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1.
Anna Dongari-Bagtzoglou Helena Kashleva Prabhat Dwivedi Patricia Diaz John Vasilakos 《PloS one》2009,4(11)
C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response. 相似文献
2.
Characterization of Candida albicans dihydrofolate reductase 总被引:3,自引:0,他引:3
D P Baccanari R L Tansik S S Joyner M E Fling P L Smith J H Freisheim 《The Journal of biological chemistry》1989,264(2):1100-1107
Dihydrofolate reductase from Candida albicans was purified 31,000-fold and characterized. In addition, the C. albicans dihydrofolate reductase gene was cloned into a plasmid vector and expressed in Escherichia coli, and the enzyme was purified from this source. Both preparations showed a single protein-staining band with a molecular weight of about 25,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymes were stable and had an isoelectric point of pH 7.1 on gel isoelectric focusing. Kinetic characterization showed that the enzymes from each source had similar turnover numbers (about 11,000 min-1) and Km values for NADPH and dihydrofolate of 3-4 microM. Like other eukaryotic dihydrofolate reductases, the C. albicans enzyme exhibited weak binding affinity for the antibacterial agent trimethoprim (Ki = 4 microM), but further characterization showed that the inhibitor binding profile of the yeast and mammalian enzymes differed. Methotrexate was a tight binding inhibitor of human but not C. albicans dihydrofolate reductase; the latter had a relatively high methotrexate Ki of 150 pM. The yeast and vertebrate enzymes also differed in their interactions with KCl and urea. These two agents activate vertebrate dihydrofolate reductases but inhibited the C. albicans enzyme. The sequence of the first 36 amino-terminal amino acids of the yeast enzyme was also determined. This portion of the C. albicans enzyme was more similar to human than to E. coli dihydrofolate reductases (50% and 30% identity, respectively). Some key amino acid residues in the C. albicans sequence, such as E-30 (human enzyme numbering), were "vertebrate-like" whereas others, such as I-31, were not. These results indicate that there are physical and kinetic differences between the eukaryotic mammalian and yeast enzymes. 相似文献
3.
This paper further characterizes a protein we have demonstrated in Candida albicans which has the ability to bind corticosterone and related steroid hormones. Fungal cells are disrupted and cytosol is incubated with [3H]corticosterone for 3 h at which time peak steady state binding is achieved. Bound hormone is separated from free using Sephadex G-50 minicolumns or dextran-coated charcoal. Binding was found to be a linear function of protein concentration. The bound hormone co-migrates with authentic corticosterone in thin layer chromatographic systems indicating no metabolism of the radioprobe. Scatchard analysis of the binding in the pseudohyphal form of C. albicans yielded values of 6.3 nM for the Kd and a binding capacity of about 650 fmol/mg of cytosol protein; both determinations are comparable to our findings in the yeast form of this organism. A series of sterols were tested for their ability to displace [3H]corticosterone from the yeast binder, and the results show that the binder is remarkably selective and stereo specific. Physical-chemical studies show the binder to be degraded at high temperatures and that binding is destroyed by trypsin and sulfhydryl blockers. The protein sediments at 4 S on sucrose gradients and does not exhibit ionic dependent aggregation. The molecular weight is estimated to be approximately 43,000 daltons by gel chromatography. We hypothesize that this intracellular protein may represent a primitive form of either the mammalian glucocorticoid receptor or the plasma corticosteroid-binding globulin. 相似文献
4.
The human pathogenic fungus Candida albicans has traditionally been classified as a diploid, asexual organism. However, mating-competent forms of the organism were recently described that produced tetraploid mating products. In principle, the C.albicans life cycle could be completed via a sexual process, via a parasexual mechanism, or by both mechanisms. Here we describe conditions in which growth of a tetraploid strain of C.albicans on Saccharomyces cerevisiae 'pre-sporulation' medium induced efficient, random chromosome loss in the tetraploid. The products of chromosome loss were often strains that were diploid, or very close to diploid, in DNA content. If they inherited the appropriate MTL (mating-type like) loci, these diploid products were themselves mating competent. Thus, an efficient parasexual cycle can be performed in C.albicans, one that leads to the reassortment of genetic material in this organism. We show that this parasexual cycle-consisting of mating followed by chromosome loss-can be used in the laboratory for simple genetic manipulations in C.albicans. 相似文献
5.
Candida albicans is among the most common fungal pathogens. Infections caused by C. albicans and other Candida species can be life threatening in individuals with impaired immune function. Genetic analysis of C. albicans pathogenesis is complicated by the diploid nature of the species and the absence of a known sexual cycle. Through a combination of parasexual techniques and molecular approaches, an effective genetic system has been developed. The close relationship of C. albicans to the more extensively studied Saccharomyces cerevisiae has been of great utility in the isolation of Candida genes and development of the C. albicans DNA transformation system. Molecular methods have been used for clarification of taxonomic relationships and more precise epidemiologic investigations. Analysis of the physical and genetic maps of C. albicans and the closely related Candida stellatoidea has provided much information on the highly fluid nature of the Candida genome. The genetic system is seeing increased application to biological questions such as drug resistance, virulence determinants, and the phenomenon of phenotypic variation. Although most molecular analysis to data has been with C. albicans, the same methodologies are proving highly effective with other Candida species. 相似文献
6.
