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K J Kwon-Chung

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

At least 55 records · Page 3Linked to original sources
PubMed1998

Fate of transforming DNA in pathogenic fungi.

Genetic engineering is an important tool in helping us to define the molecular basis of pathogenicity and is also useful in helping us to identify new therapeutic targets in pathogenic fungi. Molecular genetic manipulation of micro-organisms requires the development of plasmid-mediated transformation systems that include: (i) infusion of exogenous DNA into recipient cells, (ii) expression of genes present on the incoming DNA, and (iii) stable maintenance and replication of the inserted DNA leading to expression of the desired phenotypic trait. Transformation systems have been developed for only a handful of fungi that are pathogenic to humans including several species of Candida, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces derrmatitidis, Aspergillus fumigatus, Wangiella dermatitidis (Exophiala dermatitidis) and Coccidioides immitis. Except for Candida species and A. fumigatus, where passage of exogenous DNA into recipient cells has been achieved readily using methods developed for transformation of Saccharomyces cerevisiae and Aspergillus nidulans, respectively, development of transformation systems in other pathogenic fungi has been delayed considerably and has only been possible recently with the introduction of electroporation and biolistic methods. Conventional spheroplasting methods or cell wall permeabilization methods using lithium acetate have not been successful for transformation of C. neoformans and work with only low efficiency in H. capsulatum. The fate of incoming DNA varies greatly in these pathogenic species regardless of their phylogenetic relationships. Understanding the fate of incoming DNA is critical for the construction of transforming vectors and the molecular manipulation of the organisms. In this symposium, recent advances in molecular genetic systems including transformation systems, the fate of incoming DNA and strategies for targeted integration are discussed in relation to four pathogenic fungi.

DNA, Fungal↗
PubMed1998

Cryptococcus neoformans: virulence and host defences.

Cryptococcus neoformans represents a model organism for the study of virulence and the host response. In this discussion, there is a focus on the genetic, molecular, and biochemical aspects of C. neoformans as it interacts with the host. Investigations into direct and indirect virulence phenotypes are now possible. The molecular aspects of two major virulence factors, capsule and melanin, are characterized. Yeast polyol metabolism through mannitol is examined as a potential biochemical pathway for virulence. The concept of C. neoformans differentially expressed genes within the host or in response to certain environmental cues can be used indirectly to identify potential virulence genes. However, despite significant progress in molecular pathogenesis with C. neoformans, the future of research in this area will require a certain critical mass of investigators to help share in the developmental costs which continue to occur.

Cryptococcosis↗
PubMed1997

Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition.

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish-pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish-green conidial pigment. The bluish-green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1, was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8-dihydroxynaphthalene-melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea. Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish-pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish-green pigmentation of conidia as well as complement deposition.

Amino Acid Sequence↗
PubMed1997

Stereoselective interaction of the azole antifungal agent SCH39304 with the cytochrome P-450 monooxygenase system isolated from Cryptococcus neoformans.

We investigated the stereoselective inhibition of growth and ergosterol biosynthesis by SCH39304 in the pathogenic fungus Cryptococcus neoformans obtained from four AIDS patients who failed fluconazole therapy and compared the results to those obtained with a wild-type strain. For all strains, the MICs of the RR isomer were approximately half those of the racemate, with the SS enantiomer showing no inhibitory activity. The 50% inhibitory concentrations for in vitro ergosterol biosynthesis correlated with the MIC data, indicating stereoselective inhibition of their target P-450 enzyme, sterol 14alpha-demethylase, as the cause of this difference. The RR enantiomer produced classical type II spectra on addition to microsomal extracts of the strains, whereas the SS enantiomer showed an absence of binding. Stereo- and regio-specific localization of N-1 substituent groups of SCH39304 within the active site of the enzyme determined the unique discrimination between its two enantiomers, and the inability to bind to sterol 14alpha-demethylase is also true of other P-450 enzymes contained in the microsomal fraction. As previously observed for other antifungal azoles, isolates obtained following failure of fluconazole therapy showed resistance to SCH39304 and its RR enantiomer. This resistance could be associated with an alteration in the sensitivity of ergosterol biosynthesis in vitro. These alterations did not cause any changes allowing the SS enantiomer to bind to the P-450 mediating sterol 14alpha-demethylation.

