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B L Wickes

Publications and source records attributed to B L Wickes.

36 records · Page 2Linked to original sources
PubMed2000

Direct PCR of Cryptococcus neoformans MATalpha and MATa pheromones to determine mating type, ploidy, and variety: a tool for epidemiological and molecular pathogenesis studies.

Cryptococcus neoformans MATalpha and MATa pheromones were amplified by direct PCR. Nucleotide sequence analyses revealed unique restriction enzyme sites. Sixty strains were used to devise a restriction fragment length polymorphism typing scheme that yielded three variety-specific patterns. Additionally, pheromone-specific PCR allowed easier identification of diploid C. neoformans strains than flow cytometry.

Cryptococcosis↗
PubMed1997

The Cryptococcus neoformans STE12alpha gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific.

Cryptococcus neoformans possesses two mating types, MATalpha and MATa. Alpha-Cells are more virulent than a-cells and are also, unlike a-cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFalpha, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12alpha gene, however, has one striking difference from other fungal STE12 genes; it is found only in alpha-cells. The existence of STE12alpha in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

Amino Acid Sequence↗
PubMed1996

Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type.

Cryptococcus neoformans is a major opportunistic fungal pathogen in AIDS and other immunosuppressed patients. We have shown that wild-type haploid C. neoformans can develop an extensive hyphal phase under appropriate conditions. Hyphae produced under these conditions are monokaryotic, possess unfused clamp connections, and develop basidia with viable basidiospores. The ability to undergo this transition is determined by the presence of the alpha-mating type locus and is independent of serotype. The association of the hyphal phase with the alpha-mating type may explain the preponderance of this mating type in the environment and the nature of the infectious propagule of C. neoformans.

AIDS-Related Opportunistic Infections↗
PubMed1996

Genomic variation in C. albicans.

Candida albicans displays many types of variation which affect a broad spectrum of phenotypes. Among them are antigenic, chromosomal, morphologic, and biochemical variation. The ability to modulate many phenotypes is clearly an important factor in the success of this fungus as a pathogen and variation at the genomic level may be the common denominator among the different systems. Genomic variation in C. albicans has been studied by many researchers and a number of different mechanisms have been identified. Among them are ploidy fluctuations, which allow the organism to cycle from 2n chromosome number to 4n or higher; translocation, which has been demonstrated to involve many different chromosomes and affects many phenotypes including virulence; mitotic recombination, which has been demonstrated to increase resistance to certain drugs; and nondisjunction, which has been shown to have morphological consequences. The number and diversity of these mechanisms combine to make C. albicans a highly successful organism. Although normally a commensal of humans, when invasive, C. albicans can inhabit almost any site in the body. It is not known what governs the transition of C. albicans from a commensal to pathogenic invader, however, variation at the genomic level likely plays a role. One possible consequence of variation is the generation of atypical strains, further expanding the documented phenotypic plasticity of this organism. The exposure of patients to cytotoxic drugs during treatment of such diseases as AIDS or cancer increases the selective pressure and has exacerbated both the frequency and degree of variability observed in C. albicans. The molecular analysis of genomic variation in C. albicans is proving to be a fertile area of research and future investigations can only be expected to add to the mechanisms documented in this review.

Candida albicans↗
PubMed1995

Further analysis of the CAP59 locus of Cryptococcus neoformans: structure defined by forced expression and description of a new ribosomal protein-encoding gene.

Cryptococcus neoformans (Cn) produces an extracellular polysaccharide capsule that is an essential factor for virulence. We previously isolated a gene, CAP59, which is necessary for capsule formation. To dissect the functional region of CAP59, we placed it under control of the Cn GAL7 promoter (pGAL7). Among the several pGAL7::CAP59 fusion constructs, only the one containing the entire open reading frame of CAP59 was able to complement the acapsular phenotype under galactose induction. A missense mutation in the coding region abolished complementation by the fusion construct. We also found that the CAP59 locus is contiguous to a convergently transcribed L27 ribosomal protein-encoding gene (CL27). The distance between the cDNA ends of these two genes is only 25 bp. CL27 has two introns near its N terminus. The translated CL27 protein is 183 amino acids (aa) in length with an estimated molecular mass of 20 kDa, and the first 34 aa at the N terminus may be a targeting peptide for mitochondria. A high degree of restriction-fragment-length polymorphism was detected in the DNA sequence containing CAP59 and CL27.

Amino Acid Sequence↗
PubMed1995

The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter.

A Cryptococcus neoformans galactose auxotroph was created by ultraviolet light mutagenesis and complemented with a C. neoformans genomic library. The translated sequence of the complementing DNA revealed a high degree of similarity to a number of UDP glucose-D-galactose-1-phosphate uridylyltransferases. Expression of C. neoformans GAL7 mRNA followed a pattern similar to Saccharomyces cerevisiae expression; it was first observed within 2.5 min of induction and fully induced by 30 min. The gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two genes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MF alpha, which encodes a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, beta-glucuronidase (gusA), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7p::GUS fusion was used to quantify inducibility of the GAL7 promoter, the level of enzyme activity was at least 500-fold greater for cells grown in galactose than for cells grown in glucose. The GAL7 promoter is the first inducible promoter characterized in C. neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.

Amino Acid Sequence↗
PubMed1994

Comparison of the electrophoretic karyotypes and chromosomal location of ten genes in the two varieties of Cryptococcus neoformans.

We compared multiple isolates of the two varieties of Cryptococcus neoformans, as well as previously characterized representative isolates, for their electrophoretic karyotypes using pulsed-field electrophoresis. The two varieties could be clearly distinguished based upon the size of the smallest chromosome. The smallest chromosome for isolates of the gattii variety (serotypes B and C) was found to be 400-700 kb in size. The smallest chromosome for isolates of the neoformans variety was consistently found to be larger, approximately 770 kb in size. Isolates of the gattii variety averaged 13 chromosomes while the neoformans variety averaged 12. The size of the Cryptococcus genome was found to be approximately 23 megabases. Isolates of C. neoformans var. neoformans tended to be more conserved than those of var. gattii with regard to gene position.

Chromosomes, Fungal↗
PubMed1992

The molecular analysis of synonymy among medically important yeasts within the genus Candida.

Three sets of medically important yeasts, Candida albicans, C. tropicalis, and C. krusei, were compared with their putative synonyms (C. langeronii and C. claussenii, C. paratropicalis, and Itssatchenkia orientalis, respectively) to determine if these synonyms are genetically distinguishable from each other. Pulsed-field electrophoresis and hybridization to species-specific probes were used to accomplish this goal. The species-specific probes for C. albicans and C. tropicalis have been previously described (27A and CT13.8, respectively) whereas the probe for C. krusei (CK3) was cloned in this study. No distinguishing characteristics between synonyms were identified, thus supporting the current taxonomic treatment of these organisms.

Candida↗
PubMed1992

Genetic association of mating types and virulence in Cryptococcus neoformans.

A pair of congenic Cryptococcus neoformans var. neoformans strains, B-4476 (a mating type) and B-4500 (alpha mating type), that presumably differ only in mating type was constructed. This pair and their progeny, five alpha type and five a type, were tested for virulence in mice. In the parent strains as well as the progeny, alpha type was clearly more virulent than a type. In addition, death tended to occur earlier among the alpha-strain-infected mice that died than among the mice that died by infection caused by a strains. These data strongly suggest the genetic association of virulence with mating type in this human fungal pathogen.

Animals↗
PubMed1992

Virulence, serotype, and molecular characteristics of environmental strains of Cryptococcus neoformans var. gattii.

Four strains of Cryptococcus neoformans var. gattii originating from Eucalyptus camaldulensis, three from Australia and one from San Francisco, were tested for their serotype, virulence for mice, and a number of genetic and molecular characteristics. All were found to be serotype B and showed significantly higher virulence for mice than did the type strains of C. neoformans var. gattii and Filobasidiella neoformans var. bacillispora, which were obtained from human cryptococcosis cases. Electrophoretic karyotypes of the strains from Australia were identical, although they were collected from sites at least 15 to 500 km apart. The electrophoretic karyotype of the strain from San Francisco was the same as that of the Australian isolates except for the mobility of one chromosome. On the contrary, no two isolates of serotype B (of a total of 11) from clinical sources were the same, regardless of their geographic origin. Furthermore, none of the clinical isolates showed a chromosomal banding pattern identical to that of Eucalyptus-originated strains. The Eucalyptus-originated strains failed to form dikaryons when crossed with the tester strains of the two varieties of F. neoformans. Hybridization analysis with a nucleic acid probe (AccuProbe C. neoformans Culture Confirmation Test; Gen-Probe Inc., San Diego, Calif.), however, showed signals of equal intensity for clinical strains and the Eucalyptus-originated strains. Various fungi phylogenetically related to C. neoformans, including a phenol oxidase-positive strain of Cryptococcus laurentii obtained from E. camaldulensis, were negative in the nucleic acid hybridization test. These observations confirm that, in spite of karyotypic differences and the lack of dikaryon formation with the tester strains of F. neoformans, Eucalyptus-originated C. neoformans var. gattii is the same organism as those isolated from cases of human infection. Furthermore, the C. neoformans culture confirmation test using a commercial nucleic acid probe is specific for C. neoformans.

Animals↗
PubMed1991

Chromosomal rearrangement in Candida stellatoidea results in a positive effect on phenotype.

When type I Candida stellatoidea is plated onto sucrose agar at levels in excess of 10(8) cells, some isolates spontaneously form sucrose-positive colonies. These isolates do not display typical type I phenotypes but instead exhibit phenotypes intermediate between type I C. stellatoidea and C. albicans. Also, this phenotypic change only occurs in conjunction with a chromosomal rearrangement. These rearrangements have been studied in a strain naturally marked for methionine auxotrophy. Chromosome-size DNA bands separated by pulsed-field gel electrophoresis were probed with genes cloned from C. albicans. The hybridization pattern indicated that the genes on several chromosomes underwent extensive rearrangement.

Candida↗
PubMed1990

Is Candida stellatoidea disappearing from the vaginal mucosa?

A total of 681 vaginal isolates of germ tube-positive or germ tube-untested white, yeastlike fungi obtained from patients in various cities of the United States were tested for the presence of Candida stellatoidea (type I). Only 1 of the 681 isolates was identified as C. stellatoidea.

Candida↗
PubMed1989

Genetic differences between type I and type II Candida stellatoidea.

Genetic similarities and differences between type I and type II Candida stellatoidea were studied. The electrophoretic karyotype, mitochondrial DNA (mtDNA) restriction patterns, and midrepeat sequence of nuclear DNA in type I C. stellatoidea were clearly distinguishable from those of a reference culture of Candida albicans. The karyotype and the major bands of the midrepeat sequence of type II C. stellatoidea were indistinguishable from those of the reference C. albicans. The mtDNA restriction patterns of four type I isolates were homogeneous regardless of the endonucleases and probes used. The mtDNA restriction patterns of type II C. stellatoidea varied from strain to strain. Some of them were identical to that of C. albicans, while others were the same as that of type I C. stellatoidea. Immunofluorescence with C. albicans serotype A-specific monoclonal antibody indicated that the four isolates of type I C. stellatoidea were serotype B (non-A), whereas all three type II isolates studied were serotype A. Taken together, these results support the hypothesis that the isolates of C. stellatoidea type II studied are sucrose-negative mutants of serotype A C. albicans. Since C. stellatoidea type I differs from C. albicans in several major genetic characteristics, it cannot be viewed as a simple mutant derived from C. albicans. Hybrids produced by protoplast fusion of type I and type II cells were capable of assimilating sucrose, indicating that the sucrose-negative phenotypes of the parents are due to different mutations.

Candida↗
PubMed1989

Taxonomic clarification of Cladosporium trichoides Emmons and its subsequent synonyms.

Cladosporium trichoides Emmons has been treated by some mycologists as a synonym of Cladosporium bantianum (Sacc.) Borelli and has been transferred to the genus Xylohypha (Fr.) Mason. In the present study, a herbarium specimen of C. bantianum (Torula bantiana Sacc.) Borelli, prepared by Saccardo, was compared with a herbarium specimen and a living type culture of C. triochoides by light and scanning electron microscopy (SEM) and was found to be dissimilar. Herbarium specimens and living cultures of Xylohypha nigrescens, the type species of the genus Xylohypha, were also compared with those of C. trichoides and other pathogenic Cladosporium species. Fundamental differences were found between X. nigrescens and Cladosporium species, in colony morphology, manner of sporulation and conidial morphology. All Cladosporium isolates produced olive-black colonies regardless of environmental conditions, bore brown pigment on the walls of the vegetative hyphae as well as on the walls of the fruiting structures and produced branched chains of conidia either from well differentiated or poorly differentiated conidiophores, or directly from the hyphae. By SEM, conidia showed strong to moderately protruded hila, and the basal contour of the conidia was always truncated. On germination, hyphal tubes were produced randomly from the surface of the conidia. In contrast, X. nigrescens produced white colonies with or without brown centres, depending on the culture medium, bore pigment on the conidial walls and on conidiogenous cells but not on the vegetative hyphae and produced infrequently branched conidial chains, usually from intercalary conidiogenous cells which were globose to hat-shaped. Conidial hila were nonprotruding but, instead, were deeply concave and pore-like. The basal contour of the conidia was round and germ tubes were produced only from the pore-like hila. These results indicate that C. triochoides Emmons is different from C. bantianum (Sacc.) Borelli and that the reclassification of C. trichoides into the genus Xylohypha was not warranted.

Animals↗
PubMed1988

Association of electrophoretic karyotype of Candida stellatoidea with virulence for mice.

Seven isolates of Candida stellatoidea were studied for their electrophoretic karyotype, virulence for mice, sensitivity to UV radiation, growth rate in vitro, reaction on cycloheximide-indicator medium, and proteinase activity. The isolates exhibited one of two distinct electrophoretic karyotypes as determined by orthogonal field alternating gel electrophoresis (OFAGE). Four isolates, including the type culture of C. stellatoidea, belonged to electrophoretic karyotype type I by OFAGE, showing eight to nine bands of which at least two bands were less than 1,000 kilobases in size as estimated by comparison with the DNA bands of Saccharomyces cerevisiae. These isolates failed to produce fatal infection in mice within 20 days when 5 X 10(5) cells were injected intravenously. The yeasts were cleared from the kidneys of two of three mice tested by day 30. Type I showed proteinase activity on bovine serum albumin agar at pH 3.8 and produced a negative reaction on cycloheximide-bromcresol green medium within 48 h. The three grouped in type II by OFAGE showed banding patterns similar to those of a well-characterized isolate of Candida albicans. The isolates of type II had an electrophoretic karyotype of six to seven bands approximately 1,200 kilobases or greater in size. All three type II isolates were highly virulent for mice, producing fatality curves similar to those of a previously studied C. albicans isolate. From 80 to 90% of the mice injected with 5 X 10(5) cells intravenously died within 20 days. The type II isolates produced a positive reaction on cycloheximide-bromcresol green agar and showed no proteinase activity on bovine serum albumin agar at the low pH. In addition, the type II isolates grew faster and were significantly more resistant to UV irradiation than the type I isolates. These results indicated that type II, but not type I, isolates can be considered simply as sucrose-negative C. albicans.

Animals↗
PubMed1987

Ploidy and DNA content of Candida stellatoidea cells.

Three isolates of Candida stellatoidea contained approximately the same amount of DNA per blastospore (38.3 to 41.9 fg) as did a known diploid isolate of Candida albicans and about twice as much as did a haploid isolate of Saccharomyces cerevisiae. The diploidy of C. stellatoidea was supported by demonstration of mitotic segregation of an ade marker.

Candida↗
PubMed1987

Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans (74 isolates) and C. neoformans var. gattii (44 isolates) were used to test urease activity after growth on both yeast extract-glucose-peptone agar (YEPG) and on YEPG supplemented with 100 microM EDTA. Every isolate grown on YEPG agar for 48 h at 30 degrees C produced a positive reaction within 1 h in a modified rapid urease assay at 37 degrees C. However, isolates grown on YEPG with 100 microM EDTA showed a distinct pattern which corresponded to their varietal status. All but 1 of 74 C. neoformans var. neoformans isolates (98.7%) produced a positive reaction within 1 to 4 h, while none of 44 C. neoformans var. gattii isolates produced a positive reaction within the same period. The urease inhibition results and the canavanine-glycine-bromthymol blue agar test results showed 100% correlation among isolates of C. neoformans var. gattii and 98.7% correlation among isolates of C. neoformans var. neoformans. Two representative isolates of C. neoformans var. gattii (serotypes B and C) were further tested for urease during a prolonged incubation period in urea broth. These isolates failed to show a positive reaction even after 11 h of incubation. The uptake of EDTA was negligible in the two varieties. Extracts of cells grown on YEPA agar showed a high level of urease activity in both varieties. Extracts of cells grown on the agar with 100 microM EDTA showed a marked reduction (86%) of urease activity in one isolate of C. neoformans var. gattii but showed only a 30% reduction in one isolate of C. neoformans var. neoformans. Based on these results, the differential effect of EDTA on the two varieties of C. neoformans appeared to be due to greater inhibition of urease synthesis in C. neoformans var. gattii.

Cryptococcus↗