The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes. 相似文献
7.
cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system. 相似文献
8.
Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents 总被引:1,自引:0,他引:1
Several antifungal agents, at concentrations of 10 micrograms/ml, were shown to suppress ATP concentrations very rapidly in intact cells and spheroplasts of Candida albicans. The highest ATP-suppressing activity was shown by the highly lipophilic imidazole derivatives difonazole, clotrimazole, econazole, isoconazole, miconazole, oxiconazole and tioconazole, which all caused a reduction of cellular ATP content of more than 50% in 10 min. Relatively hydrophilic imidazole derivatives such as ketoconazole were essentially inactive in the test, as were the triazole derivatives fluconazole, ICI 153066, itraconazole and terconazole, and 5-fluorocytosine. Amphotericin B and terbinafine possessed intermediate ATP-suppressing activity, and the dose-response and pH-response curves for these compounds suggested their mechanism of ATP suppression differed from that of the active imidazole derivatives. ATP suppression by azole antifungals did not involve leakage of ATP from the cells and the effect was entirely abrogated by the presence of serum. Intact cells and spheroplasts of yeast-form and hyphal-form C. albicans were generally equally sensitive to ATP suppression, but stationary-phase cells of both morphological forms were less sensitive than exponential-phase cells. The extent of ATP suppression was significantly reduced in stationary-phase yeast cells of a C. albicans strain with known resistance to azole antifungals, but exponential-phase cells of resistant and susceptible strains were equally sensitive. The effect is tentatively ascribed to membrane damage caused directly by the antifungals. 相似文献
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11.
Characterization of the inverted duplication in the mitochondrial DNA of Candida albicans. 总被引:1,自引:1,他引:1
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J A Shaw W B Troutman B A Lasker M M Mason W S Riggsby 《Journal of bacteriology》1989,171(11):6353-6356
The mitochondrial DNA (mtDNA) of Candida albicans contains a large inverted duplication. As is the case with most chloroplast DNAs and one other mtDNA, the nonduplicated regions of the molecule occur in two orientations with respect to each other, indicating that internal recombination occurs. Like some other mtDNAs, the C. albicans mtDNA contains a single SalI restriction site located near one end of the large rRNA gene. In contrast to other cases, however, the inverted duplication does not appear to contain any sequences coding for rRNA. 相似文献
12.
S A Klotz 《Applied microbiology》1989,55(9):2119-2122
Cell surface hydrophobicity may be an important factor contributing to the virulence of Candida yeast cells. Surface hydrophobic and surface polar groups would be required for a yeast cell to act as a surface-active agent. In this report, the surface activities of whole yeast cells were measured. Yeast cells added at 10(8)/ml reduced the surface tension (gamma s) of saline by 20% as determined by the du Nouy method. A 1% suspension of yeast cell wall fragments reduced gamma s of saline by 36%. Whole yeast cells caused a reduction in interfacial tension (gamma I) between hexadecane and saline. The reduction of gamma I was proportional to the surface hydrophobicity of the yeasts. Yeast cells grown in glucose as the sole carbon source (thus possessing a relatively more hydrophilic cell surface) reduced gamma I by 30%, whereas yeast cells grown in hexadecane (thus possessing a more hydrophobic cell surface) reduced gamma I by 41%. The reduction of gamma I was reversed upon the addition of a strong surfactant. It was also demonstrated that yeast cells blended with nonionic surfactants during growth in a glucose broth in order to change their cell surface hydrophobicity adhered to solid surfaces in direct proportion to their cell surface hydrophobicity. Thus, the surface-active properties of Candida yeast cells may significantly contribute to the accumulation of yeast cells at various biological interfaces such as liquid-solid, liquid-liquid, and liquid-air, leading to their eventual adhesion to solid or tissue surfaces. 相似文献
13.
Enzyme cytochemistry of Candida albicans. 总被引:2,自引:0,他引:2
The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed. 相似文献
14.
