Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30°C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37°C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3⌬ and the csr2⌬ mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.

Yeast DNA polymerase ␦ (Pol␦) has three subunits of 125, 58, and 55 kDa. The gene for the 125-kDa catalytic subunit (POL3) has been known for several years. Here we describe the cloning of the genes for the 58-and 55-kDa subunits using peptide sequence analysis and searching of the yeast genome data base. The 58-kDa subunit, encoded by the POL31 gene, shows 23-28% sequence similarity to the 48-kDa subunit of human Pol␦ and to S. pombe Cdc1. POL31 is allelic to HYS2 and SDP5. The 55-kDa subunit is encoded by the POL32 gene (ORF YJR043c in the yeast data base). Very limited sequence similarity was observed between Pol32p and Schizosaccharomyces pombe Cdc27, the functionally analogous subunit in S. pombe Pol␦. The POL32 gene is not essential, but a deletion mutant shows cold sensitivity for growth and is sensitive to hydroxyurea and DNA damaging agents. In addition, lethality was observed when the POL32 deletion mutation was combined with conditional mutations in either the POL3 or POL31 gene. Pol32⌬ strains are weak antimutators and are defective for damage-induced mutagenesis. The POL32 gene product binds proliferating cell nuclear antigen. A gel filtration analysis showed that Pol32p is a dimer in solution. When POL31 and POL32 were co-expressed in Escherichia coli, a tetrameric (Pol31p⅐Pol32p) 2 species was detected by gel filtration, indicating that the two subunits form a complex. DNA polymerase ␦ (Pol␦)1 is the major replicative DNA polymerase in the eukaryotic cell. This insight is based on extensive in vitro studies using the simian 40 virus as a model system, and on genetic and biochemical studies in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe (reviewed in Refs. 1 and 2). These genetic studies have not only shown a role for Pol␦ in bulk DNA replication but also for maintaining genome fidelity via the proofreading exonuclease activity of this enzyme (3, 4). A similar, but perhaps less defined role has also been identified for DNA polymerase ⑀, whereas the synthetic function of DNA polymerase ␣-primase appears to be limited to that of initiator RNA-DNA synthesis for priming Okazaki fragments on the lagging strand of the DNA replication fork (5-7).The best characterized Pol␦ from mammalian cells is the enzyme purified from fetal calf thymus tissue, a heterodimer with a catalytic subunit of 125 kDa and a second subunit of 48 kDa (8). The small subunit is required for efficient stimulation of the polymerase processivity by the proliferating cell nuclear antigen (PCNA) (9, 10).The forms of Pol␦ isolated from bakers' and fission yeast are more complex. Our previous studies of S. cerevisiae Pol␦ indicated that the enzyme might consist of three or more subunits (11). Very recently, Pol␦ has been isolated from S. pombe as an enzyme with five distinct subunits, four of which also appear to be subunits based on genetic arguments (12).In this paper we describe an improved purification of S. cerevisiae Pol␦ as a three-subunit enzyme, the cloning of the two small subunits of Pol␦, and a genetic and ...

SummaryCell wall biogenesis and integrity are crucial for fungal growth, pathogenesis and survival, and are attractive targets for antifungal therapy. In this study, we identify, delete and analyse mutant strains for 10 genes involved in the PKC1 signal transduction pathway and its regulation in Cryptococcus neoformans . The kinases Bck1 and Mkk2 are critical for maintaining integrity, and deletion of each of these causes severe phenotypes different from each other. In stark contrast to results seen in Saccharomyces cerevisiae , a deletion in LRG1 has severe repercussions for the cell, and one in ROM2 has little effect. Also surprisingly, the phosphatase Ppg1 is crucial for cell integrity. These data indicate that the mechanisms of maintaining cell integrity differ between the two fungi. Deletions in SSD1 and PUF4 , potential alternative regulators of cell integrity, also exhibit phenotypes. This is the first comprehensive analysis examining genes involved the maintenance of cell integrity in C. neoformans and sets the foundation for future biochemical and virulence studies.

Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a "top-down" approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1⌬ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

Yeast DNA polymerase ␦ (Pol␦) consists of three subunits encoded by the POL3, POL31, and POL32 genes. Each of these genes was cloned under control of the galactose-inducible GAL1-10 promoter and overexpressed in various combinations. Overexpression of all three genes resulted in a 30-fold overproduction of Pol␦, which was identical in enzymatic properties to Pol␦ isolated from a wild-type yeast strain. Whereas overproduction of POL3 together with POL32 did not lead to an identifiable Pol3p⅐Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 and POL31. This two-subunit complex, designated Pol␦*, is structurally and functionally analogous to mammalian Pol␦. The properties of Pol␦* and Pol␦ were compared. A gel filtration analysis showed that Pol␦* is a heterodimer (Pol3p⅐Pol31p) and Pol␦ a dimer of a heterotrimer, (Pol3p⅐Pol31p⅐Pol32p) 2 . In the absence of proliferating cell nuclear antigen (PCNA), Pol␦* showed a processivity of 2-3 on poly(dA)⅐oligo(dT) compared with 5-10 for Pol␦. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Pol␦* on a natural DNA template was dependent on PCNA and on replication factor C. However, Pol␦*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Pol␦ was achieved upon addition of Pol32p to Pol␦*.

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SummaryThe polysaccharide b-1,6-glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in b-1,6-glucan synthesis. The H99 deletion mutants kre5D and kre6Dskn1D contained less cell wall b-1,6-glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI-anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5D and kre6Dskn1D. Our results indicate that KRE5, KRE6 and SKN1 are involved in b-1,6-glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.