids required for silencing in the Saccharomyces cerevisiae Sir3 protein. Our data show that there is a large variability in adhesin expression and adherence to epithelial cells among different C. glabrata clinical isolates.     

Glycan microarray analysis of Candida glabrata adhesin ligand specificity

The Candida glabrata genome encodes at least 23 members of the EPA ( ep ithelial a dhesin) family responsible for mediating adherence to host cells. To better understand the mechanism by which the Epa proteins contribute to pathogenesis, we have used glycan microarray analysis to characterize their carbohydrate-binding specificities. Using Saccharomyces cerevisiae strains surface-expressing the N-terminal ligand-binding domain of the Epa proteins, we found that the three Epa family members functionally identified as adhesins in Candida glabrata (Epa1, Epa6 and Epa7) bind to ligands containing a terminal galactose residue. However, the specificity of the three proteins for glycans within this class varies, with Epa6 having a broader specificity range than Epa1 or Epa7. This result is intriguing given the close homology between Epa6 and Epa7, which are 92% identical at the amino acid level. We have mapped a five-amino-acid region within the N-terminal ligand-binding domain that accounts for the difference in specificity of Epa6 and Epa7 and show that these residues contribute to adherence to both epithelial and endothelial cell lines in vitro .