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K J Kwon-Chung

Publications and source records attributed to K J Kwon-Chung.

At least 73 records · Page 4Linked to original sources

Phylogenetic spectrum of fungi that are pathogenic to humans.

Recent phylogenetic studies based on ribosomal RNA sequences have confirmed that the organisms traditionally treated as fungi include those that have evolved from several different lines (multiphyletic organisms), as has been suspected. Even organisms causing disease in humans represent at least two evolutional lines. Pythium insidiosum and Prototheca species are both believed to have evolved from one line, while the rest of the pathogens have evolved from another line. P. insidiosum is more closely related to red algae and diatoms than to fungi. Prototheca species, as has been previously postulated, are closer to blue-green algae and plants than to fungi. Pythiosis and protothecosis, however, will still be dealt with by medical mycologists because of the morphological and in vivo staining characteristics of the causative organisms. Molecular genetic studies have revealed that Pneumocystis carinii can best be categorized as a fungus, although questions regarding its fungal status may remain unanswered until additional information becomes available on its life cycle, nuclear division, cell-wall chemistry, nutritional uptake pattern, and lysine biosynthetic pathway as well as the ultrastructural characteristics of its cellular components such as the Golgi complex. The phylogeny of the agents of lobomycosis and rhinosporidiosis, although they are treated as fungi, remains unknown. Although there is no in vitro culture system for Loboa loboi and Rhinosporidium seeberi at present, a molecular approach would allow us to reveal their phylogenetic relationship, and we can hope that such attempts are forthcoming.

DNA, Fungal↗

Gene arrangement and sequence of the 5S rRNA in Filobasidiella neoformans (Cryptococcus neoformans) as a phylogenetic indicator.

We cloned the 5S rRNA gene and determined its organization in the four genes encoding rRNAs in a ribosomal DNA repeat unit of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans. The 5S rRNA gene contained 118 nucleotides and was located 1 kb upstream from the 18S rRNA gene within the 8.6-kb fragment of the ribosomal DNA repeat unit. The sequence of the 5S rRNA gene from F. neoformans was more similar to the sequence of the 5S rRNA gene from Tremella mesenterica than to the sequences of the 5S rRNA genes from Filobasidium species. The arrangement of the rRNA genes in F. neoformans closely resembles the arrangement of the rRNA genes in mushrooms such as Schizophyllum commune, Agaricus bisporus, and Coprinus cinereus in that the 5S rRNA-coding region not only is located within the repeat unit that encodes the other rRNAs but also is transcribed in the same direction as the other rRNA genes. This is the first description of the arrangement of rRNA genes in a species belonging to the Heterobasidiomycetes.

Base Sequence↗

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↗

Epidemiology studies of clinical isolates of Cryptococcus neoformans of Japan by restriction fragment length polymorphism.

Previous epidemiological studies of Cryptococcus neoformans infection in Japan showed that only C. neoformans var. neoformans is present and serotype A is the most common with frequencies in excess of 95%. A DNA fingerprinting method, using a genomic DNA probe (UT-4p), has become available recently which discriminates between individual isolates in a population that are morphologically and serologically indistinguishable. Fifty-two serotype A isolates of C. neoformans were obtained from three different institutions (in Nagasaki, Chiba, and Tokyo) in Japan. Only two of these strains were isolated from AIDS patients and one from pigeon excreta. Of the nine reported finger-printing patterns in serotype A, only three types (IV, V and VII) were observed in Japanese isolates. Pattern IV was almost exclusively observed in Nagasaki isolates (21/22) with only one of pattern VII. In Chiba, however, patterns VII and IV appeared to be equally distributed. In Tokyo, patterns IV and V (which included two isolates from AIDS patients) were observed at similar frequencies. Restriction fragment length polymorphism analysis of four isolates of serotype AD showed a typical serotype A pattern which also contained a serotype D-specific band. This finding suggests the independence of serotype AD. These data could enhance the survey of the epidemiology of cryptococcosis.

Cryptococcosis↗

HYP1, a hydrophobin gene from Aspergillus fumigatus, complements the rodletless phenotype in Aspergillus nidulans.

Aspergillus fumigatus produces conidia that are highly dispersable and resistant to degradation. We have sought to analyze these properties by studying the rodlets which form the outer spore coat protein. Degenerate primers based on hydrophobins in other fungi were applied to genomic DNA from A. fumigatus in PCR. A product of this reaction with similarity to an Aspergillus nidulans gene as judged by Southern hybridization was chosen for further study. Cloning and sequencing revealed a gene with two introns which encodes a protein of 159 amino acids. Structural characteristics consistent with those of other fungal hydrophobin genes, especially conserved cysteine residues, are present. The expression of the gene is limited to the developmental stages in which maturing conidiophores are present. This A. fumigatus gene, HYP1, was used to transform a mutant strain of A. nidulans that lacks rodlets. Transformants with a single copy of HYP1 expressed a rodlet layer on their conidia as observed by freeze-fracture electron microscopy.

Amino Acid Sequence↗

Double-stranded RNA virus in the human pathogenic fungus Blastomyces dermatitidis.

Double-stranded RNA viruses were detected in a strain of Blastomyces dermatitidis isolated from a patient in Uganda. The viral particles are spherical (mostly 44 to 50 nm in diameter) and consist of about 25% double-stranded RNA (5 kb) and 75% protein (90 kDa). The virus contains transcriptional RNA polymerase activity; it synthesized single-stranded RNA in vitro in a conservative manner. The newly synthesized single-stranded RNA was a full-length strand, and the rate of chain elongation was approximately 170 nucleotides per min. The virus-containing strain shows no morphological difference from virus-free strains in the mycelial phase. Although the association with the presence of the virus is unclear, the virus-infected strain converts to the yeast form at 37 degrees C, but the yeast cells fail to multiply at that temperature.

