Characterization of Aspergillus nidulans α‐glucan synthesis: roles for two synthases and two amylases

Cell walls are essential for fungal survival and growth. Fungal walls are ～ 90% carbohydrate, mostly types not found in humans, making them promising targets for anti‐fungal drug development. Echinocandins, which inhibit the essential β‐glucan synthase, are already clinically available. In contrast, α‐glucan, another abundant fungal cell wall component has attracted relatively little research attention because it is not essential for most fungi. Aspergillus nidulans has two α‐glucan synthases (AgsA and AgsB) and two α‐amylases (AmyD and AmyG), all of which affect α‐glucan synthesis. Gene deletion showed that AgsB was the major synthase. In addition, AmyG promoted α‐glucan synthesis whereas AmyD had a repressive effect. The lack of α‐glucan had no phenotypic impact on solid medium, but reduced conidial adhesion during germination in shaken liquid. Moreover, α‐glucan level correlated with resistance to Calcofluor White. Intriguingly, overexpression of agsA could compensate for the loss of agsB at the α‐glucan level, but not for phenotypic defects. Thus, products of AgsA and AgsB have different roles in the cell wall, consistent with agsA being mainly expressed at conidiation. These results suggest that α‐glucan contributes to drug sensitivity and conidia adhesion in A. nidulans, and is differentially regulated by two synthases and two amylases.     

Functional characterization of Pneumocystis carinii brl1 by transspecies complementation analysis

Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ectopic expression of this gene rescues null alleles of essential nuclear membrane proteins of the Brr6/Brl1 family in both yeasts.     

Correlation Between Pneumocystis jirovecii Mitochondrial Genotypes and High and Low Fungal Loads Assessed by Single Nucleotide Primer Extension Assay and Quantitative Real‐Time PCR

We designed a single nucleotide primer extension (SNaPshot) assay for Pneumocystis jirovecii genotyping, targeting mt85 SNP of the mitochondrial large subunit ribosomal RNA locus, to improve minority allele detection. We then analyzed 133 consecutive bronchoalveolar lavage (BAL) fluids tested positive for P. jirovecii DNA by quantitative real‐time PCR, obtained from two hospitals in different locations (Hospital 1 [n = 95] and Hospital 2 [n = 38]). We detected three different alleles, either singly (mt85C: 39.1%; mt85T: 24.1%; mt85A: 9.8%) or together (27%), and an association between P. jirovecii mt85 genotype and the patient's place of hospitalization (p = 0.011). The lowest fungal loads (median = 0.82 × 10³ copies/μl; range: 15–11 × 10³) were associated with mt85A and the highest (median = 1.4 × 10⁶ copies/μl; range: 17 × 10³–1.3 × 10⁷) with mt85CTA (p = 0.010). The ratios of the various alleles differed between the 36 mixed‐genotype samples. In tests of serial BALs (median: 20 d; range 4–525) from six patients with mixed genotypes, allele ratio changes were observed five times and genotype replacement once. Therefore, allele ratio changes seem more frequent than genotype replacement when using a SNaPshot assay more sensitive for detecting minority alleles than Sanger sequencing. Moreover, because microscopy detects only high fungal loads, the selection of microscopy‐positive samples may miss genotypes associated with low loads.     

<Emphasis Type="BoldItalic">Pneumocystis Jirovecii</Emphasis> Pneumonia: Current Knowledge and Outstanding Public Health Issues

Antiretroviral therapy and cotrimoxazole prophylaxis have caused a decline in Pneumocystis jirovecii incidence in developing countries since the 1990s. Nevertheless, Pneumocystis jirovecii pneumonia (PCP) remains the primary AIDS-defining disease around the world. Much about the disease remains unknown, including its global burden, best diagnostic practices, the frequency of drug resistance, and the risks associated with Pneumocystis colonization. This review describes current knowledge about Pneumocystis infection and highlights areas where new research could benefit public health.     

Structural characterization of CA1462, the <Emphasis Type="Italic">Candida albicans</Emphasis> thiamine pyrophosphokinase

Background  

In search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential. Since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria.     

Composition of the cell walls of several yeast species

Cell walls, representing 26%–32% of the cell dry weight, were prepared from several strains of the yeasts Kloeckera apiculata, Debaryomyces hansenii, Zygosaccharomyces bailii,Kluyveromyces marxianus and Saccharomyces cerevisiae. Extraction of the walls with potassium hydroxide at 4 °C, followed by saturation of the alkali-soluble extract with ammonium sulphate gave fractions of mannoprotein, alkali-soluble glucan and alkali-insoluble glucan. Chitin was associated with the alkali-insoluble glucan. The proportions of the different fractions within the walls varied with the species and strain. Mannoprotein comprised between 25% and 34% of the walls, the content of alkali-insoluble glucan ranged from 15% to 48%, and the content of alkali-soluble glucan ranged from 10% to 48%. There was significant variation in the physical appearance of the alkali-soluble glucans and the relative viscosity of suspensions of these glucans. The yeasts could represent novel sources of polysaccharides with industrial and medical applications. Received: 30 December 1997 / Received revision: 24 March 1998 / Accepted: 27 March 1998     

Multiple β-1,3 Glucanases in the Lytic Enzyme Complex of Bacillus circulans WL 12

The lytic enzyme complex produced by Bacillus circulans WL 12 upon induction with the myceiia of Piricularia oryzae P₂ was analyzed by large scale polyacrylamide gel electrophoresis, Six β-1,3 glucanases were separated. Activity profiles were obtained of these multiple β-1,3 glucanases against P. oryzae P₂ cell walls, Saccharomyces cerevisiae walls, laminarin, oat glucan, Phytophthora glucan and pachyman.     

Callose (β-1,3 glucan) is essential for <Emphasis Type="Italic">Arabidopsis</Emphasis>pollen wall patterning,but not tube growth

Background  

Callose (β-1,3 glucan) separates developing pollen grains, preventing their underlying walls (exine) from fusing. The pollen tubes that transport sperm to female gametes also contain callose, both in their walls as well as in the plugs that segment growing tubes. Mutations in CalS5, one of several Arabidopsis β-1,3 glucan synthases, were previously shown to disrupt callose formation around developing microspores, causing aberrations in exine patterning, degeneration of developing microspores, and pollen sterility.     

Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3 and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromise Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90‐dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network and reveals new targets for development of antifungal drugs.     

New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.     

Deletion of individual mRNA capping genes is unexpectedly not lethal to Candida albicans and results in modified mRNA cap structures

Eukaryotic mRNAs are modified at the 5′ end with a cap structure. In fungal cells, the formation of the mRNA cap structure is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and methyltransferase. Fungal capping enzymes have been proposed to be good antifungal targets because they differ significantly from their human counterparts and the genes encoding these enzymes are essential in Saccharomyces cerevisiae. In the present study, Candida albicans null mutants were constructed for both the mRNA triphosphatase-encoding gene (CET1) and the mRNA methyltransferase encoding gene (CCM1), proving that these genes are not essential in C. albicans. Heterozygous deletions were generated, but no null mutants were isolated for the guanylyltransferase-encoding gene (CGT1), indicating that this gene probably is essential in C. albicans. Whereas these results indicate that Cet1p and Ccm1p are not ideal molecular targets for development of anticandidal drugs, they do raise questions about the capping of mRNA and translation initiation in this fungus. Southern blot analysis of genomic DNA indicates that there are not redundant genes for CET1 and CCM1 and analysis of mRNA cap structures indicate there are not alternative pathways compensating for the function of CET1 or CCM1 in the null mutants. Instead, it appears that C. albicans can survive with modified mRNA cap structures.     

Metabolite profiling studies in Saccharomyces cerevisiae: an assisting tool to prioritize host targets for antiviral drug screening

Background  

The cellular proteins Pat1p, Lsm1p, and Dhh1p are required for the replication of some positive-strand viruses and therefore are potential targets for new antiviral drugs. To prioritize host targets for antiviral drug screening a comparative metabolome analysis in Saccharomyces cerevisiae reference strain BY4742 Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 and deletion strains pat1Δ, lsm1Δ and dhh1Δ was performed.     

Roles for the Conserved Spc105p/Kre28p Complex in Kinetochore-Microtubule Binding and the Spindle Assembly Checkpoint

Background

Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain ∼70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores.

Principal Findings

We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells.

Conclusions/Significance

Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.     

Arabinoxylan, β‐glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo‐SEM

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo‐SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of β‐glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo‐SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with β‐glucan depositions on the inside. During germination, arabinoxylan and β‐glucan are hydrolysed, but unlike β‐glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.     

Comparing the van Oosterhout and Chybicki‐Burczyk methods of estimating null allele frequencies for inbred populations

In spite of the usefulness of codominant markers in population genetics, the existence of null alleles raises challenging estimation issues in natural populations that are characterized by positive inbreeding coefficients (F > 0). Disregarding the possibility of F > 0 in a population will generally lead to overestimates of null allele frequencies. Conversely, estimates of inbreeding coefficients (F) may be strongly biased upwards (excess homozygotes), in the presence of nontrivial frequencies of null alleles. An algorithm has been presented for the estimation of null allele frequencies in inbred populations (van Oosterhout method), using external estimates of the F‐statistics. The goal of this study is to introduce a modification of this method and to provide a formal comparison with an alternative likelihood‐based method (Chybicki‐Burczyk). Using simulated data, we illustrate the strengths and limitations of these competing methods. Under most circumstances, the likelihood method is preferable, but for highly inbred organisms, a modified van Oosterhout method offers some advantages.     

Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.Abbreviations BSA bovine serum albumin - Con A concanavalin A - SDS sodium dodecyl sulphate