Biomedical subjects

K J Kwon-Chung

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

At least 37 records · Page 2Linked to original sources

PubMed2000

A novel episomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive selection marker in bacteria.

We report the engineering of a new shuttle vector featuring its episomal maintenance in Cryptococcus neoformans and the lethal Escherichia coli ccdB gene for positive selection in bacteria. Telomere-like sequences from C. neoformans and the STAB fragment confer episomal maintenance to the vector (pPM8) upon transformation in C. neoformans. The vector generated high transformation frequencies and each transformant was estimated to harbor thirty copies of the plasmid. The plasmids recovered in E. coli from the C. neoformans transformants showed no evidence of rearrangement. This construct will be very useful for cloning and studying the regulation of genes in C. neoformans.

Bacterial Proteins↗

PubMed2000

Cryptococcus neoformans STE12alpha regulates virulence but is not essential for mating.

The Cryptococcus neoformans STE12alpha gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)alpha cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12alpha. The ability to form hyphae, however, was not affected by deletion of STE12alpha when convergently growing MATa strains were present. Furthermore, ste12alpha disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12alpha disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12alpha locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12alpha cells than those of mice infected with STE12alpha cells. Using reporter gene constructs, we found that STE12alpha controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans.

Animals↗

PubMed2000

Characterization of the L41 gene in Cryptococcus neoformans: its application as a selectable transformation marker for cycloheximide resistance.

A transformation system using resistance to the antibiotic cycloheximide as a dominant selectable marker was developed for the pathogenic yeast Cryptococcus neoformans. A 3.5 kb DNA fragment containing a gene encoding the ribosomal protein L41 was cloned from a wild-type strain of C. neoformans which is sensitive to cycloheximide. The open reading frame of the L41 gene contains five introns and encodes a protein of 107 amino acids, which is similar to those reported for other yeasts. The cycloheximide resistance gene to be used as a marker was constructed by replacing a DNA segment of the wild-type L41 gene, which contained the amino acid proline at its 56th position with a homologous DNA segment from a mutant strain resistant to cycloheximide that contained leucine in that position. Cycloheximide resistant transformants were obtained by electroporation on YEPD plates, supplemented with 10-20 microg/ml cycloheximide, at a maximum efficiency of 300 transformants/microg plasmid DNA. While with other genes, most transformants of serotype D in C. neoformans maintain the transforming DNA as episomes, the cycloheximide-resistant transformants were all the result of ectopic genomic integration events.

Amino Acid Sequence↗

PubMed2000

Mapping of the Cryptococcus neoformans MATalpha locus: presence of mating type-specific mitogen-activated protein kinase cascade homologs.

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.

Chemoreceptor Cells↗

PubMed2000

Temperature-sensitive strain of Cryptococcus neoformans producing hyphal elements in a feline nasal granuloma.

We report the isolation of a temperature-sensitive, serotype A, mating type alpha strain of Cryptococcus neoformans from a case of nasal cryptococcosis in a cat. The strain grew extremely slowly at 35 degrees C and failed to grow at 37 degrees C in vitro. Histopathological sections of the infected tissue revealed yeast cells producing hyphae up to several hundred micrometers in length, in addition to numerous encapsulated yeast cells typical of C. neoformans. The cultures grown on yeast extract-peptone-glucose agar at 35 degrees C also produced some yeast cells with germ tube-like hyphal elements up to 100 microm in length.

Animals↗

PubMed2000

Cryptococcosis: clinical and biological aspects.

The incidence of cryptococcosis rose dramatically with the advent of the acquired immune deficiency syndrome (AIDS) epidemic in the early 1980s until the early 1990s. The frequency of cryptococcosis has been declining since mid 1990s in Europe and America due to the development of more effective antiretroviral therapy and prophylactic treatment regimens designed to prevent fungal infections. The disease, however, is still recognized as one of the most common life-threatening opportunistic fungal infections in immunocompromised patients, particularly among those infected with human immunodeficiency virus (HIV). For this reason, research interest in clinical and biological aspects of the disease remains high. In addition to previously embarked areas of research, the cryptococcal research community has taken advantage of the current sequencing technology and initiated genome sequencing of Cryptococcus neoformans var. neoformans. This review includes various areas of research interest ranging from pathobiology, biochemistry and immunology, to genomics.

AIDS-Related Opportunistic Infections↗

PubMed1999

Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene [correction of glyceraldehyde-3-phosphate gene] and the use of its promoter for heterologous expression in Cryptococcus neoformans, a human pathogen.

The GPD gene encoding glyceraldehyde-3-phosphate dehydrogenase was isolated from Cryptococcus neoformans, a heterobasidiomycetous yeast that is pathogenic to humans. The gene contains 11 introns, differing from the conserved intron positions found in the GPD genes of Basidiomycetes. The predicted amino-acid sequence of this gene is extremely similar to that reported from GPD proteins of other basidiomycetes. The promoter region of the C. neoformans GPD gene was similar to those of other basidiomycetes. Plasmid constructs containing up to 1600 base pairs upstream of the native GPD open reading frame were used to express either the native URA5 gene in a ura5 mutant or the heterologous hphI gene (a bacterial gene that confers resistance to the aminoglycoside hygromycin) in a wild-type strain of C. neoformans. Transformation frequencies resulting from the plasmid-borne Gpdp::URA5 gene were at levels similar to those of the native URA5, which suggested that all the sequences necessary for proper expression were present. Transformation frequencies using the Gpdp::hphI gene constructs were poor. However, addition of DNA sequences flanking the 3'-end of an native C. neoformans gene significantly improved the transformation frequencies resulting from the expression of the heterologous hphI gene.

Amino Acid Sequence↗

PubMed1999

On the origins of congenic MATalpha and MATa strains of the pathogenic yeast Cryptococcus neoformans.

The basidiomycetous yeast Cryptococcus neoformans infects humans and causes a meningoencephalitis that is uniformly fatal if untreated. The organism has a defined sexual cycle involving mating of haploid MATa and MATalpha strains, gene disruption by transformation and homologous recombination is now readily accomplished, and robust animal models for infection have been well established. In addition, a pair of congenic MATalpha and MATa haploid strains have been constructed that permit detailed studies on physiology and virulence by classical genetic approaches. These strains represent a valuable resource for further studies in this organism, and the genomic sequence of one of these strains, JEC21 (=B-4500), was recently chosen to be sequenced by an international consortium. Because of the importance of these strains for genetic studies in C. neoformans and the fact that the genomic sequence of one of these strains is in progress, we review here how these congenic strains were originally constructed.

Animals↗

PubMed1999

Heteroresistance to fluconazole and voriconazole in Cryptococcus neoformans.

Cryptococcus neoformans isolates that exhibited unusual patterns of resistance to fluconazole and voriconazole were isolated from seven isolates from two different geographical regions: one isolate from an Israeli non-AIDS patient and six serial isolates from an Italian AIDS patient who had suffered six recurrent episodes of cryptococcal meningitis. Each isolate produced cultures with heterogeneous compositions in which most of the cells were susceptible, but cells highly resistant to fluconazole (MICs, >/=64 microg/ml) were recovered at a variable frequency (7 x 10(-3) to 4.6 x 10(-2)). Evidence showed that this type of resistance is innate and is unrelated to drug exposure since the Israeli patient had never been treated with azoles or any other antimycotic agents. Analysis of clonal subpopulations of these two strains showed that they exhibited heterogeneous patterns of resistance. The number of subpopulations which grew on fluconazole or voriconazole agar declined progressively with increasing azole concentration without a sharp cutoff point. For the Italian serial isolates, the number of clonal populations resistant to fluconazole (64 microg/ml) and voriconazole (1 microg/ml) increased steadily, yielding the highest number for the isolate from the last episode. Attempts to purify a sensitive subpopulation failed, but clones highly resistant to fluconazole (100 microg/ml) and moderately resistant to voriconazole (1 microg/ml) always produced a homogeneous population of resistant cells. Upon maintenance on drug-free medium, however, the majority of the homogeneously resistant cells of these subclones lost their resistance and returned to the stable initial heteroresistant phenotype. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared to be suppressed at 35 degrees C and was completely abolished at 40 degrees C. Although heterogeneity in azole resistance among subpopulations of single isolates has been reported for Candida species, the transient changes in expression of resistance under different growth conditions reported here have not been observed in fungal pathogens.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Isolation, characterization, and localization of a capsule-associated gene, CAP10, of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene, CAP10, which is required for capsule formation. Complementation of the acapsular cap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of CAP10 are modulated by the transcriptional factor STE12alpha. Deletion of STE12alpha downregulated the expression levels of CAP10 while overexpression of STE12alpha upregulated the expression levels of CAP10. Animal model studies indicate that deletion of the CAP10 gene results in the loss of virulence, and complementation of the acapsular phenotype of cap10 restores virulence. Thus, CAP10 is required for capsule formation and virulence.

Animals↗

PubMed1999

A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus.

Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.

Amino Acid Sequence↗

PubMed1998

Virulence of catalase-deficient aspergillus nidulans in p47(phox)-/- mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare genetic disorder in which phagocytes fail to produce superoxide because of defects in one of several components of the NADPH oxidase complex. As a result, patients develop recurrent life-threatening bacterial and fungal infections. The organisms to which CGD patients are most susceptible produce catalase, regarded as an important factor for microbial pathogenicity in CGD. To test the role of pathogen-derived catalase in CGD directly, we have generated isogenic strains of Aspergillus nidulans in which one or both of the catalase genes (catA and catB), have been deleted. We hypothesized that catalase negative mutants would be less virulent than the wild-type strain in experimental animal models. CGD mice were produced by disruption of the p47(phox) gene which encodes the 47-kD subunit of the NADPH oxidase. Wild-type A. nidulans inoculated intranasally caused fatal infection in CGD mice, but did not cause disease in wild-type littermates. Surprisingly, wild-type A. nidulans and the catA, catB, and catA/catB mutants were equally virulent in CGD mice. Histopathological studies of fatally infected CGD mice showed widely distributed lesions in the lungs regardless of the presence or absence of the catA and catB genes. Similar to the CGD model, catalase-deficient A. nidulans was highly virulent in cortisone-treated BALB/c mice. Taken together, these results indicate that catalases do not play a significant role in pathogenicity of A. nidulans in p47(phox)-/- mice, and therefore raise doubt about the central role of catalases as a fungal virulence factor in CGD.

Acatalasia↗

PubMed1998

Construction of stable episomes in Cryptococcus neoformans.

We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker.

Blotting, Southern↗

PubMed1998

Aspergillus nidulans infection in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of the NADPH oxidase complex in which phagocytes are defective in generating reactive oxidants. As a result, patients with CGD suffer from recurrent bacterial and fungal infections. The most common fungal infections are caused by Aspergillus species. Aspergillus nidulans is a rare pathogen in most patient populations with quantitative or qualitative neutrophil defects. We have reviewed all cases in which A. nidulans was isolated from patients at the National Institutes of Health (Bethesda, MD) between 1976 and 1997. A. nidulans infection occurred in 6 patients with CGD, but was not a pathogen in any other patient group. Aspergillus fumigatus was a more common pathogen in CGD compared with A. nidulans, but A. nidulans was more virulent. A. nidulans was significantly more likely to result in death compared with A. fumigatus, to involve adjacent bone, and to cause disseminated disease. Patients with A. nidulans received longer courses of amphotericin B therapy than patients with A. fumigatus, and were treated with surgery more often. In contrast to A. fumigatus, A. nidulans was generally refractory to intensive antifungal therapy, suggesting that early surgery may be important. These data show that A. nidulans is a distinct pathogen in CGD and its isolation carries more severe implications than that of A. fumigatus.

Adolescent↗

PubMed1998

Isolation of the third capsule-associated gene, CAP60, required for virulence in Cryptococcus neoformans.

A polysaccharide capsule is one of the most important virulence factors for the pathogenic fungus Cryptococcus neoformans. We previously characterized two capsule-associated genes, CAP59 and CAP64. To further dissect the molecular mechanism of capsule synthesis, 16 acapsular mutants induced by 4-nitroquinoline-1-oxide were obtained. The acapsular phenotype of one of these mutants was complemented. The cloned gene was designated CAP60, and deletion of this newly described capsule-associated gene resulted in an acapsular phenotype. The proposed 67-kDa Cap60p contains 592 amino acids and appears to have a putative transmembrane domain close to the N terminus. DNA sequence analysis revealed that CAP60 has similarity to CAP59 at the center portion of its coding regions. Contour-clamped homogeneous electric field blot analysis suggested that these two genes are on the same chromosome. CAP60 and CAP59, however, could not be functionally substituted for each other by direct complementation or by domain swap experiments. In addition, CAP60 is closely linked to a gene which is similar to a cellulose growth-specific gene of Agaricus bisporus, CEL1. Immunogold electron microscopy studies of the epitope-tagged CAP60 gene revealed that Cap60p was primarily localized to the nuclear membrane. Animal model studies indicated that CAP60 is essential for virulence. Thus, CAP60 is required for both capsule formation and virulence.

Amino Acid Sequence↗

PubMed1998

The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence.

Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene, alb1, which is required for conidial pigmentation. The alb1 gene encodes a putative polyketide synthase, and disruption of alb1 resulted in an albino conidial phenotype. Expression of alb1 is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. The alb1 mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating that alb1 is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that the alb1 disruptant exhibited a smooth conidial surface, whereas complementation of the alb1 deletion restored the echinulate wild-type surface. Disruption of alb1 resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of the alb1 disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. The alb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, the alb1 disruptant had a statistically significant loss of virulence compared to the wild-type and alb1-complemented strains in a murine model. These results suggest that disruption of alb1 causes pleiotropic effects on conidial morphology and fungal virulence.

Amino Acid Sequence↗

PubMed1998

Invasive infection with Fusarium chlamydosporum in a patient with aplastic anemia.

We report the first case of invasive disease caused by Fusarium chlamydosporum. The patient had aplastic anemia with prolonged neutropenia and was treated with immunosuppressive therapy. While she was receiving empirical amphotericin B, a dark crusted lesion developed on her nasal turbinate. Histologic analysis revealed invasive hyaline hyphae and some darkly pigmented structures that resembled conidia of dematiaceous molds. Only after the mold was grown in culture were characteristic colonial morphology, phialides, conidia, and chlamydospores evident, thus permitting the identification of F. chlamydosporum. This case illustrates the ever-increasing spectrum of pathogenic Fusarium spp. in immunocompromised patients and emphasizes the potential pitfalls in histologic diagnosis, which may have important treatment implications.

Adult↗

PubMed1998

Concentrations of airborne Aspergillus compared to the incidence of invasive aspergillosis: lack of correlation.

Air sampling of the rooms and corridors of the oncology wards of the hospital was carried out over a 54-week period to assess the concentration of viable Aspergillus conidia. A. fumigatus and A. flavus were recovered at a mean of 1.83 cfu m-3 air sampled. Individual samplings yielded concentrations of up to 11.6 cfu m-3. Other Aspergillus spp. were recovered at a mean of 2.38 cfu m-3 (maximum 32.6 cfu m-3). Concentration was not correlated with season or hospital ward. Review of autopsy results showed an average of 6.6 cases of aspergillosis annually over a 22-year period. No seasonal variation in case incidence was found. Six cases of invasive aspergillosis were diagnosed on the three cancer wards during the air-sampling period, but no association was seen linking these cases with changes in recovery of airborne Aspergillus. A seasonal pattern was not observed in the overall incidence of aspergillosis cases nor concentrations of airborne conidia.

Air Microbiology↗