Blastomyces↗

Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.

Amino Acid Sequence↗

Structural variability in the glucuronoxylomannan of Cryptococcus neoformans serotype A isolates determined by 13C NMR spectroscopy.

Cryptococcus neoformans, the etiologic agent of cryptococcal meningoencephalitis, produces glucuronoxylomannan (GXM) as the major capsule component. Purified GXMs obtained from eight serotype A isolates of C. neoformans were treated by ultrasonic irradiation and then O-deacetylated prior to their comprehensive chemical analysis by GLC, GLC-MS, and 13C NMR spectroscopy. The average xylose: mannose: glucuronic acid molar ratio of the eight isolates is 1.96 +/- 0.25: 3.00: 0.58 +/- 0.10. Methylation analyses and 13C NMR spectroscopy show a general structure for GXM that is comprised of a linear (1----3)-alpha-D-mannopyranan substituted with beta-D-GlcpA and with beta-D-Xylp at O-2. Variable quantities of unsubstituted (1----3)-alpha-D-Manp were observed between the eight isolates studied. In several isolates some of the (1----3)-alpha-D-Manp residues are disubstituted with beta-D-GlcpA at O-2 and with beta-D-Xylp at O-4; this type of substitution was not previously thought to occur in serotype A isolates. Heterogeneity, between isolates, in the disposition of the substituents along the mannopyranan backbone was revealed by 13C NMR spectroscopy. The eight isolates, and three isolates previously studied, were each assigned to one of four distinct groups based on the 13C NMR chemical shifts of the anomeric carbons. Six of the eleven isolates gave identical spectra (Group I). The six major anomeric resonances from Group I were assigned to specific glycosidic linkages present in GXM. The remaining five isolates gave more complex spectra that are indicative of additional linkages and comprise the remaining three groups. Three of these five isolates contain substantial amounts of linkages previously thought to be distinctive of serotypes B and C, i.e., Manp residues that are 4-O-glycosylated with beta-D-Xylp. Methylation analyses only predicted an average repeating unit, whereas 13C NMR spectroscopy demonstrated that GXM from each isolate may be categorized into four groups by the occurrence of distinct sequences of carbohydrate residues.

Carbohydrate Sequence↗

Successful treatment of systemic Exophiala dermatitidis infection in a patient with chronic granulomatous disease.

Exophiala dermatitidis, one of the saprophytic dematiaceous fungi, is a rare cause of human infection that, when invasive, is nearly always fatal. Besides the more common subcutaneous infection usually caused by traumatic inoculation, infection can also spread hematogenously, in which case the organism has a distinct neurotropism. A patient with autosomal recessive chronic granulomatous disease of childhood who was found to have a progressive pulmonary and central nervous system infection with E. dermatitidis responded to an aggressive, multifaceted therapeutic approach. Scanning electron microscopy of the cultured conidiogenous cells confirmed that the manner of conidiogenesis is typical of the genus Exophiala. We report the first successful treatment of an infection involving the lungs and central nervous system by a combination of surgical resection of the pulmonary source and medical therapy with amphotericin B, flucytosine or ketoconazole, and transfused white cells, followed by a prolonged course of fluconazole.

Adult↗

Successful treatment of Paecilomyces varioti infection in a patient with chronic granulomatous disease and a review of Paecilomyces species infections.

An 8-year-old boy who had chronic granulomatous disease developed a soft tissue infection of the right heel after riding on a motor scooter. Infection was insidious, and minor heel pain and fevers occurred only on the day interferon-gamma was injected. Soft tissue biopsy showed hyphal elements, and Paecilomyces varioti grew in culture. The infection was treated with amphotericin B for 7 weeks (total dose, 40 mg/kg) followed by 1 year of therapy with itraconazole (100 mg twice daily). Complete cure was achieved during the follow-up period of 10 months.

Amphotericin B↗

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↗

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↗

Molecular and genetic analysis of URA5 transformants of Cryptococcus neoformans.

Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.

Chromosome Mapping↗

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↗

A zinc finger protein from Candida albicans is involved in sucrose utilization.

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.

Amino Acid Sequence↗

DNA probe for strain typing of Cryptococcus neoformans.

A 7-kb linear plasmid, harbored by a URA5 transformant, hybridized to all the chromosomes of Cryptococcus neoformans separated by contour-clamped homogeneous electric field electrophoresis. Its linear maintenance was determined to have been facilitated by the presence of telomere-like sequences at its free ends. Hybridization of this plasmid to AccI-digested genomic DNAs of 26 C. neoformans strains generated 21 unique DNA fingerprints. The DNA fingerprints of isolates within the same serotype were more similar to one another than to those from different serotypes. An acapsular clinical isolate, strain 602, widely used in immunological studies and previously thought to be in serotype D, showed DNA fingerprints typical of serotype A isolates. Isogenic strains of C. neoformans exhibited DNA fingerprints that were identical to one another. The DNA fingerprints were stable and reproducible in spite of repeated transfers in the laboratory on either complex (1% yeast extract, 2% Bacto Peptone, 2% glucose) or minimal (yeast nitrogen base) medium. The DNA fingerprints of isolates recovered from primary blood and cerebrospinal fluid cultures of patients for whom AIDS had been diagnosed showed that the original infection in each of these patients contained a homogeneous population of C. neoformans. The DNA fingerprints of isolates recovered from different tissues of infected mice and from patients undergoing different drug therapy regimens were also found to be very stable.

AIDS-Related Opportunistic Infections↗