In-vitro susceptibility of Candida albicans and Candida stellatoidea to sulfamethoxazole

The susceptibility of organisms of the speciesCandida to sulfamethoxazole were tested in-vitro using the disc sensitivity method.Under specified conditions a zone of growth inhibition surrounding sulfamethoxazole tablet sensi disc was consistently produced in cultures ofCandida albicans, Candida stellatoidea andSaccharomyces cervisiae.Diameters of growth inhibition zone were measured and found to be greatest in cultures ofCandida stellatoidea andSaccharomyces, but growth inhibition inCandida albicans cultures was observed to a lesser degree.The correlation between in vivo pathogenicity of organisms of the speciesCandida, and degree of growth inhibition by sulfamethoxazole sensi disc presents an interesting relationship.     

Simple rapid identification of Candida albicans with emphasis on the differentiation between Candida albicans and Candida stellatoidea

Summary Subcultures ofC. albicans, made from Sabouraud agar, grown at room temperature for 48 hours, were inoculated into a 10 times saline dilution of Sabouraud liquid medium and left in the incubator for 45–60 minutes at 37° C, transferred to corn meal agar plates and incubated at 37° C for 18–24 hours.Small portions of the surface agar containing the yeasts from these plates were pressed under cover glasses and examined under the oil immersion lens.Under these conditions,C. albicans cultures were observed to produce only yeast-like cells, whereasC. stellatoidea cultures contained predominantly abundant, long, thin mycelia.     

Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.     

Pigment Formation for Differentiating Cryptococcus neoformans from Candida albicans

When 2,3- or 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxycinnamic acid are added to growth media, they are converted to a characteristic brown pigment by Cryptococcus neoformans. This pigment formation has hitherto been encountered only when this microorganism was cultivated on media containing Guizotia abyssinica seed. This phenomenon can be used for differentiating Cryptococcus neoformans from Candida albicans. Possible precursors of these o-diphenols (quinic acid, aromatic monohydroxy acids, or tyrosine) do not give rise to the brown pigmentation.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

A secreted beta-glucan-branching enzyme from Candida albicans.

A Mr 34,000 wall protein was isolated as a by-product of the purification of an endo-(1-3)-beta-glucanase from the culture filtrate of Candida albicans. The purified fraction contained no exo- or endo-beta-glucanase activity, and analysis by SDS poly-acrylamide gel electrophoresis (SDS-PAGE) showed one protein band at Mr 34,000. Analysis by gel filtration high performance liquid chromatography (HPLC) of reaction products from incubations of the protein fraction with laminarioligosaccharides of five glucosyl units or greater revealed a unique glucanosyl transferase activity. The enzyme specifically cleaved laminaribiaose (G2) from the reducing-end of a linear beta-(1-3)-glucan and transferred the remainder to another laminarioligosaccharide. The reaction with laminaripentaose (G5) produced G2 and a product eluting at the position of G8. Analysis of the latter transferase product by 13C- and 1H-nuclear magnetic resonance (NMR) spectroscopy shows it to be a branched molecule containing a beta-(1-3)-beta-(1-6)-branchpoint. It is suggested that the Mr 34,000 wall protein is a glucan branching enzyme, perhaps the key enzyme responsible for the transformation of the initial linear beta-(1-3)-glucan into the branched beta-(1-3)-beta-1-6)-glucan as found in the cell wall of C. albicans.     

DNA homology between Candida albicans strains: Evidence to justify the synonymous status of C. stellatoidea

Genetic relatedness between strains of C. albicans and C. stellatoidea was studied by measuring G + C content and overall sequence homology. G + C contents determined by high-performance liquid chromatography were 32.6 to 34.2% for 26 strains of C. albicans and 33.0 to 33.9% for eight strains of C. stellatoidea. DNA-DNA hybridization with two C. albicans and two C. stellatoidea probes revealed that all 34 test strains formed a single cluster in which the extents of hybridization with the heterologous probes ranged between 77.9 and 105.6% of those with the homologous probes. These results give support to the unification of C. albicans and C. stellatoidea into a single species.     

A Green Pigment from North Dakota Burial Mounds

Abstract

A clayey, green pigment was found in association with human interments in 3 mounds at the Boundary Mound site (32S11) on the Missouri River 16 miles downstream from Fort Yates, North Dakota.

Samples submitted to the Freer Gallery of Art, Washington, D.C. were identified by microscopic and chemical analysis as Greensand, a sedimentary deposit containing greenish grains of glauconite. The nearest present day exposure appears to be just south of Wessington Springs, in Jerauld County, South Dakota. This report is the first identifi - cation of greensand as a pigment mineral.     

A cyclic AMP-independent protein kinase from Candida albicans.

A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents.     

