Genomic Discovery and Structure-Activity Exploration of a Novel Family of Enzyme-Activated Covalent Cyclin-Dependent Kinase Inhibitors.

Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD3), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2, which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43, with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD3 to identify mechanistically and structurally novel molecules for the development of new medicines.

The lncRNA RZE1 Controls Cryptococcal Morphological Transition.

In the fungal pathogen Cryptococcus neoformans, the switch from yeast to hypha is an important morphological process preceding the meiotic events during sexual development. Morphotype is also known to be associated with cryptococcal virulence potential. Previous studies identified the regulator Znf2 as a key decision maker for hypha formation and as an anti-virulence factor. By a forward genetic screen, we discovered that a long non-coding RNA (lncRNA) RZE1 functions upstream of ZNF2 in regulating yeast-to-hypha transition. We demonstrate that RZE1 functions primarily in cis and less effectively in trans. Interestingly, RZE1's function is restricted to its native nucleus. Accordingly, RZE1 does not appear to directly affect Znf2 translation or the subcellular localization of Znf2 protein. Transcriptome analysis indicates that the loss of RZE1 reduces the transcript level of ZNF2 and Znf2's prominent downstream targets. In addition, microscopic examination using single molecule fluorescent in situ hybridization (smFISH) indicates that the loss of RZE1 increases the ratio of ZNF2 transcripts in the nucleus versus those in the cytoplasm. Taken together, this lncRNA controls Cryptococcus yeast-to-hypha transition through regulating the key morphogenesis regulator Znf2. This is the first functional characterization of a lncRNA in a human fungal pathogen. Given the potential large number of lncRNAs in the genomes of Cryptococcus and other fungal pathogens, the findings implicate lncRNAs as an additional layer of genetic regulation during fungal development that may well contribute to the complexity in these "simple" eukaryotes.

Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

Transcriptome Analysis of a Ustilago maydis ust1 Deletion Mutant Uncovers Involvement of Laccase and Polyketide Synthase Genes in Spore Development.

Ustilago maydis, causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic budding haploid and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing tumorous structures, and only within these does the fungus sporulate, producing melanized sexual teliospores. Previously we identified Ust1, an APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) transcription factor, whose deletion led to filamentous haploid growth and the production of highly pigmented teliospore-like structures in culture. In this study, we analyzed the transcriptome of a ust1 deletion mutant and functionally characterized two highly upregulated genes with potential roles in melanin biosynthesis: um05361, encoding a putative laccase (lac1), and um06414, encoding a polyketide synthase (pks1). The Δlac1 mutant strains showed dramatically reduced virulence on maize seedlings and fewer, less-pigmented teliospores in adult plants. The Δpks1 mutant was unaffected in seedling virulence but adult plant tumors generated hyaline, nonmelanized teliospores. Thus, whereas pks1 appeared to be restricted to the synthesis of melanin, lac1 showed a broader role in virulence. In conclusion, the ust1 deletion mutant provided an in vitro model for sporulation in U. maydis, and functional analysis supports the efficacy of this in vitro mutant analysis for identification of genes involved in in planta teliosporogenesis.

Non-coding RNAs in the development and pathogenesis of eukaryotic microbes.

RNA has long been regarded as the important intermediary in the central dogma of gene expression. Recently, the importance of RNAs in the regulation of gene expression became evident with the identification and characterization of non-protein coding transcripts named non-coding RNAs (ncRNAs). The ncRNAs, small and long, are ubiquitously present in all three domains of life and are being recognized for their important roles in genome defense and development. Some of the ncRNAs have been associated with diseases, and therefore, they offer diagnostic and therapeutic potential. In this mini-review, we have highlighted some recent research on the ncRNAs identified in eukaryotic microbes, with special emphasis on fungi that are pathogenic to humans or plants when possible. It is our contention that further elucidation and understanding of ncRNAs will advance our understanding of the development and pathogenesis of eukaryotic microbes and offer alternatives in the diagnosis and treatment of the diseases caused by these pathogens.

Deletion of the Ustilago maydis ortholog of the Aspergillus sporulation regulator medA affects mating and virulence through pheromone response.

Mating of compatible haploid cells of Ustilago maydis is essential for infection and disease development in the host. For mating and subsequent filamentous growth and pathogenicity, the transcription factor, prf1 is necessary. Prf1 is in turn regulated by the cAMP and MAPK pathways and other regulators like rop1 and hap1. Here we describe the identification of another putative Prf1 regulator, med1, the ortholog of the Aspergillus nidulans medusa (medA) transcription factor and show that it is required for mating and full virulence in U. maydis. med1 deletion mutants show both pre- and post-mating defects and are unresponsive to external pheromone. The expression of prf1 is down-regulated in Δmed1 compared to the wild type, suggesting that med1 is upstream of prf1. Additionally, indicative of a role in secondary metabolism regulation, deletion of the med1 gene de-represses the production of glycolipids in U. maydis.