Antifungal Agents↗
PubMed1997

A gene homologous to Saccharomyces cerevisiae SNF1 appears to be essential for the viability of Candida albicans.

The SNF1 gene of Saccharomyces cerevisiae (ScSNF1) is essential for the derepression of catabolic repression. We report here the isolation and characterization of an SNF1 homolog from Candida albicans (CaSNF1) which is apparently essential for the viability of this organism. The putative amino acid sequence of CaSNF1 has 68% identity with that of ScSNF1 and can restore the S. cerevisiae snf1 delta mutant's ability to utilize sucrose. Disruption of one of the CaSNF1 alleles resulted in morphological changes and decreased growth rates but did not modify the carbon source utilization pattern. Repetitive unsuccessful attempts to generate a snf1/snf1 homozygote by disruption of the second allele, using various vectors and approaches, suggest the lethal nature of this mutation. Integration into the second allele was possible only when a full-length functional SNF1 sequence was reassembled, further supporting this hypothesis and indicating that the indispensability of Snf1p prevented the isolation of snf1/snf1 mutants. The mutant bearing two disrupted SNF1 alleles and the SNF1 functional sequence maintained its ability to utilize sucrose and produced stellate colonies with extensive hyphal growth on agar media. It was demonstrated that in a mouse model, the virulences of this mutant and the wild-type strain are similar, suggesting that hyphal growth in vitro is not an indicator for higher virulence.

Amino Acid Sequence↗
PubMed1997

Structure and biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.

The extracellular polysaccharide capsule of Cryptococcus neoformans is a well-recognized virulence factor. Strain 602 is an acapsular clinical isolate of unknown serotype which has been widely used in studies of virulence and host-parasite interactions. In previous studies, strain 602 was compared with genetically unrelated strains of various serotypes because the wild-type equivalent of strain 602 was not available. We created an encapsulated strain, TYCC38-602, by transforming strain 602 with the CAP64 gene which was isolated from a serotype D strain. Serological tests and chemical analysis of the major polysaccharide capsule of TYCC38-602 indicated that strain 602 was originally derived from a serotype A strain. Restoration of the ability to produce a capsule enabled strain 602 to cause fatal infection in mice, whereas the acapsular strain 602 remained avirulent. Capsule-restored yeast cells of strain 602 activated the human complement system and bound C3 fragments in a manner that is characteristic of encapsulated cryptococci. In addition, the capsule in TYCC38-602 masked the ability of the organism to induce tumor necrosis factor alpha and subsequent nitric oxide synthase production in primed macrophage-like cells. These results indicate that the lack of capsule in strain 602 is the reason for its inability to cause fatal infection. Moreover, the acapsular phenotype accounts for differences in various biological activities of strain 602 compared to encapsulated strains. The results also indicate that the gene product of CAP64 does not contribute to serotype specificity of capsules in C. neoformans.

Animals↗
PubMed1996

Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.

To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper-binding site. When this base change was introduced into CNLAC1 by site-directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.

Animals↗
PubMed1996

Structure of the O-deacetylated glucuronoxylomannan from Cryptococcus neoformans Cap70 as determined by 2D NMR spectroscopy.

