Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts.

Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.

Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans.

We showed previously that a DeltafluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG ( approximately 400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of DeltafluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from DeltaflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

Conserved cAMP signaling cascades regulate fungal development and virulence.

Two well characterized signal transduction cascades regulating fungal development and virulence are the MAP kinase and cAMP signaling cascades. Here we review the current state of knowledge on cAMP signaling cascades in fungi. While the processes regulated by cAMP signaling in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved. Fungal cAMP signaling cascades are also quite versatile, which is apparent from the differential regulation of similar biological processes. In this review we compare and contrast cAMP signaling pathways that regulate development in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and differentiation and virulence in the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. We also present examples of interaction between the cAMP and MAP kinase signaling cascades in the regulation of fungal development and virulence.

Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans.

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.

Effect of hyperthermia on the transcription rate of heat-shock genes in the rabbit cerebellum and retina assayed by nuclear run-ons.

The induction of heat-shock protein 70 (hsp70) mRNA in the hyperthermic rabbit brain has been examined previously by using Northern blotting and in situ hybridization procedures that measure steady-state levels of mRNA, which may be influenced by transcript stability and transcription rate. In the present investigation, the in vivo transcription rate of hsp70 has been examined by using run-on transcription assays on isolated brain nuclei. A major up-regulation in the transcription rate of hsp70 was observed between 0.75 and 1.50 hours after hyperthermia in the cerebellum and the retina. Gel-mobility shift assays revealed that the time course of conversion of heat-shock transcription factor (HSF1) to a DNA-binding form paralleled the transcriptional induction profile of hsp70. The transcription rates of several nonheat-shock genes were also studied in the hyperthermic brain, and little change was noted relative to the induction of hsp70. Thus, a physiologically relevant increase in temperature of 2.5 degrees C induces a major up-regulation in the in vivo transcription rate of hsp70 in the nervous system with little affect on the transcription rates of other genes.

Formation of reactive metabolites of phenacetin in humans and rats.

The metabolism of phenacetin to reactive intermediates in humans was estimated from the excretion of thio adducts in urine. N-Hydroxyphenacetin, a precursor of reactive metabolites, was also quantified. Following an oral dose of phenacetin (10 mg/kg) to humans, these metabolites in 24 h urine were: paracetamol-3-cysteine, 4.4% dose; paracetamol-3-mercapturate, 3.9%; 3-thiomethylparacetamol, 0.4%; N-hydroxyphenacetin, 0.5%. Rats showed a considerable increase in N-hydroxyphenacetin excretion after chronic dosing with phenacetin at high dosage (500 mg/kg) for one month. chronic dosing with a low dose (50 mg/kg) did not increase N-hydroxyphenacetin excretion, but a marked increase occurred on concomitant administration of aspirin and caffeine.

An audit of therapeutic drug monitoring procedures--use of theophylline assay services.

Monitoring therapy involving drugs exhibiting high intersubject variation and/or narrow therapeutic index has become more generally accepted in hospitals. This paper describes an audit conducted jointly by clinical pharmacists and clinical biochemists into the use and therapeutic value of a drug monitoring service in a general hospital using theophylline as a model. Assay values were categorized as 'subtherapeutic', 'therapeutic' or 'potentially toxic'. The reporting procedures involving the two departments were evaluated along with action taken by the requesting clinicians. The study showed that 60% of the theophylline assay results fell within the therapeutic range at the time of sampling. However only 38% of samples were considered to have been taken under steady state conditions. There was a significant delay in relaying the results to the clinicians. Procedural changes involving sampling times, analytical techniques, recording and conveying results to clinicians and co-operative policies between the departments of pharmacy and clinical chemistry have been implemented as a result of this study.