Hrk1 Plays Both Hog1-Dependent and -Independent Roles in Controlling Stress Response and Antifungal Drug Resistance in Cryptococcus neoformans

The HOG (High Osmolarity Glycerol response) pathway plays a central role in controlling stress response, ergosterol biosynthesis, virulence factor production, and differentiation of Cryptococcus neoformans, which causes fatal fungal meningoencephalitis. Recent transcriptome analysis of the HOG pathway discovered a Hog1-regulated gene (CNAG_00130.2), encoding a putative protein kinase orthologous to Rck1/2 in Saccharomyces cerevisiae and Srk1 in Schizosaccharomyces pombe. Its function is not known in C. neoformans. The present study functionally characterized the role of Hrk1 in C. neoformans. Northern blot analysis confirmed that HRK1 expression depends on the Hog1 MAPK. Similar to the hog1Δ mutant, the hrk1Δ mutant exhibited almost complete resistance to fludioxonil, which triggers glycerol biosynthesis via the HOG pathway. Supporting this, the hrk1Δ mutant showed reduced intracellular glycerol accumulation and swollen cell morphology in response to fludioxonil, further suggesting that Hrk1 works downstream of the HOG pathway. However, Hrk1 also appeared to have Hog1-independent functions. Mutation of HRK1 not only further increased osmosensitivity of the hog1Δ mutant, but also suppressed increased azole-resistance of the hog1Δ mutant in an Erg11-independent manner. Furthermore, unlike the hog1Δ mutant, Hrk1 was not involved in capsule biosynthesis. Hrk1 was slightly involved in melanin production but dispensable for virulence of C. neoformans. These findings suggest that Hrk1 plays both Hog1-dependent and –independent roles in stress and antifungal drug susceptibility and virulence factor production in C. neoformans. Particularly, the finding that inhibition of Hrk1 substantially increases azole drug susceptibility provides a novel strategy for combination antifungal therapy

The Two-Component Sensor Kinase TcsC and Its Role in Stress Resistance of the Human-Pathogenic Mold Aspergillus fumigatus

Two-component signaling systems are widespread in bacteria, but also found in fungi. In this study, we have characterized TcsC, the only Group III two-component sensor kinase of Aspergillus fumigatus. TcsC is required for growth under hyperosmotic stress, but dispensable for normal growth, sporulation and conidial viability. A characteristic feature of the ΔtcsC mutant is its resistance to certain fungicides, like fludioxonil. Both hyperosmotic stress and treatment with fludioxonil result in a TcsC-dependent phosphorylation of SakA, the final MAP kinase in the high osmolarity glycerol (HOG) pathway, confirming a role for TcsC in this signaling pathway. In wild type cells fludioxonil induces a TcsC-dependent swelling and a complete, but reversible block of growth and cytokinesis. Several types of stress, such as hypoxia, exposure to farnesol or elevated concentrations of certain divalent cations, trigger a differentiation in A. fumigatus toward a “fluffy” growth phenotype resulting in white, dome-shaped colonies. The ΔtcsC mutant is clearly more susceptible to these morphogenetic changes suggesting that TcsC normally antagonizes this process. Although TcsC plays a role in the adaptation of A. fumigatus to hypoxia, it seems to be dispensable for virulence

RNA editing signature during myeloid leukemia cell differentiation

Adenosine deaminases acting on RNA (ADARs) are key proteins for hematopoietic stem cell self-renewal and for survival of differentiating progenitor cells. However, their specific role in myeloid cell maturation has been poorly investigated. Here we show that ADAR1 is present at basal level in the primary myeloid leukemia cells obtained from patients at diagnosis as well as in myeloid U-937 and THP1 cell lines and its expression correlates with the editing levels. Upon phorbol-myristate acetate or Vitamin D3/granulocyte macrophage colony-stimulating factor (GM-CSF)-driven differentiation, both ADAR1 and ADAR2 enzymes are upregulated, with a concomitant global increase of A-to-I RNA editing. ADAR1 silencing caused an editing decrease at specific ADAR1 target genes, without, however, interfering with cell differentiation or with ADAR2 activity. Remarkably, ADAR2 is absent in the undifferentiated cell stage, due to its elimination through the ubiquitin–proteasome pathway, being strongly upregulated at the end of the differentiation process. Of note, peripheral blood monocytes display editing events at the selected targets similar to those found in differentiated cell lines. Taken together, the data indicate that ADAR enzymes play important and distinct roles in myeloid cells