A middle repetitive DNA element, Candida albicans repetitive element-1 (CARE-1) has been isolated from the pathogenic yeast C. albicans. CARE-1 appears to be species-specific and constitutes approx. 0.045% of total C. albicans DNA, or a reiteration frequency of about two to twelve copies per haploid genome. The CARE-1 element has been detected on several C. albicans chromosomes separated by field-inversion gel electrophoresis, suggesting that the element is dispersed. Interstrain variation was observed in the number and distribution of hybridizing bands. The element is well conserved, since no nucleotide (nt) heterogeneity was observed when the sequences of two CARE-1 family members isolated from two different chromosomes (A and B) of C. albicans were compared. CARE-1 possesses 467 bp and is characterized by several stretches of A's and T's, short direct repeats and shows no significant homology to any known nt sequence. 相似文献
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Fine structural studies on the septa of Candida albicans in vitro (when leading a saprophytic existence) and the organism in its invasive form (as a pathogen in oral candidosis) have shown that in the former the septum exhibits a unique central perforation resembling an aggregate of fine canaliculi connecting one cell to the other. In the invasive form the septum is non-perforated and appears as a solid structure. Septal ultrastructure is well characterised in many pathogenic fungi. Our observations on Candida albicans do not resemble any previous studies carried out on other deutromycetous fungi. 相似文献
17.
Phospholipase A and lysophospholipase have been identified as the enzymes responsible for phospholipid hydrolysis by Candida albicans. The method used to identify and measure the activity of these enzymes is described, and the probable significance of phospholipase in the invasion of the epithelium by Candida albicans discussed. 相似文献
18.
We subjected 16 Candida albicans clinical isolates to ultraviolet radiation and tested the survivors for auxotrophy. Six isolates displayed strongly biased auxotroph spectra: three yielded methionine auxotrophs, two yielded both isoleucine-valine and adenine auxotrophs, and one yielded lysine auxotrophs. We present evidence that auxotrophs arise by segregation from naturally occurring heterozygous states. The remaining isolates yielded few or no auxotrophs in an arbitrary sample (greater than 2,500) of survivors of irradiation. Our experiments indicate that C. albicans is diploid, although aneuploidy (2n + i) cannot be rigorously excluded. We discuss the possible utility of heterozygosity as a marker in epidemiological studies, and we discuss a rationale for the frequent occurrence of heterozygosity. 相似文献
19.
H Tsushima H Mine K Hoshika Y Kawakami F Hyodoh A Ueki 《Journal of bacteriology》1992,174(14):4807-4810
A cysteine proteinase inhibitor was found in culture media of Candida albicans. Purification to homogeneity of the inhibitor was performed by carboxymethyl-papain-Sepharose affinity, DE-52 ion-exchange, and reverse-phase high performance liquid chromatographies. The purified inhibitor had an M(r) of 15 kDa and a pI of 4.9. It was more stable to heat and pH than most proteins. The N-terminal sequence of the first 30 residues demonstrated high similarity with that of human cystatin A. Thus, C. albicans cysteine proteinase inhibitor seems to belong to the cystatin superfamily. The inhibitor activity of the yeast cellular form was 4.0 times higher than that of the hyphal cellular form in 7-day culture media. It is suggested that the inhibitor has regulatory functions similar to those of its counterpart proteinases in the invasion of host cells. 相似文献
20.
P J Gallagher D E Bennett M C Henman R J Russell S R Flint D B Shanley D C Coleman 《Journal of general microbiology》1992,138(9):1901-1911
Approximately 50% (15/28) of a selection of oral isolates of Candida albicans from separate individuals infected with the human immunodeficiency virus (HIV) exhibited low susceptibility to ketoconazole as determined by hyphal elongation assessment. Nine of these isolates exhibited colony morphology variation or switching at 37 degrees C, of which six expressed low ketoconazole susceptibility. To determine whether colony morphology variation could give rise to derivatives with reduced azole susceptibility, several high-frequency switching variants of three HIV-patient isolates were recovered and assessed. All but one of the variants expressed similar azole susceptibility profiles to their respective parental strains. However, the C. albicans derivative 132ACR expressed significantly reduced susceptibility to ketoconazole in comparison to its parental strain 132A. In whole cells, on the basis of total growth the switched derivative 132ACR was markedly less susceptible than its parental isolate 132A to ketoconazole at 10 microM. A much smaller difference was observed with fluconazole at 10 microM, with the switched derivative 132ACR exhibiting a threefold lower susceptibility compared with the parental isolate 132A. The incorporation of [14C]acetate in control and azole-treated cells of both organisms was higher for the parental strain. When cell lysates of strain 132A and its derivative 132ACR were incubated with [14C]mevalonic acid and ketoconazole, the IC50 for 14C-label incorporation into C-4 demethyl sterols was fivefold higher for lysates of the switched derivative 132ACR compared with those of the parental strain 132A. With fluconazole the IC50 value for the derivative 132ACR was 25-fold higher than for strain 132A. The 14-sterol demethylase of the switched derivative 132ACR was possibly less sensitive to azole inhibition than that of the enzyme of strain 132A. These studies indicated that colony morphology variation in vitro can generate derivatives with stable, reduced azole susceptibility without prior exposure to azoles. 相似文献