Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ∼20-megabase genome, which contains ∼6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes.

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.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a "leaky melanin" phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets.

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.

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.

Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic lne. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

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.

Cryptococcus neoformans is a major cause of systemic fungal infection in immunocompromised patients. Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyzes the transfer of myristate (C14:0) from myristoyl-CoA to the N-terminal glycine of a subset of cellular proteins produced during vegetative growth of C. neoformans. A Glyw --Asp mutation was introduced into C. neoformans NMT by targeted gene replacement. The resulting strains are temperaturesensitive myristic acid auxotrophs. They are killed at 37C when placed in medium lacking myristate and, in an immunosuppressed animal model ofcryptococcal meningitis, are completely eliminated from the subarachnoid space within 12 days of initial infection. C. neoformans and human Nmts exhibit differences in their peptide substrate specificities. These differences can be exploited to develop a new class of fungicidal drugs.Cryptococcus neoformans var. neoformans is an opportunistic pathogen which has emerged as a serious cause of systemic fungal infection in immunocompromised humans (1-3). Persistent C. neoformans infections are common in AIDS patients after completion of primary therapy with amphotericin B or fluconazole for meningitis. This leads to relapse rates of 50-60%o and a shortened lifespan unless patients receive lifelong suppressive therapy, generally with fluconazole (4, 5). Both amphotericin B and fluconazole target ergosterol, the principal sterol in the organism's plasma membrane. Amphotericin B, a fungicidal macrolide, also binds to cholesterol in animal cell membranes, resulting in renal and other toxicities. Chronic suppressive therapy with the fungistatic drug fluconazole in severely immunocompromised hosts will most likely result in emergence of resistant strains (6). The search for alternative therapeutic targets in this organism would be helped by the ability to use targeted gene disruption to establish whether a given gene product is essential for viability. Protocols for high-efficiency transformation of C. neoformans have been reported (7-9). Gene replacement has remained elusive and may be strain-or locus-specific (9, 10).Myristoyl-CoA:protein N-myristoyltransferase (Nmt) catalyzes the cotranslational transfer of myristate (C14:0) from myristoyl-CoA to the amino-terminal glycine of a subset of eukaryotic cellular and viral proteins (11). Saccharomyces cerevisiae and human Nmts have an ordered bi-bi reaction mechanism: the apoenzymes bind to myristoyl-CoA, forming a high-affinity binary complex. Formation of this complex allows generation of a functional peptide binding site and subsequent generation of a myristoyl-CoA/Nmt/peptide ternary complex. After catalysis, CoA and then the myristoylpeptide product are released (12-14).NMTgenes have been isolated from S. cerevisiae, Candida albicans, Histoplasma capsulatum, C. neoformans, and humans (15-18). All are present in a single copy per haploid genome. Several S. cerevisiae N-myristoylproteins are essential for viability and depend upon a covalently bound myristoyl group for expression ...