Cryptococcus neoformans, an opportunistic pathogen, is the fourth leading cause of death among AIDS patients. The yeast's capsule is a major virulence factor, and serotype is related to the chemical structure of glucuronoxylomannan (GXM), its capsular polysaccharide. The GXM from Cap70, a hypocapsular mutant of serotype D isolate B-3501, was investigated by chemical analysis and 2D NMR spectroscopy. The assignment of 1H and 13C chemical shifts for the O-deacetylated polysaccharide was accomplished from the analysis of DQF-COSY, TOCSY, and gradient-enhanced HSQC spectra. The sequence and linkage positions of glycosyl residues were determined by NOESY and ROESY spectra. Two repeating polysaccharide components were identified as having the following structures in approximately equal proportions: [formula: see text] It is not known if these repeating units comprise a single or two separate polymer chains. Pentasaccharide 2 has been known to be the major GXM polymer of B-3501 and other serotype D isolates. Hexasaccharide 1 is identified for the first time although it has subsequently been identified in other C. neoformans isolates. The presence of 1 in the GXM of Cap70 is consistent with the extra xylose found relative to that in isolate B-3501. The mannose:xylose:glucuronic acid:O-acetyl molar ratio of Cap70 GXM is 3.00:1.73:0.78:1.75, while the same ratio for B-3501 and other serotype D isolates is approximately 3.00:1.00:0.80:1.75. Methylation analysis confirmed that the GXM of Cap70 contains unsubstituted, monosubstituted (2-linked), and disubstituted (2- and 4-linked) mannose in a ratio of 0.87:1.75:0.38. Dot blot immunoassay indicates that Cap70 is a serotype D isolate like its parent strain.

Acetylation↗
PubMed1996

Disruption of the SNF1 gene abolishes trehalose utilization in the pathogenic yeast Candida glabrata.

The SNF1 gene product, a serine/threonine protein kinase, is a global regulatory protein which has been isolated from several organisms. In Saccharomyces cerevisiae the SNF1 gene product is essential for the derepression of glucose repression since snf1 strains are unable to utilize sucrose, galactose, maltose, melibiose, or nonfermentable carbohydrates. Moreover, the SNF1 gene product was suggested to interact with additional regulatory pathways and to affect the expression of multiple target genes as reflected by the pleiotropic nature of the snf1 mutation. Here we report the characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S. cerevisiae. The carbon utilization spectrum of C. glabrata is considerably narrower than that of other pathogenic yeasts, and the majority of the strains utilize solely glucose and trehalose from among 20 of the most commonly tested carbohydrates. Disruption of the C. glabrata SNF1 homolog resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved.

Amino Acid Sequence↗
PubMed1996

The second capsule gene of cryptococcus neoformans, CAP64, is essential for virulence.

The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, (AP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.

Amino Acid Sequence↗
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

IL-12 prevents mortality in mice infected with Histoplasma capsulatum through induction of IFN-gamma.

Histoplasma capsulatum is a pathogenic fungus found in discrete geographic locations throughout the world. The fungus invades the reticuloendothelial organs such as the spleen and liver of immunocompetent hosts where it is usually controlled. However, in individuals with immune deficiency, histoplasmosis is a severe and potentially fatal disease. Resistance to this infection is due primarily to a cellular immune response mediated by T cells and macrophages. Moreover, IFN-gamma is critical in activating macrophages to kill the organism. Herein we study the regulation of cytokine induction in mice infected with H. capsulatum and the effects of IL-12 in the course of infection. Mice infected with H. capsulatum and treated with neutralizing Abs to IFN-gamma, TNF-alpha, or IL-12 experienced accelerated mortality, indicating that endogenous production of these cytokines plays an important role in response to infection. In contrast, mice treated with IL-12 or a neutralizing Ab to IL-4 at the initiation of infection had substantially diminished mortality. Moreover, mice infected and treated with IL-12 show a two- to threefold increase in the amount of IFN-gamma following in vitro stimulation with specific H. capsulatum Ag compared with the control infected mice. The protective effect of IL-12 could be abrogated if a neutralizing Ab to IFN-gamma was given at the same time, demonstrating that the role of IL-12 in protection was mediated by IFN-gamma. Additionally, infected mice treated with IL-12 had a severalfold decrease in the colony counts of H. capsulatum in spleen cells after 5 days of infection as compared with control animals. Lastly, spleen cells from infected animals treated with IL-12 showed a striking decrease in their proliferative response to mitogen or H. capsulatum Ag. Responses could be restored by adding inhibitors of IFN-gamma or of nitric oxide to the in vitro cultures. The above observations suggest that IL-12 may be useful in immunologic intervention against this opportunistic pathogen.

Animals↗
PubMed1995

Suppurative cutaneous granulomata caused by Microascus cinereus in a patient with chronic granulomatous disease.