Fungal Adenylyl Cyclase Acts As a Signal Sensor and Integrator and Plays a Central Role in Interaction with Bacteria

10.1371/journal.ppat.1003612PLoS Pathogens910

N-Acetylglucosamine Induces White to Opaque Switching, a Mating Prerequisite in Candida albicans

To mate, the fungal pathogen Candida albicans must undergo homozygosis at the mating-type locus and then switch from the white to opaque phenotype. Paradoxically, opaque cells were found to be unstable at physiological temperature, suggesting that mating had little chance of occurring in the host, the main niche of C. albicans. Recently, however, it was demonstrated that high levels of CO2, equivalent to those found in the host gastrointestinal tract and select tissues, induced the white to opaque switch at physiological temperature, providing a possible resolution to the paradox. Here, we demonstrate that a second signal, N-acetylglucosamine (GlcNAc), a monosaccharide produced primarily by gastrointestinal tract bacteria, also serves as a potent inducer of white to opaque switching and functions primarily through the Ras1/cAMP pathway and phosphorylated Wor1, the gene product of the master switch locus. Our results therefore suggest that signals produced by bacterial co-members of the gastrointestinal tract microbiota regulate switching and therefore mating of C. albicans

β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3−) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions

Canonical A-to-I and C-to-U RNA Editing Is Enriched at 3′UTRs and microRNA Target Sites in Multiple Mouse Tissues

RNA editing is a process that modifies RNA nucleotides and changes the efficiency and fidelity of the central dogma. Enzymes that catalyze RNA editing are required for life, and defects in RNA editing are associated with many diseases. Recent advances in sequencing have enabled the genome-wide identification of RNA editing sites in mammalian transcriptomes. Here, we demonstrate that canonical RNA editing (A-to-I and C-to-U) occurs in liver, white adipose, and bone tissues of the laboratory mouse, and we show that apparent non-canonical editing (all other possible base substitutions) is an artifact of current high-throughput sequencing technology. Further, we report that high-confidence canonical RNA editing sites can cause non-synonymous amino acid changes and are significantly enriched in 3′ UTRs, specifically at microRNA target sites, suggesting both regulatory and functional consequences for RNA editing

Comparative Genomics of Bordetella pertussis Reveals Progressive Gene Loss in Finnish Strains

BACKGROUND: Bordetella pertussis is a gram-negative bacterium that infects the human respiratory tract and causes pertussis or whooping cough. The disease has resurged in many countries including Finland where the whole-cell pertussis vaccine has been used for more than 50 years. Antigenic divergence has been observed between vaccine strains and clinical isolates in Finland. To better understand genome evolution in B. pertussis circulating in the immunized population, we developed an oligonucleotide-based microarray for comparative genomic analysis of Finnish strains isolated during the period of 50 years. METHODOLOGY/PRINCIPAL FINDINGS: The microarray consisted of 3,582 oligonucleotides (70-mer) and covered 94% of 3,816 ORFs of Tohama I, the strain of which the genome has been sequenced. Twenty isolates from 1953 to 2004 were studied together with two Finnish vaccine strains and two international reference strains. The isolates were selected according to their characteristics, e.g. the year and place of isolation and pulsed-field gel electrophoresis profiles. Genomic DNA of the tested strains, along with reference DNA of Tohama I strain, was labelled and hybridized. The absence of genes as established with microarrays, was confirmed by PCR. Compared with the Tohama I strain, Finnish isolates lost 7 (8.6 kb) to 49 (55.3 kb) genes, clustered in one to four distinct loci. The number of lost genes increased with time, and one third of lost genes had functions related to inorganic ion transport and metabolism, or energy production and conversion. All four loci of lost genes were flanked by the insertion sequence element IS481. CONCLUSION/SIGNIFICANCE: Our results showed that the progressive gene loss occurred in Finnish B. pertussis strains isolated during a period of 50 years and confirmed that B. pertussis is dynamic and is continuously evolving, suggesting that the bacterium may use gene loss as one strategy to adapt to highly immunized populations

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

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives