Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae

Summary An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2 ^–) to Leu⁺ at a frequency of 2.15 × 10³ transformants per pg DNA, and transformed C. albicans SGY-243 (ura3) to Ura⁺ at a frequency of 1.91 × 10³ transformants per g DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5TTTTATGTTTT3) which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 by from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes. A sub-fragment (494 bp) containing the 5S rRNA gene (but not the region containing the ARS cores) hybridized to genomic DNAs from a number of yeast species, including S. cerevisiae, C. tropicalis, C. pseudotropicalis, C. parapsilosis, C. kruseii, C. (Torulopsis) glabrata and Neurospora crassa. The 709-bp ARS element (but not the 5S rRNA gene) was necessary for high-frequency transformation and autonomous plasmid replication in both S. cerevisiae and C. albicans.EMBL/GenBank database accession number: X16634 (5S rRNA)     

beta-Galactosidase of Kluyveromyces lactis (Lac4p) as reporter of gene expression in Candida albicans and C. tropicalis.

Summary Vectors containing fusions of the Candida albicans ACT promoter to heterologous genes were constructed and transformed into a C. albicans host strain. -Galactosidase (Lac4p) activity was detected in transformants carrying an ACT fusion to the Kluyveromyces lactis LAC4 gene, while fusions to the Escherichia coli lacZ gene and to other heterologous genes were not expressed. Lac4p was also produced by C. tropicalis transformants carrying the ACT/LAC4 fusion. Plasmids in transformed C. albicans strains were present either as free multimers in high copy number or, more frequently, integrated into the genome in low copy number yielding high and low LAC4 mRNA and Lac4p expression levels, respectively. Lac4p-expressing transformants of C. tropicalis, but not of C. albicans, were able to utilize lactose as sole carbon source. An ACT/LAC4 fusion was not differentially expressed during the yeast and hyphal growth phases of C. albicans, indicating that the ACT promoter is not regulated during morphogenesis. These results define the first reporter gene system for convenient monitoring of gene expression in Candida species.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

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.     

Recombinagenicity of caffeine for Candida albicans

Caffeine at concentrations of 0.5 × 10^–2 M or higher inhibited cell replication and induced gene segregations in Candida albicans cultured on defined complete medium. Both responses increased incrementally with increasing caffeine concentrations, and were more severe during incubation at 37 °C than 25 °C; at 37 °C, caffeine levels above 1.5 × 10^–2 M caused cellular inactivation. Caffeine effects occurred only under conditions permitting cell growth, and their magnitudes were greater for unbudded than budding cells, were influenced by cellular genetic backgrounds, and were unaffected by the presence of adenine in the medium. Evaluations of segregations for recessive auxotrophic markers of a four member linkage group carried heterozygously in a cis arrangement in treated cells established that induced segregants arise through either reciprocal or nonreciprocal recombinations. The frequency distributions of classes of reciprocal and nonreciprocal recombinants for these markers conformed with those previously obtained following induction by ultraviolet radiation, indicating that the probabilities of recombinational events within the chromosomal regions defined by the markers are not biased by the differences in kinds of initial DNA lesions caused by the two recombinagens. A panel of four protoplast fusion hybrids considered deficient for DNA repair because of enhanced susceptibilities to UV induced cellular inactivation and mitotic recombination exhibited corresponding increased sensitivities to caffeine, signifying that DNA damage induced by caffeine is subject to repair. Caffeine did not affect behavior of a variant strain exhibiting high frequency phenotypic switching between minute smooth and large rough colonial forms, and no evidence for mutagenicity of the drug was obtained with systems for detection of forward or reverse mutations. The mechanism of caffeine's recombinagenicity, and the implications of that property for genetic studies of C. albicans are discussed.     

Detection of Candida albicans in disseminated candidosis by immunofluorescence staining

An indirect immunofluorescence (IF) method using rabbit anti-Candida albicans was used to detect C. albicans in blood samples of 12 patients with systemic candidosis defined clinically, histologically and by blood cultures. Positive staining of C. albicans could be detected in all of the patients. The findings suggest that IF-method offers a more rapid method in the diagnosis of disseminated candidosis.     

Cell-associated collagenolytic activity by Candida albicans

Cell associated collagenolytic activity of Candida albicans was quantified by measuring the degradation of synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which is a specific substrate for collagenase, by the freeze-thaw procedure method. This collagenolytic activity was enhanced by cells cultured in the presence of bovine serum albumin (BSA) in culture medium. However, this activity was inhibited in the presence of ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), but not by the serine proteinase inhibitor p-amidinophenyl methanesulfonyl fluoride (APMSF), nor the aspartyl proteinase inhibitor pepstatin A. These results suggested the presence of a metalloenzyme on pericellular C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mannan and D-arabinitol concentrations in serum from a patient with Candida albicans endocarditis

In an attempt to clarify the comparative values of serological and microbiological examinations for the early diagnosis of systemic candidiasis, antibodies against Candida albicans, serum mannan, and the D-arabinitol creatinine ratio were investigated in a patient with aortic valve endocarditis associated with carcinoma of the bile duct. Candida precipitins and the antibody titer against Candida cell wall mannan were examined by an immunodiffusion technique and hemagglutination test, respectively. Serum mannan was tested by enzyme-linked immunosorbent assay (ELISA) using the biotin-streptavidin procedure. The upper limit of negativity of the assay was determined by adding 0.06 to the absorbance of pooled serum from healthy laboratory workers. This value ws about 0.8 ng/ml with ELISA. The D-arabinitol concentration in serum was examined by an enzymatic fluorometric method. Rising antibody titers against C. albicans, mannan antigenemia, and an elevated D-arabinitol creatinine ratio were first observed between the 11th and 12th hospital days. Blood cultures obtained on 8th, 9th, and 11th hospital days grew C. albicans after 3 to 4 days of incubation. Of 11 serum samples, 5 were positive for mannan, whereas D-arabinitol creatinine ratio was positive in 7 of 9 samples. Blood cultures was the earliest evidence of Candida infections in our cases. However, because of saprophytic nature of Candida species, tests for antibodies, antigenemia, and the D-arabinitol creatinine ratio in combination with blood cultures are necessary to confirm systemic candidiasis at an early stage of infection.Abbreviations ELISA enzyme-linked immunosorbent assay     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

Antibody formation in experimental immunizations with Candida albicans ribosomal fractions

Sera of mice immunized with ribosomal fractions of Candida albicans showed the presence of anti-C. albicans antibodies, detected by the gel-immunodiffusion, agglutination and immune adherence tests.Candida infections are among the most prevalent opportunistic yeast infections, attacking debilitated individuals, and against which there is no effective prophylactic treatment currently available (1, 2, 3, 4, 5). In view of the succes reported in experimental immunizations with ribosomal fractions from various bacteria and some fungi, as summarized by Youmans and Tewari (7, 8), a similar approach for immunization in experimental candidiasis appears reasonable. The present work describes preliminary results on circulating antibodies elicited in the course of immunizations with ribosomal fractions of Candida albicans.Ribosomal preparations were obtained from mechanically disrupted cell-pellets of C. albicans by differential centrifugation and purification in a 15% sucrose and 5% ammonium sulfate solution (in sodium-magnesium-Tris buffer), using a modification of the procedure described by Rubin (6). Concentration of ribosomal-RNA was determined by the absorbance at 260 nm; ribosomal-protein concentration by the Lowry reaction; and purity of the ribosomal preparation checked by the ratio of absorbance at 260 nm to 280, and at 260 to 235 nm. Mice (ICR strain) were immunized with these ribosomal preparations in amounts of 50–100 g ribosomalprotein/mouse, by 2–3 subcutaneous inoculations with Frend's adjuvant, with a 10–21 day interval between the inoculations.This work constitutes part of Ruth Levy's research study towards the Ph.D. degree.     

Partial purification and characterization of a mannosyl transferase involved in O-linked mannosylation of glycoproteins in Candida albicans

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.     

Cytochemical and ultrastructural studies of Candida albicans

Ultrastructural modifications of the cell wall coat of Candida albicans during adherence to host cells were investigated using various cytochemical techniques. Attachment of the fungus to buccal epithelial cells appeared to involve spatial rearrangement of their cell wall surface. In particular adhering yeast developed a fibrogranular surface layer visualized by the periodic acid — thiocarbohydrazide silver proteinate technique (a polysaccharide detectron technique); Concanavalin A binding sites detected on their cell wall coat were highly increased. Attachment of yeasts to epithelial cells appeared mediated by fibrillar structures or polysaccharidic granules distributed on the cell wall coat. But free extra-cell wall material containing mannoproteins released from the yeast surface suggested additional mechanisms.Abbreviations Con A Concanavalin A - Man-fer mannosyl ferritin - PATAg Periodic acid-thiocarbohydrazide-silver proteinate     

Non-glucan Attached Proteins of Candida albicans Biofilm Formed on Various Surfaces

Non-glucan attached proteins of the cell surface and extracellular matrix of Candida albicans biofilms formed on two catheter surfaces and denture acrylic were examined. The SDS-PAGE protein profiles of these proteins compared with that obtained from planktonic yeast cells and germ tubes were generally similar. This observation suggested that this class of biofilm surface proteins is not composed of a unique set of extracellular proteins or that one or a few proteins dominate the non-glucan attached proteins of biofilm. However, differences were observed in the proteins obtained from biofilm formed on one catheter surface and two proteins, Grp2p and ORF19.822p, identified by mass spectrometry following two-dimensional separation. These proteins have previously been associated with drug resistance and their presence or abundance appeared to be influenced by the surface on which the biofilm was formed.     

Fungispecificity of fluconazole against Candida albicans

Candida albicans is an opportunistic pathogen of human mucosal surfaces. Colonization of oral and vaginal mucosa by this yeast is antagonized by the resident normal bacterial population. However, antibacterial therapy can alter the normal flora to allow fungal cells to attach, grow and invade host tissues. We studied the antimicrobic activity of fluconazole against clinical isolates of oral and vaginal bacteria and Candida albicans in vitro and in vivo by scanning and transmission electron microscopy; we also compared the bactericidal activity of fluconazole with clotrimazole in vitro by microbiologie assay. Fluconazole lysed fungi but did not change the ultrastructure of bacteria. Clotrimazole, but not fluconazole, was bactericidal against lactobacillus and streptococcus, the principal species of the oral and vaginal cavities. We conclude that Candida albicans, but not oral and vaginal bacteria, is susceptible to fluconazole. These observations help explain the antimycotic specificity of fluconazole and its efficacy against candidiasis in humans.     

Development of amphotericin B liposomes bearing antibody specific to Candida albicans

Summary Liposomes expressing external antibody specific for Candida albicans and encapsulating amphotericin B were developed and characterized in this study. Antibody was first modified by the covalent attachment of palmitic acid residues. Liposomes were produced by reverse-phase evaporation and modified antibody was incorporated into these liposomes via the hydrophobic interaction between the palmitic acid and the phospholipids composing the liposomes. The liposomes were characterized as to the amount of amphotericin B by spectroscopy and for the presence of antibody by protein analysis and secondary immunolabeling by fluorescent and electron microscopic methods. Immunogold labeling showed that the antibody was being expressed externally on the liposomes in the electron microscopic studies and the specificity of these liposomes for C. albicans was observed by secondary immunofluorescence.     

Effect of Candida albicans dsDNA in Gastrointestinal Candida Infection

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-γ. In infected DNA-treated mice, an enhanced production of IFN-γ by Peyer’s patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.     

Immune responses to yeast and mycelial forms of Candida albicans in intraperitoneally infected mice

Candida albicans E-139 produced pure mycelial and yeast cultures in a low sulphate medium at different temperatures. The influence of the morphological phase, dose and viability of the fungi on the kinetic of delayed-type hypersensitivity (DTH) and anti-mycelial and anti-yeast antibodies have been studied in mice injected intraperitoneally. The mycelial form elicited higher DTH levels than the yeast phase. This effect seems to be related to its antigenic properties. The effect of dose on the immune response depends on the viability of the fungus. The mycelial cytoplasmic antigens were more effective than the yeast ones in detecting antibodies induced during the experiments, particularly during the later stages of the observation periods, suggesting that such antigens may be useful in the serodiagnosis of Candida infections.     

Anaerobically induced production of hybrid monokaryons by heterokaryons of Candida albicans

Summary During aerobic replication, balanced heterokaryons (hets) of Candida albicans produced by fusing protoplasts of complementing auxotrophic strains characteristically segregate low frequencies of prototrophic monokaryons bearing hybrid nuclei formed either through karyogamy or unidirectional internuclear genetic transfers within het cells. Anaerobic growth causes exponential inactivation of hets and induces their production of hybrid monokaryons. Both responses are functions of heterokaryosis as such and not the genetic backgrounds of hets. Evidence is presented that (i) the nuclei of anaerobically generated hybrids arise through induction in hets of karyogamy not internuclear genetic transfers and that (ii) the events underlying that induction are different from those responsible for inactivation of the cells.