NORMAL GENOMIC

• Idnurm A, Heitman J
• Ferrochelatase is a conserved downstream target of the blue light-sensingWhite collar complex in fungi.
• Microbiology. 2010; 156: 2393-407
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• Light is a universal signal perceived by organisms, including fungi, inwhich light regulates common and unique biological processes depending onthe species. Previous research has established that conserved proteins,originally called White collar 1 and 2 from the ascomycete Neurosporacrassa, regulate UV/blue light sensing. Homologous proteins function indistant relatives of N. crassa, including the basidiomycetes andzygomycetes, which diverged as long as a billion years ago. Here weconducted microarray experiments on the basidiomycete fungus Cryptococcusneoformans to identify light-regulated genes. Surprisingly, only a singlegene was induced by light above the commonly used twofold threshold. Thisgene, HEM15, is predicted to encode a ferrochelatase that catalyses thefinal step in haem biosynthesis from highly photoreactive porphyrins. TheC. neoformans gene complements a Saccharomyces cerevisiae hem15Deltastrain and is essential for viability, and the Hem15 protein localizes tomitochondria, three lines of evidence that the gene encodesferrochelatase. Regulation of HEM15 by light suggests a mechanism by whichbwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. Weshow that ferrochelatase is also light-regulated in a whitecollar-dependent fashion in N. crassa and the zygomycete Phycomycesblakesleeanus, indicating that ferrochelatase is an ancient target ofphotoregulation in the fungal kingdom.

• Idnurm A, Walton FJ, Floyd A, Reedy JL, Heitman J
• Identification of ENA1 as a virulence gene of the human pathogenic fungusCryptococcus neoformans through signature-tagged insertional mutagenesis.
• Eukaryot Cell. 2009; 8: 315-26
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• A library of more than 4,500 signature-tagged insertion mutants of thehuman pathogenic fungus Cryptococcus neoformans was generated, and asubset was screened in a murine inhalation model to identify genesrequired for virulence. New genes that regulate aspects of C. neoformansvirulence were also identified by screening the entire library for invitro phenotypes related to the ability to cause disease, includingmelanin production, growth at high temperature, and growth underconditions of nutrient limitation. A screen of 10% of the straincollection in mice identified an avirulent mutant strain with an insertionin the ENA1 gene, which is predicted to encode a fungus-specific sodium orpotassium P-type ATPase. The results of the deletion of the gene andcomplementation experiments confirmed its key role in mammalian virulence.ena1 mutant strains exhibited no change in sensitivity to high saltconcentrations but were sensitive to alkaline pH conditions, providingevidence that the fungus may have to survive at elevated pH duringinfection of the mammalian host. The mutation of the well-characterizedvirulence factor calcineurin (CNA1) also rendered C. neoformans strainssensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutantstrains were upregulated in response to high pH, and cna1 ena1 doublemutant strains exhibited increased sensitivity to elevated pH, indicatingthat at least two pathways in the fungus mediate survival under alkalineconditions. Signature-tagged mutagenesis is an effective strategy for thediscovery of new virulence genes in fungal pathogens of animals.

• Narasipura SD, Chaturvedi V, Chaturvedi S
• Characterization of Cryptococcus neoformans variety gattii SOD2 revealsdistinct roles of the two superoxide dismutases in fungal biology andvirulence.
• Mol Microbiol. 2005; 55: 1782-800
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• We studied superoxide dismutases (SODs) in the encapsulated yeastCryptococcus neoformans (Cn) variety gattii to analyse the role ofmitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 wascloned from a Cn cosmid library, sod2 mutant and sod2 + SOD2 reconstitutedstrains were constructed by homologous recombination, and two sod1sod2double mutants were constructed by replacing SOD2 in the sod1 mutant withthe sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids,with 36-66% identity with other fungal SOD2ps. SOD2 deletion rendered Cnhighly growth-defective at 37 degrees C in 19-20% oxygen (normal air), andthis defect was reversed by limiting oxygen to 1.3% as well in thepresence of antioxidant, ascorbic acid. The sod2 mutant accumulatedsignificantly more reactive oxygen species (ROS) at 37 degrees C as wellat 30 degrees C in the presence of antimycin A, suggesting that SOD2p isthe primary defence of Cn against the superoxide anion (O(2) (.-)) in themitochondria. The sod2 was also highly susceptible to redox-cyclingagents, high salt and nutrient limitations. The sod2 mutant was avirulentin intranasally infected mice and markedly attenuated in its virulence inintravenously infected mice. The virulence defect of sod2 mutant appearedrelated to its growth defects in high oxygen environment, but notresulting from increased sensitivity to oxidative killing by phagocytes.The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2double mutants showed a marked reduction in the activities of other knownCn virulence factors; and they were more susceptible to PMN killing thanwas the sod2 single mutant. Previously, we reported that the attenuationof sod1 mutant in mice was resulting from enhanced susceptibility tophagocyte killing, combined with a reduction in the activities of a numberof virulence factors. Thus, SOD1p and SOD2p play distinct roles in thebiology and virulence of Cn var. gattii via independent modes of action.

• Tsai CT, Ting JW, Wu MH, Wu MF, Guo IC, Chang CY
• Complete genome sequence of the grouper iridovirus and comparison ofgenomic organization with those of other iridoviruses.
• J Virol. 2005; 79: 2010-23
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• The complete DNA sequence of grouper iridovirus (GIV) was determined usinga whole-genome shotgun approach on virion DNA. The circular form genomewas 139,793 bp in length with a 49% G + C content. It contained 120predicted open reading frames (ORFs) with coding capacities ranging from62 to 1,268 amino acids. A total of 21% (25 of 120) of GIV ORFs areconserved in the other five sequenced iridovirus genomes, including DNAreplication, transcription, nucleotide metabolism, protein modification,viral structure, and virus-host interaction genes. The whole-genomenucleotide pairwise comparison showed that GIV virus was partiallycolinear with counterparts of previously sequenced ranaviruses (ATV andTFV). Besides, sequence analysis revealed that GIV possesses severalunique features which are different from those of other complete sequencediridovirus genomes: (i) GIV is the first ranavirus-like virus which hasbeen sequenced completely and which infects fish other than amphibians,(ii) GIV is the only vertebrate iridovirus without CpG sequencemethylation and lacking DNA methyltransferase, (iii) GIV contains a purinenucleoside phosphorylase gene which is not found in other iridoviruses orin any other viruses, (iv) GIV contains 17 sets of repeat sequence, withbasic unit sizes ranging from 9 to 63 bp, dispersed throughout the wholegenome. These distinctive features of GIV further extend our understandingof molecular events taking place between ranavirus and its hosts and theiridovirus evolution.

• Kraus PR et al.
• Identification of Cryptococcus neoformans temperature-regulated genes witha genomic-DNA microarray.
• Eukaryot Cell. 2004; 3: 1249-60
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• The ability to survive and proliferate at 37 degrees C is an essentialvirulence attribute of pathogenic microorganisms. A partial-genomemicroarray was used to profile gene expression in the human-pathogenicfungus Cryptococcus neoformans during growth at 37 degrees C. Genes withorthologs involved in stress responses were induced during growth at 37degrees C, suggesting that a conserved transcriptional program is used byC. neoformans to alter gene expression during stressful conditions. A geneencoding the transcription factor homolog Mga2 was induced at 37 degrees Cand found to be important for high-temperature growth. Genes encodingfatty acid biosynthetic enzymes were identified as potential targets ofMga2, suggesting that membrane remodeling is an important component ofadaptation to high growth temperatures. mga2Delta mutants were extremelysensitive to the ergosterol synthesis inhibitor fluconazole, indicating acoordination of the synthesis of membrane component precursors.Unexpectedly, genes involved in amino acid and pyrimidine biosynthesiswere repressed at 37 degrees C, but components of these pathways werefound to be required for high-temperature growth. Our findings demonstratethe utility of even partial-genome microarrays for delineating regulatorycascades that contribute to microbial pathogenesis.

• Lengeler KB et al.
• Mating-type locus of Cryptococcus neoformans: a step in the evolution ofsex chromosomes.
• Eukaryot Cell. 2002; 1: 704-18
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• The sexual development and virulence of the fungal pathogen Cryptococcusneoformans is controlled by a bipolar mating system determined by a singlelocus that exists in two alleles, alpha and a. The alpha and a mating-typealleles from two divergent varieties were cloned and sequenced. The C.neoformans mating-type locus is unique, spans >100 kb, and contains morethan 20 genes. MAT-encoded products include homologs of regulators ofsexual development in other fungi, pheromone and pheromone receptors,divergent components of a MAP kinase cascade, and other proteins with noobvious function in mating. The alpha and a alleles of the mating-typelocus have extensively rearranged during evolution and strain divergencebut are stable during genetic crosses and in the population. The C.neoformans mating-type locus is strikingly different from the other knownfungal mating-type loci, sharing features with the self-incompatibilitysystems and sex chromosomes of algae, plants, and animals. Our studyestablishes a new paradigm for mating-type loci in fungi with implicationsfor the evolution of cell identity and self/nonself recognition.