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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dictyostelium discoideum is a molecularly amenable host model system for several human pathogens, including Legionella pneumophila, Mycobacterium avium, Mycobacterium marinum, Pseudomonas aeruginosa, and Cryptococcus neoformans. Dictyostelium wild-type cells have proven useful in screening and identifying numerous bacterial und fungal virulence factors. Moreover, Dictyostelium mutant cells can be used to identify genetic host determinants of susceptibility and resistance to infections. Marker genes such as the green fluorescence protein (GFP) gene allow the in vivo monitoring of infection-relevant host factors. Here, we present methods that have already contributed to the deciphering of important aspects of the Dictyostelium-Legionella interaction. Moreover, the described phagocytosis assay, infection assay, and the confocal in vivo monitoring of GFP-tagged host factors can easily be adapted to other host-pathogen interactions.
Methods Mol Biol 2006
PMID:Dictyostelium discoideum as a model to study host-pathogen interactions. 1695 11

Cryptococcus neoformans, which causes fatal infection in immunocompromised individuals, has an elaborate polysaccharide capsule surrounding its cell wall. The cryptococcal capsule is the major virulence factor of this fungal organism, but its biosynthetic pathways are virtually unknown. Extracellular polysaccharides of eukaryotes may be made at the cell membrane or within the secretory pathway. To test these possibilities for cryptococcal capsule synthesis, we generated a secretion mutant in C. neoformans by mutating a Sec4/Rab8 GTPase homolog. At a restrictive temperature, the mutant displayed reduced growth and protein secretion, and accumulated approximately 100-nm vesicles in a polarized manner. These vesicles were not endocytic, as shown by their continued accumulation in the absence of polymerized actin, and could be labeled with anti-capsular antibodies as visualized by immunoelectron microscopy. These results indicate that glucuronoxylomannan, the major cryptococcal capsule polysaccharide, is trafficked within post-Golgi secretory vesicles. This strongly supports the conclusion that cryptococcal capsule is synthesized intracellularly and secreted via exocytosis.
Mol Biol Cell 2006 Dec
PMID:A eukaryotic capsular polysaccharide is synthesized intracellularly and secreted via exocytosis. 1702 Dec 52

Hsp70 proteins are a well-known class of chaperones that have also been described to have roles in cellular regulation. Here, we show that a Cryptococcus neoformans Hsp70 homologue Ssa1 acts as a DNA-binding transcriptional co-activator of the fungal virulence factor, laccase, via binding to a GC-rich element within the 5'-UAS in response to glucose starvation, iron, copper, calcium and temperature. In addition, Ssa1 forms a regulatory complex with heat shock transcription factor and TATA-binding protein during laccase induction. Furthermore, deletion of Ssa1 results in reduced laccase and attenuated virulence using a mouse model. These results indicate that Hsp70 functions as a stress-related transcriptional co-activator required for fungal virulence.
Mol Microbiol 2006 Nov
PMID:The Hsp70 member, Ssa1, acts as a DNA-binding transcriptional co-activator of laccase in Cryptococcus neoformans. 1704 Apr 92

Pathogenic microorganisms must precisely regulate morphogenesis to survive and proliferate within an infected host. This regulation is often controlled by conserved signal transduction pathways that direct morphological changes in varied species. One such pathway, whose components include Ras proteins and the PAK kinase Ste20, allows the human fungal pathogen Cryptococcus neoformans to grow at high temperature. Previously, we found that Ras1 signalling is required for differentiation, thermotolerance and pathogenesis in C. neoformans. We show here that the guanine nucleotide exchange factor Cdc24 is a Ras1 effector in C. neoformans to mediate the ability of this fungus to grow at high temperature and to cause disease. In addition, we provide evidence that the Ras1-Cdc24 signalling cascade functions specifically through one of the three Cdc42/Rac1 homologues in C. neoformans. In conclusion, our studies illustrate how components of conserved signalling cascades can be specialized for different downstream functions, such as pathogenesis.
Mol Microbiol 2007 Feb
PMID:A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans. 1723 29