Microsomal chitinase activity from Candida albicans

Chitinase (E.C. 3.2.1.14) was characterized in microsomal fractions from yeast cells of Candida albicans. Following six washes with buffer (50 mM Bis-Tris.Cl, pH 6.5), enzyme activity of microsomes fell markedly to 0.3% of total and 6% of the specific activity detected in the low-speed supernatant (9000 X g) of a cell lysate. An apparently zymogenic, microsomal chitinase activity became more readily detectable with washing and after six washes enzyme activity was activated 1.7-fold following pre-incubation with trypsin. The following properties of microsomal chitinase were closely comparable with those for cytosolic chitinase (indicated in parentheses): Km = 2.1 mg chitin per ml (2.9 mg chitin per ml); temperature optimum = 45 degrees C (45 degrees C); inhibition by allosamidin competitive, Ki = 0.29 microM (competitive, Ki = 0.23 microM). A range of detergents solubilized and activated microsomal chitinase in a highly specific manner. Following density gradient centrifugation of microsomes, chitinase was distributed approximately evenly throughout the gradient suggesting that microsomal chitinase is not associated exclusively with any one membrane component. The possible morphogenetic role of microsomal chitinase is discussed in relation to the potential of this enzyme as a target for highly specific antifungal agents.     

Structure of the D-mannan of Candida stellatoidea IFO 1397 strain. Comparison with that of the phospho-D-mannan of Candida albicans NIH B-792 strain

The structure of the D-mannan of Candida stellatoidea IFO 1397 strain, which has properties identical to those of the phospho-D-mannan of C. albicans serotype B strain, does not contain phosphate groups, and its 1H- and 13C-n.m.r. spectra are quite similar to those of the phospho-D-mannan of C. albicans NIH B-792 strain. However, the 1H-n.m.r. and 1H-13C-correlation n.m.r. spectra of the products obtained by digestion with alpha-D-mannosidase of C. stellatoidea D-mannan considerably differed from those of the corresponding digestion products of the C. albicans phospho-D-mannan. Additionally, the enzyme-linked immunosorbent assay, by means of a monoclonal antibody corresponding to (1----2)-linked beta-D-oligomannosyl residues, of the phospho-D-mannan of the same C. albicans strain indicated that the C. stellatoidea D-mannan does not contain any (1----2)-linked beta-D-oligomannosyl residues. The absence of these residues may be used as one of the criteria of chemotaxonomical identification of C. stellatoidea spp.     

