Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human pathogenic fungus Cryptococcus neoformans secretes a phospholipase enzyme that demonstrates phospholipase B (PLB), lysophospholipase hydrolase and lysophospholipase transacylase activities. This enzyme has been postulated to be a cryptococcal virulence factor. We cloned a phospholipase-encoding gene (PLB1) from C. neoformans and constructed plb1 mutants using targeted gene disruption. All three enzyme activities were markedly reduced in the mutants compared with the wild-type parent. The plb1 strains did not have any defects in the known cryptococcal virulence phenotypes of growth at 37 degrees C, capsule formation, laccase activity and urease activity. The plb1 strains were reconstituted using the wild-type locus and this resulted in restoration of all extracellular PLB activities. In vivo testing demonstrated that the plb1 strain was significantly less virulent than the control strains in both the mouse inhalational model and the rabbit meningitis model. We also found that the plb1 strain exhibited a growth defect in a macrophage-like cell line. These data demonstrate that secretory phospholipase is a virulence factor for C. neoformans.
Mol Microbiol 2001 Jan
PMID:Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. 1112 98

The aim of this study was to enable the polymerase chain reaction (PCR) amplification of DNA fragments within endoglucanase gene(s) of Torula thermophila, by using degenerate primers so that the amplified fragment(s) could be used as homologous probe(s) for cloning of full-length endoglucanase gene(s). The design of the degenerate PCR primers was mainly based on the endoglucanase sequences of other fungi. The endoglucanase gene sequence of Humicola insolens was the only sequence from a thermophilic fungus publicly available in the literature. Therefore, the endoglucanase sequences of the two Trichoderma species, Trichoderma reesei and Trichoderma longibrachiatum, were used to generalize the primers. PCR amplification of T. thermophila genomic DNA with these primers resulted in a specific amplification. The specificity of the amplified fragment was shown by Southern hybridization analysis using egl3 gene of T. reesei as probe. This result suggested that the degenerate primers used in this study may be of value for studies aimed at cloning of endoglucanase genes from a range of related fungi.
Mol Biotechnol 2000 Oct
PMID:PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus. 1113 71

Calcineurin is a Ca2+-calmodulin-regulated protein phosphatase that is the target of the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is a heterodimer composed of a catalytic A and a regulatory B subunit. In previous studies, the calcineurin A homologue was identified and shown to be required for growth at 37 degrees C and hence for virulence of the pathogenic fungus Cryptococcus neoformans. Here, we identify the gene encoding the calcineurin B regulatory subunit and demonstrate that calcineurin B is also required for growth at elevated temperature and virulence. We show that the FKR1-1 mutation, which confers dominant FK506 resistance, results from a 6 bp duplication generating a two-amino-acid insertion in the latch region of calcineurin B. This mutation was found to reduce FKBP12-FK506 binding to calcineurin both in vivo and in vitro. Molecular modelling based on the FKBP12-FK506-calcineurin crystal structure illustrates how this mutation perturbs drug interactions with the phosphatase target. In summary, our studies reveal a central role for calcineurin B in virulence and antifungal drug action in the human fungal pathogen C. neoformans.
Mol Microbiol 2001 Feb
PMID:Calcineurin regulatory subunit is essential for virulence and mediates interactions with FKBP12-FK506 in Cryptococcus neoformans. 1125 6

The identification of yeast and yeastlike organisms in tissue sections can be very difficult. Biopsy tissues may be limited, with only occasional organisms present. In addition, several common species have overlapping histologic features. Deoxyribonucleic acid probes were designed to detect both the 18S and 28S ribosomal ribonucleic acid sequences of five fungal organisms with a high degree of specificity for each fungus. Each of these organisms--Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, and Sporothrix schenckii--can be manifested histologically as round, yeastlike structures, often within a similar size range. Probes were tested against 98 archived, formalin-fixed, paraffin-embedded tissue specimens, each of which had culture-proved involvement by one of these organisms. Assessment of accuracy was based on the presence of yeastlike organisms in consecutive Grocott's methanemine silver (GMS)-stained tissue sections, and agreement with culture results. The results indicated that GMS had a greater overall sensitivity in detecting fungal organisms (95.9%) compared with in situ hybridization (ISH; 82.7%). ISH with oligonucleotide deoxyribonucleic acid probes, however, was more specific, with all species-specific probes yielding 100% specificity (compared with 96.2-100% specificity based on morphology alone). ISH also had a higher positive predictive value (100% in all cases) compared with GMS (83.3-100%). In addition, four cases with rare organisms present (4.1% of cases tested) were detected by ISH but not by GMS staining. These results show that ISH, directed against ribosomal ribonucleic acid, provides a rapid, accurate technique for the identification of yeastlike organisms in histologic tissue sections. Its primary strength lies in the ability to speciate organisms accurately that are too few or atypical to identify based solely on morphologic features.
Diagn Mol Pathol 2001 Mar
PMID:In situ hybridization for the identification of yeastlike organisms in tissue section. 1127 90

Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the Galpha protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a Galpha protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.
Mol Cell Biol 2001 May
PMID:Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. 1128 22

Partial sequence analysis of the Cryptococcus neoformans MATalpha mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11alpha, showed the highest degree of similarity to the Neurospora crassa nrc-1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction-mediated sib-selection technique was successfully adapted for the purpose of disrupting STE11alpha. C. neoformans ste11alphaDelta mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11alphaDelta mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild-type STE11alpha strain with a ste11alphaDelta disruptant for virulence using the mouse model showed that the ste11alphaDelta strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11alpha, linkage analysis showed that STE11alpha is only found in mating type alpha strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.
Mol Microbiol 2001 Apr
PMID:The Cryptococcus neoformans STE11alpha gene is similar to other fungal mitogen-activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific. 1129 87

The polysaccharide capsule surrounding Cryptococcus neoformans comprises manose, xylose and glucuronic acid, of which mannose is the major constituent. The GDP-mannose biosynthesis pathway is highly conserved in fungi and consists of three key enzymes: phosphomannose isomerase (PMI), phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP). The MAN1 gene, encoding for the PMI enzyme, was isolated and sequenced from C. neoformans, and a disruption of the MAN1 gene was generated. One MAN1 disruption mutant, man1, which showed poor capsule formation, reduced polysaccharide secretion and morphological abnormalities, was chosen for virulence studies. In both the rabbit and the mouse models of invasive cryptococcosis, man1 was shown to be severely impaired in its virulence, with complete elimination of the yeast from the host. A reconstituted strain of man1 was constructed using gene replacement at the native locus. The wild-type and reconstituted strains were significantly more virulent than the knock-out mutant in both animal models. Our findings reveal that PMI activity is essential for the survival of C. neoformans in the host. The fact that the man1 mutant was not pathogenic suggests that blocking mannose synthesis could be fungicidal in the mammalian host and thus an excellent target for antifungal drug development.
Mol Microbiol 2001 May
PMID:Identification and characterization of the Cryptococcus neoformans phosphomannose isomerase-encoding gene, MAN1, and its impact on pathogenicity. 1135 67

The pathogenic yeast Cryptococcus neoformans is distinguished by an extensive polysaccharide capsule, which impedes host defences and is absolutely required for fungal virulence. Despite the biological importance of the capsule, nothing is known about how it is assembled. Substantial capsule growth occurs in two distinct situations relevant to cryptococcal pathogenesis: formation of new buds and induction of capsule on mature cells. We developed pulse-chase protocols to examine these events in a dynamic way using a variety of microscopy techniques. We show that the capsule overlying buds is newly synthesized and differs physically from the corresponding parental material. New capsule formed by mature cells upon induction of synthesis is added at the inner aspect of the existing structure, displacing pre-existing material outwards. Surprisingly, new polysaccharide material is also deposited throughout the capsule, yielding a progressively denser structure. These results yield the first model of capsule synthesis and open new lines of investigation into the underlying mechanisms.
Mol Microbiol 2001 Jul
PMID:Spatial and temporal sequence of capsule construction in Cryptococcus neoformans. 1145 4

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule-structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the PROPSEARCH software classified Cas1p as a putative glycosyltransferase. Cas1p is a well-conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O-acetylation.
Mol Microbiol 2001 Oct
PMID:Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide. 1170 67

In the phytopathogenic fungus Ustilago maydis, the switch to filamentous growth and pathogenic development is controlled by a heterodimeric transcription factor consisting of the bW and bE homeodomain proteins. To identify genes in the regulatory cascade triggered by the bW/bE heterodimer, we have constructed strains in which transcription of the b genes is inducible by either arabinose or nitrate. At different time-points after induction, genes that are switched on or off were identified through a modified, non-radioactive RNA fingerprint procedure. From 348 gene fragments isolated initially, 48 fragments representing 34 different genes were characterized in more detail. After eliminating known genes, false positives and genes influenced in their expression profile by media conditions, 10 new b-regulated genes were identified. Of these, five are upregulated and five are downregulated in presence of the b heterodimer. Two do not share significant similarity to database entries, whereas the other eight show similarity to disulphide isomerases, exochitinases, cation antiporters, plasma membrane (H+)-ATPases, acyl transferases, a capsular associated protein of Cryptococcus neoformans, DNA polymerases X, as well as to a potential protein of Neurospora crassa. We demonstrate that in one of the early upregulated genes, the promoter can be bound by a bW/bE fusion protein in vitro. Interestingly, three out of the four genes that are downregulated by the b heterodimer appear upregulated after pheromone stimulation, suggesting a connection to the mating process.
Mol Microbiol 2001 Nov
PMID:Identification of genes in the bW/bE regulatory cascade in Ustilago maydis. 1173 46


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