Yeast cell walls are critical for maintaining cell integrity, particularly in the face of challenges such as growth in mammalian hosts. The pathogenic fungus Cryptococcus neoformans additionally anchors its polysaccharide capsule to the cell surface via alpha(1-3) glucan in the wall. Cryptococcal cells disrupted in their alpha glucan synthase gene were sensitive to stresses, including temperature, and showed difficulty dividing. These cells lacked surface capsule, although they continued to shed capsule material into the environment. Electron microscopy showed that the alpha glucan that is usually localized to the outer portion of the cell wall was absent, the outer region of the wall was highly disorganized, and the inner region was hypertrophic. Analysis of cell wall composition demonstrated complete loss of alpha glucan accompanied by a compensatory increase in chitin/chitosan and a redistribution of beta glucan between cell wall fractions. The mutants were unable to grow ina mouse model of infection, but caused death in nematodes. These studies integrate morphological and biochemical investigations of the role of alpha glucan in the cryptococcal cell wall.
Mol Microbiol 2007 Mar
PMID:Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence. 1724 96

Despite significant progress, the general functional and evolutionary significance of the untranslated regions (UTRs) of eukaryotic transcripts remain mysterious. Particularly mysterious is the common occurrence of spliceosomal introns in transcript UTRs because UTR splicing is not necessary for restoration of transcript coding sequence. In general, it is not known to what extent such splicing performs an important function or merely represents spliceosomal "noise." We conducted the first analysis of evolutionary conservation of UTR splicing. Among 4 species from Cryptococcus neoformans species complex, we find high levels of conservation of UTR intron boundary sequences, strongly suggesting that UTR intron splicing is conserved by purifying selection. We estimate that 50-90% of splice boundaries are maintained by selection. Donor site sequences are more highly conserved than acceptor sequences, and splicing boundaries are more conserved in 5' UTRs than in 3' UTRs. In addition, we report a variety of differences between patterns of UTR splicing in Cryptococcus and corresponding patterns in animals and plants. These results focus attention on the functional roles of eukaryotic UTRs and deepen the mystery of UTR intron splicing.
Mol Biol Evol 2007 May
PMID:Evolutionary conservation of UTR intron boundaries in Cryptococcus. 1737 79

Cryptococcus neoformans is a pathogenic yeast that causes life-threatening meningoencephalitis and grows well on mycological media regardless of inoculum size. Interestingly, a deletion of the global repressor TUP1 in C. neoformans uncovered a density-dependent growth phenotype reminiscent of the quorum-sensing phenomenon. An inoculum size of lower than 10(3) cells of the tup1Delta strain failed to form colonies on agar media while inocula of 10(5)-10(6) cells per plate formed a lawn. This phenotype, expressed as the inability to grow at low cell densities, was rescued by the culture filtrate from a high cell density tup1Delta culture and the active molecule in this culture filtrate was identified to be an oligopeptide composed of 11 amino acids. Activity assays, using a synthetic version of the peptide with strains harbouring a deletion of the corresponding gene, proved that the oligopeptide functioned as an autoregulatory molecule responsible for the density-dependent phenotype. Although a density-dependent growth phenotype has been reported in several species of Ascomycetes, no peptide has been reported to function as an autoregulator in the Kingdom Fungi. The identification of an 11-mer peptide as an autoregulatory molecule in C. neoformans suggests that a diverse mechanism of cell-to-cell communication exists in the Kingdom Fungi.
Mol Microbiol 2007 May
PMID:TUP1 disruption in Cryptococcus neoformans uncovers a peptide-mediated density-dependent growth phenomenon that mimics quorum sensing. 1746 10