We describe a patient with chronic granulomatous disease who presented with erythematous papular skin lesions on the chest, back, and arm. Examination of biopsy specimens from the lesions on the arm and back showed suppurative granulomata in association with acute and chronic inflammation. Histopathologic examination of a specimen from the lesion on the arm revealed fungal elements, and cultures yielded Microascus cinereus. The patient was treated with 2.5 g of intravenous amphotericin B, and the lesions resolved. We report what is, to our knowledge, the first case of invasive disease due solely to M. cinereus.

Amphotericin B↗
PubMed1995

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.

Antifungal Agents↗
PubMed1995

Mucormycosis caused by Rhizopus microsporus var. microsporus: cellulitis in the leg of a diabetic patient cured by amputation.

Mucormycosis accompanied the development of bacterial infection in the leg of a diabetic African-American man. Local injury, diabetic ketoacidosis, renal insufficiency, and antimicrobial therapy were factors that contributed to the pathogenesis of the mucormycosis. The cellulitis was caused in part by Rhizopus microsporus var. microsporus and was cured by amputation. We report this unusual case of mucormycosis to emphasize the value of fungal identification, to illustrate a dramatic and successful clinical result, and to draw attention to an apparent role for bacterial infection and its treatment in the pathogenesis of mucormycosis. It is the third case report of mucormycosis in a human in which R. microsporus var. microsporus was definitively identified as the etiologic agent.

Adult↗
PubMed1995

Diversity of DNA fingerprints in Cryptococcus neoformans.

DNA fingerprint patterns of 156 Cryptococcus neoformans isolates (26 AIDS patients, 46 non-AIDS patients, and 40 environmental sources) from both varieties (126 C. neoformans var. neoformans and 30 C. neoformans var. gattii isolates) and from seven countries were analyzed by using the DNA probe UT-4p. Nine and twelve distinct DNA fingerprint patterns were observed for isolates of the C. neoformans var. neoformans and var. gattii, respectively. No pattern was unique to AIDS patients, non-AIDS patients, or the environment. Pattern II was observed more often in non-AIDS patients (8 of 23) than in AIDS patients (0 of 25). Pattern V was the most prevalent pattern (42 of 82) in clinical and environmental isolates. Isolates from three AIDS patients in Burundi and Zaire exhibited patterns identical to each other but different from those of isolates collected from their houses (i.e., dust of floors, walls, etc.) or a nearby pigeon coop. DNA fingerprint stability was determined for 53 isolates from nine non-AIDS patients at different time intervals during 5 to 128 weeks of antifungal therapy. For eight patients, the fingerprint pattern was stable while the ninth may have had a mixed infection. Pattern II was observed in 4 of 9 patients, which is similar to 4 of 14 in other non-AIDS patients as reported here. In spite of the extensive pattern heterogeneity among 15 C. neoformans var. gattii isolates in Australia, the patterns observed in seven California isolates were quite different from those in Australia. Among isolates of C. neoformans var. gattii, one fingerprint pattern (designated b) was observed in several countries of the Far East. The fingerprint patterns of two of three environmental isolates from Eucalyptus camaldulensis trees in Australia were identical to those of 2 of the 12 clinical isolates from the country.

AIDS-Related Opportunistic Infections↗
PubMed1994

Formation of a minichromosome in Cryptococcus neoformans as a result of electroporative transformation.

A minichromosome of approximately 270 kilobases was generated following complementation of a ura5 mutant strain of C. neoformans with the plasmid pURA5g2. This is the first report of the in-vivo generation of a minichromosome by the method of electroporative transformation. The minichromosome occurred at a relatively high (> 20%) frequency in transformants that were stable for uracil protoprophy. The minichromosome was maintained in linear form as a large extrachromosomal element of the normal karyotype. Gel-purified DNA from the minichromosome readily transformed the ura5 mutant of C. neoformans. Southern-blot analysis of the minichromosome revealed the presence of multiple copies of the URA5 gene and ribosomal DNA sequences in addition to containing telomere-like sequence repeats. The minichromosome was transmitted through mitosis and meiosis with extremely-high fidelity.

Base Sequence↗