Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

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

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Sulfonylureas have antifungal activity and are potent inhibitors of Candida albicans acetohydroxyacid synthase

The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans, attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(N-((4-iodo-6-methoxypyrimidin-2-yl)carbamoyl)sulfamoyl)benzoate (10c), which has a K-i value of 3.8 nM for C. albicans AHAS and an MIC90 of 0.7 mu g/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell

Comparative Genomics of Serial Isolates of Cryptococcus neoformans Reveals Gene Associated With Carbon Utilization and Virulence

The opportunistic fungal pathogen Cryptococcus neoformans is a leading cause of mortality among the human immunodeficiency virus/acquired immunodeficiency syndrome population and is known for frequently causing life-threatening relapses. To investigate the potential contribution of in-host microevolution to persistence and relapse, we have analyzed two serial isolates obtained from a patient with acquired immunodeficiency syndrome who suffered an initial and relapse episode of cryptococcal meningoencephalitis. Despite being identical by multilocus sequence typing, the isolates differ phenotypically, exhibiting changes in key virulence factors, nutrient acquisition, metabolic profiles, and the ability to disseminate in an animal model. Whole-genome sequencing uncovered a clonal relationship, with only a few unique differences. Of these, two key changes are expected to explain the phenotypic differences observed in the relapse isolate: loss of a predicted AT-rich interaction domain protein and changes in copy number of the left and right arms of chromosome 12. Gene deletion of the predicted transcriptional regulator produced changes in melanin, capsule, carbon source use, and dissemination in the host, consistent with the phenotype of the relapse isolate. In addition, the deletion mutant displayed altered virulence in the murine model. The observed differences suggest the relapse isolate evolved subsequent to penetration of the central nervous system and may have gained dominance following the administration of antifungal therapy. These data reveal the first molecular insights into how the Cryptococcus neoformans genome changes during infection of humans and the manner in which microevolution progresses in this deadly fungal pathogen

Calcineurin Targets Involved in Stress Survival and Fungal Virulence

<div><p>Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen <i>Cryptococcus neoformans</i>. Calcineurin is activated by increased Ca²⁺ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in <i>C</i>. <i>neoformans</i> by employing phosphoproteomic TiO₂ enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a <i>bona fide</i> calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in <i>Saccharomyces cerevisiae</i>. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight <i>C</i>. <i>neoformans</i> as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.</p></div

Mutations of calcineurin-dependent phosphorylation sites promotes Crz1 nuclear localization and transcriptional activity at 24°C.

<p><b>(A)</b> Schematic diagram of the Crz1 protein drawn to scale. The yellow pins indicate the locations of 7 of the 12 predicted phosphorylation sites acted upon by calcineurin and mutated in the Crz1^7S-A strain; the green and red boxes show the PolyQ domain and the zinc finger domains, respectively. <b>(B)</b> The Crz1^WT (ECt3) and Crz1^7S-A (ECt335) strains were grown at 24°C and then shifted to 37°C for 15 minutes prior to fixation with 4% formaldehyde and visualized by direct fluorescence microscopy. GFP-Nop1 served as a nucleolar marker. <b>(C)</b> Quantification of cells with Crz1 nuclear localization at 24°C and after thermal stress (37°C) for 15 minutes. Error bars represent standard deviation; quantifications were conducted using three biological replicates. <b>(D)</b> To assay Crz1 gel migration mobility, the Crz1^WT (ECt3), Crz1^4S-A (ECt223), Crz1^6S-A (ECt231) and Crz1^7S-A (ECt335) strains were grown to log phase at 24°C and then shifted to 37°C for 1 hr in the presence or absence of 1 μg/ml FK506. Protein was extracted and analysed by western blot with mCherry antibody. <b>(E)</b> Expression levels of the <i>CHS6</i> gene in WT (H99), <i>crz1</i>Δ (AFA3-3), Crz1^WT, Crz1^6S-A, and Crz1^7S-A strains employed in panel 3D were compared. Strains were grown at 24°C in YPD medium with or without 1 μg/ml FK506. RNA was isolated and the expression of the <i>CHS6</i> gene was assessed by real-time PCR. Results shown are representative of three biological replicates. <b>(F)</b> The wild-type (H99), <i>cna1</i>Δ (KK1), <i>crz1</i>Δ (AFA3-3), Crz1^7S-A (ECt335 and ECt362), and Crz1^WT (ECt3 and ECt4) strains were grown in YPD media. Five 10-fold serial dilutions of each strain were spotted onto YPD containing the indicated additives and incubated at 30°C for 48 hours. Cultures with no additives were incubated at 30°C or 37°C for 48 hours or 39°C for 72 hours. CR: Congo Red; CFW: calcofluor white.</p

Identification of calcineurin-dependent targets.

<p><b>(A)</b> Venn diagram illustrating the number of potential calcineurin targets identified by the phosphopeptide screens and target overlap between the 44 phosphorylated proteins enriched by at least 2-fold in the two calcineurin-deficient samples (FK506 exposure or <i>cna1</i> mutation). Phosphopeptide profiles from WT (H99) cells grown at 25°C and shifted to 37°C for 1 hr and exposed or not to FK506 were aligned and compared (FK506). A similar comparison was performed for phosphopeptides profiles from the WT (strain H99) culture shifted from 25°C to 37°C and a <i>cna1</i>Δ (strain KK1) culture subjected to the same temperature shift (<i>cna1Δ</i>). Biological cultures were performed in duplicate for each condition tested and following trypsin digestion each sample was divided into three to access TiO2 enrichment and analytical reproducibility. <b>(B)</b> Functional category of the forty-four putative calcineurin targets. <b>(C)</b> Summary of potential calcineurin targets identified at 37°C. Note that this table only lists a subset of the potential calcineurin targets at 37°C with at least a 2-fold or greater signal difference (T-test p-value <0.05) in the two different screens and for which a characterized <i>S</i>. <i>cerevisiae</i> ortholog is known (based on best hit blast searches against the <i>S</i>. <i>cerevisiae</i> genome database) and does not include the identified but uncharacterized ORFs. The differential fold change signal for each individual phosphopeptide (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005873#ppat.1005873.s007" target="_blank">S2</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005873#ppat.1005873.s008" target="_blank">S3</a> Tables</b> for detailed and complete individual phosphopeptide data) within the same protein observed under the FK506 or <i>cna1</i>Δ condition is indicated. * denotes proteins characterized as components of PB/SGs in <i>C</i>. <i>neoformans</i>.</p

Sulfonylureas Have Antifungal Activity and Are Potent Inhibitors of Candida albicans Acetohydroxyacid Synthase

The sulfonylurea herbicides exert their activity by inhibiting plant acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acid biosynthesis pathway. It has previously been shown that if the gene for AHAS is deleted in Candida albicans, attenuation of virulence is achieved, suggesting AHAS as an antifungal drug target. Herein, we have cloned, expressed, and purified C. albicans AHAS and shown that several sulfonylureas are inhibitors of this enzyme and possess antifungal activity. The most potent of these compounds is ethyl 2-(<i>N</i>-((4-iodo-6-methoxypyrimidin-2-yl)­carbamoyl)­sulfamoyl)­benzoate (<b>10c</b>), which has a <i>K</i>_i value of 3.8 nM for C. albicans AHAS and an MIC₉₀ of 0.7 μg/mL for this fungus in cell-based assays. For the sulfonylureas tested there was a strong correlation between inhibitory activity toward C. albicans AHAS and fungicidal activity, supporting the hypothesis that AHAS is the target for their inhibitory activity within the cell

Phenotypes of the calcineurin target mutants exposed to various stresses.

<p>Spot dilution assays with WT (H99), <i>cna1</i>Δ (HP243), <i>crz1</i>Δ (HP235), <i>lhp1</i>Δ (HP22), <i>pbp1</i>Δ (HP6), <i>puf4</i>Δ (HP17), <i>vts1</i>Δ (HP24), <i>anb1</i>Δ (HP36), <i>tif3</i>Δ (HP9), <i>gcd2</i>Δ (HP28), and <i>gwo1</i>Δ (HP1) mutants were performed under several stress conditions as indicated. Strain cultures were incubated overnight, serially diluted 10-fold, and plated on YPD medium without or with FK506, CaCl₂, Congo red (CR), dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and tunicamycin (TM) at the indicated concentrations. Cells were incubated for 2 to 3 days at 30°C, 37°C, 38°C, or 39°C as indicated and all cultures with compound additions were incubated at 30°C. Results shown are representative of two independent experimental replicates.</p