Cryptococcus neoformans is an environmental pathogen requiring atmospheric levels of oxygen for optimal growth. Upon inhalation, C. neoformans disseminates to the brain and causes meningoencephalitis, but the mechanisms by which the pathogen adapts to the low-oxygen environment in the brain have not been investigated. We found that SRE1, a homologue of the mammalian sterol regulatory element-binding protein (SREBP), functions in an oxygen-sensing pathway. Low oxygen decreased sterol synthesis in C. neoformans and triggered activation of membrane-bound Sre1p by the cleavage-activating protein, Scp1p. Microarray and Northern blot analysis demonstrated that under low oxygen, Sre1p activates genes required for ergosterol biosynthesis and iron uptake. Consistent with these regulatory functions, sre1Delta cells were hypersensitive to azole drugs and failed to grow under iron-limiting conditions. Importantly, sre1Delta cells failed to produce fulminating brain infection in mice. Our in vitro data support a model in which Sre1p is activated under low oxygen leading to the upregulation of genes required for sterol biosynthesis and growth in a nutrient-limiting environment. Animal studies confirm the importance of SRE1 for C. neoformans to adapt to the host environment and to cause fatal meningoencephalitis, thereby identifying the SREBP pathway as a therapeutic target for cryptococcosis.
Mol Microbiol 2007 May
PMID:Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in Cryptococcus neoformans. 1746 12

The polysaccharidic capsule is the main virulence factor of Cryptococcus neoformans. It primarily comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and galactoxylomannan (GalXM, 7% of the capsule mass). We constructed a large collection of mutant strains in which genes potentially involved in capsule biosynthesis were deleted. We used a new post-genomic approach to study the virulence of the strains. Primers specific for unique tags associated with the disruption cassette were used in a real-time PCR virulence assay to measure the fungal burden of each strain in different organs of mice in multi-infection experiments. With this very sensitive assay, we identified a putative UDP-glucose epimerase (Uge1p) and a putative UDP-galactose transporter (Ugt1p) essential for C. neoformans virulence. The uge1Delta and ugt1Delta strains are temperature sensitive and do not produce GalXM but synthesize a larger capsule. These mutant strains (GalXM negative, GXM positive) are not able to colonize the brain even at the first day of infection whereas GXM-negative strains (GalXM positive) can still colonize the brain, although less efficiently than the wild-type strain.
Mol Microbiol 2007 May
PMID:Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence. 1746 22

Upstream open reading frames (uORFs) are common features of eukaryotic genes, occurring in 10%-25% of 5' leader sequences. Upstream ORFs that have been subjected to experimental analysis have been generally found to decrease translational efficiency of the downstream coding sequence. Previous investigations of uORFs in mammals and yeast have detected uORFs conserved over long evolutionary distances, prompting speculation about the nature and cause of the natural selection underlying such conservation. We have analyzed uORFs in the basidiomycetous fungal pathogen Cryptococcus neoformans to discern the properties of this purifying selection. We find that uORFs in the Cryptococcus species complex are conserved at twice the expected rate, and we report 122 uORFs that are conserved among all four sequenced Cryptococcus strains. A significantly greater proportion of uORF losses occur via direct mutation to the uORF start codon than expected. This observation suggests that mutational disruption of a uORF that leaves the start codon intact may be selectively disadvantageous, perhaps because of the risk of premature translation initiation. Accounting for this constrained mode of loss and comparing the relative conservation of uORFs between the 5' leader and control sequences enables us to calculate that at least a third of uORFs may be conserved for their effects on translational efficiency. The remaining fraction may be conserved either by chance or as a result of selective pressure to prevent premature translation initiation from the uORF start codon. We find that the majority of conserved uORFs do not exhibit codon usage bias or conservation at the amino acid level, and therefore they do not likely encode bioactive peptides. Our analysis suggests that uORFs are an important and underappreciated mechanism of post-transcriptional gene regulation in eukaryotes.
Mol Biol Evol 2007 Aug
PMID:Dual modes of natural selection on upstream open reading frames. 1749 29


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