A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.The human fungal pathogen Candida albicans can cause severe infections, particularly in immunocompromised patients. Important insights into its pathogenesis have been obtained by analyzing fusions to green fluorescent protein (GFP) (8). Although GFP tagging has been very successful, many fusion proteins are not easily detected. New GFP variants with improved fluorescence and protein folding properties have been identified by genetic approaches in other organisms (2, 7, 8). However, these GFP variants have not been assessed in C. albicans and related species, presumably because of the added difficulties of attempting heterologous expression in C. albicans.To adapt GFP for effective use in C. albicans, Cormack et al. introduced three types of codon changes: the S65G S72A mutations to enhance fluorescence; the CTG codon 201 change to TTG, since CUG is translated as Ser instead of Leu in C. albicans; and the optimization of the other codons for translation in C. albicans (1). This variant, known as YeGFP3, was introduced into convenient vectors for creating gene fusions in C. albicans (4). Another version of eGFP known as mut2 (S65A V68L S72A Q80R) was adapted for C. albicans by changing the CTG codon but without further codon optimization (5). These obstacles to heterologous expression in C. albicans have presumably delayed implementation of newer versions of GFP. Therefore, in this study three different GFP variants were introduced into YeGFP3 and examined for function in C. albicans.The GFP variants were constructed using standard methods to introduce changes in the coding sequence of YeGFP3. In brief, mutagenic oligonucleotides were used to prime PCR synthesis of a plasmid carrying YeGFP3, the template DNA was then destroyed by digestion with DpnI, and then the resulting DNA was transformed into Escherichia coli. DNA sequencing (carried out by the Stony Brook University DNA Sequencing Facility) confirmed that the correct substitutions were present. The mutant GFP genes were then released as PstI-AscI fragments and then were subcloned to replace the corresponding GFP fragment of plasmid pFa-GFP-URA3 (6), which carries a PCR cassette module for creating GFP fusions in C. albicans. Because of the large number of changes, the mutants were given the more convenient names of CaGFPα (F64L S65T F99S M153T V163A), CaGFPβ (F64L S65T N149K M153T I167T; also known as emerald), and CaGFPγ (F64L S65C V163A I167T). The CaGFPγ was also introduced into vectors that contain selectable markers HIS1 and ARG4 (6). DNA sequences used to design primers for creating GFP fusions in C. albicans were as follows: forward primer, 5′ (region of homology)-GGTGCTGGCGCAGGTGCTTC-3′, and reverse primer, 5′ (region of homology)-TCTGATATCATCGATGAATTCGAG-3′.CDC11-GFP fusion genes were created in C. albicans by homologous recombination, as described previously (4, 6). In brief, long oligonucleotide primers with homology to the 3′ end of the CDC11 open reading frame were used to prime PCR synthesis of each of the corresponding GFP variant genes plus an adjacent selectable marker gene (URA3). These DNA elements were then introduced into C. albicans cells and allowed to recombine with the homologous region of the CDC11 gene in C. albicans to create the CDC11-GFP fusion genes. Sequences used for the design of PCR primers to amplify the pFa-GFP plasmids are shown above. Cells carrying the indicated CDC11-GFP fusion gene were grown overnight in log phase in synthetic medium (yeast nitrogen base plus amino acids and dextrose). Cdc11-GFP fluorescence intensity was analyzed with an Olympus BH2 microscope equipped with a Zeiss AxioCam camera run by Openlab software. The relative GFP signal was determined by measuring the intensity of GFP fluorescence of the septin ring and then subtracting the fluorescence of an area immediately adjacent to each ring. All samples were visualized under the same conditions.Samples were prepared for Western blot analysis by resuspending cells in TNE lysis buffer (10 mM Tris base, 1 mM EDTA, 100 mM NaCl) with 100× protease mix (40 mg/ml pepstatin A, 40 mg/ml aprotinin, 20 mg/ml leupeptin) and then agitating in the presence of glass beads. The supernatant was collected after low-speed centrifugation at 3,000 rpm for 1 min, protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce), and then equal amounts of protein extract were separated by gel electrophoresis and transferred to a Protran nitrocellulose membrane (Whatman GmbH). The blots were incubated with mouse anti-GFP (Millipore), rabbit anti-glucose-6-phosphate dehydrogenase (anti-G6PD; Sigma), or rabbit anti-Cdc11 (Santa Cruz Biotechnology) primary antibodies; washed; and then incubated with either goat anti-mouse IRDye 800cw or goat anti-rabbit IRDye 680 (Li-Cor Biosciences, Lincoln, NE). The immunoreactive proteins were visualized with a Li-Cor fluorescence scanner run by Odyssey software.Three new GFP variants based on YeGFP3 were constructed by introducing mutations predicted to improve either the fluorescence properties or protein folding (2, 7, 8). Because multiple changes were introduced into each variant, they were given the more convenient names of CaGFPα, CaGFPβ, and CaGFPγ (see above). The key mutations in CaGFPα and CaGFPβ have been described previously (2, 7, 8), but CaGFPγ represents a novel combination of mutations. The 3 new GFP variants plus the YeGFP3 and mut2 versions were compared by fusing them to the C terminus of the Cdc11 septin protein (3). The Cdc11 protein was selected because its restricted localization to the bud neck facilitated microscopic analysis and comparison of fluorescence properties. CDC11-GFP fusion genes were constructed in strain BWP17 (9) using PCR-generated modules with a URA3 selectable marker, as described previously (4, 6).Cells were grown in synthetic medium overnight to log phase at both 30°C and 37°C, temperatures that are commonly used to propagate C. albicans and that may affect the folding properties of GFP. GFP fluorescence was then analyzed by quantifying the intensity of the septin rings in digital images (Fig. ​(Fig.1A).1A). Septin rings were analyzed only if they were obviously in focus and at the same stage of the cell cycle (large budded). CaGFPγ gave a slightly stronger signal than the other variants, which was most obvious at 30°C (Fig. ​(Fig.1A).1A). At least two independent clones were analyzed for each CDC11-GFP variant, and the two gave similar results (data not shown).Open in a separate windowFIG. 1.Properties of Cdc11-GFP fusion proteins. Cells were grown to log phase overnight at the indicated temperature, and then Cdc11-GFP fluorescence was analyzed. (A) Signal intensity for the different versions of Cdc11-GFP was compared in three independent assays in which 50 septin rings per assay were quantified for each different Cdc11-GFP. The average fluorescence intensity was normalized to 100 for Cdc11-YeGPF3. The Cdc11-CaGFPγ variant gave a significantly stronger signal than the other variants (P < 0.001). (B) Western blot analysis comparing the levels of Cdc11-GFP produced in the indicated strains. The lane labeled “neg” refers to the negative-control strain (BWP17) that lacks GFP. Blots were probed with anti-GFP to detect Cdc11-GFP, anti-glucose-6-phosphate dehydrogenase (αG6PD) as a control, and anti-Cdc11 to detect the untagged version of Cdc11.The levels of the Cdc11-GFP proteins at both 30°C and 37°C were compared on two independent Western blots using anti-GFP antibody (Fig. ​(Fig.1B).1B). The relative levels of Cdc11-mut2GFP and Cdc11-CaGFPα were the lowest, consistent with their lower fluorescence intensity. The lower levels of Cdc11-mut2GFP are consistent with the fact that the codons in the mut2 version of GFP were not optimized for expression in C. albicans (5). The Cdc11-YeGFP3 and Cdc11-CaGFPγ were present at higher levels, and the Cdc11-CaGFPβ was produced at even slightly higher levels, consistent with reports that this latter version of GFP (also known as emerald) has improved folding properties (7). The Cdc11-GFP variants did not affect the production of the untagged Cdc11 protein (Fig. ​(Fig.1B1B).Photobleaching is also an important factor for GFP (7), especially in time-lapse studies or Z-stack analysis of different optical sections of cells. Photostability of the GFP variants was examined by taking pictures at 4-s intervals during 1 min of continuous exposure to the fluorescence excitation lamp (Fig. 2A and B). The fluorescence of YeGFP3, mut2GFP, and CaGFPα fused to Cdc11 decayed to 50% of original intensity within 15 to 30 s, and the rate of photobleaching was even higher for CaGFPβ. In contrast, Cdc11-CaGFPγ showed extended photostability at both 30°C and 37°C (half-life [t_1/2] of ∼2 min). Similar results were also obtained for CaGFPγ fused to the Golgi protein Vrg4 (data not shown), although the standard deviations were larger because the mobile Golgi compartments frequently moved out of the focal plane during the time course (data not shown). On a practical level, the Cdc11-GFPγ fluorescence was readily detectable after several minutes of continuous exposure (Fig. ​(Fig.2C),2C), demonstrating its clear advantage for allowing more time to observe protein localization before photobleaching becomes significant.Open in a separate windowFIG. 2.Photostability of GFP variants. (A and B) Relative fluorescence intensity of the GFP variants at 4-s intervals over a time course of 1 min of continuous exposure to the fluorescence excitation lamp after growth at 30°C (A) and at 37°C (B). CaGFPγ showed the best photostability (t_1/2 of ∼2 min). The relative fluorescence was normalized to 100 for each Cdc11-GFP variant at the start of the time course. The results represent the average of three independent assays in which three septin rings were analyzed for each mutant. Error bars indicate standard deviations. (C) Cells carrying Cdc11 fused to YeGFP3 or CaGFPγ were continuously exposed to the fluorescence excitation lamp, and then images of septin rings were captured at the indicated times.Altogether, Cdc11-CaGFPγ had the best overall properties based on protein levels, signal intensity, and photostability in C. albicans. The higher level of Cdc11-CaGFPβ production was apparently offset by increased photobleaching, resulting in no overall advantage for this variant. The Cdc11-CaGFPα was produced at relatively low levels, and it was less photostable compared to the other versions. Thus, CaGFPγ is a novel GFP variant that offers improved features for the study of protein localization in C. albicans and will likely also be useful for expression in other species.     

A dihydrofolate reductase gene from Candida albicans: molecular cloning

The dihydrofolate reductase gene from Candida albicans has been cloned and partially characterized. A genomic bank from C. albicans strain 10127/5 was constructed in Escherichia coli and screened for trimethoprim resistance. A plasmid pMF1, carrying the resistance marker was isolated and characterized by restriction mapping and Southern blotting. Cells harbouring pMF1 were as sensitive as the parental cells to a wide spectrum of antibacterial agents, except for trimethoprim; the dihydrofolate reductase activity from these cells was trimethoprim resistant.     

A glucose sensor in Candida albicans

The Hgt4 protein of Candida albicans (orf19.5962) is orthologous to the Snf3 and Rgt2 glucose sensors of Saccharomyces cerevisiae that govern sugar acquisition by regulating the expression of genes encoding hexose transporters. We found that HGT4 is required for glucose induction of the expression of HGT12, HXT10, and HGT7, which encode apparent hexose transporters in C. albicans. An hgt4Delta mutant is defective for growth on fermentable sugars, which is consistent with the idea that Hgt4 is a sensor of glucose and similar sugars. Hgt4 appears to be sensitive to glucose levels similar to those in human serum ( approximately 5 mM). HGT4 expression is repressed by high levels of glucose, which is consistent with the idea that it encodes a high-affinity sugar sensor. Glucose sensing through Hgt4 affects the yeast-to-hyphal morphological switch of C. albicans cells: hgt4Delta mutants are hypofilamented, and a constitutively signaling form of Hgt4 confers hyperfilamentation of cells. The hgt4Delta mutant is less virulent than wild-type cells in a mouse model of disseminated candidiasis. These results suggest that Hgt4 is a high-affinity glucose sensor that contributes to the virulence of C. albicans.