Mojca Benčina

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.

Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe

ABSTRACT A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 μm of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with N-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone or cyanide p-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.

Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit

The gene pkaC encoding the catalytic subunit of cAMP-dependent protein kinase has been isolated from the industrially important filamentous fungus Aspergillus niger. A probe for screening A. niger phage libraries was generated by a polymerase chain reaction using degenerate primers. cDNA and genomic DNA clones were isolated and sequenced. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with a calculated molecular mass of 53813 Da. The cAMP-dependent protein kinase catalytic subunit (PKA-C) from A. niger has a 126 amino acid extension at the N-terminus compared to the PKA-C of higher eukaryotes that-except for the first 15 amino acids, which are homologous to the Magnaporthe grisea PKA-C-shows no significant similarity to the N-terminal extension of PKA-C of other lower eukaryotes. The catalytic core of PKA-C of A. niger shows extensive homology with the PKA-C isolated from all other eukaryotes. Low-stringency hybridization did not reveal any other pkaC homologue in A. niger. The cloned pkaC was used for transformation of A. niger, leading to increased levels of pkaC mRNA and PKA-C activity. Transformants overexpressing pkaC were phenotypically different with respect to growth, showing a more compact colony morphology, accompanied by a more dense sporulation, especially on media containing trehalose and glycerol. A number of transformants also showed a strongly reduced or complete absence of sporulation. This phenotype was quickly lost upon propagation of the strains.

The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis

BackgroundThe antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger.ResultsWe determined an AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the α-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi.Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells.ConclusionsThe present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.

Cross-talk between cAMP and calcium signalling in Aspergillus niger

Very little is known about cross‐talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)‐dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon‐optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl‐cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP‐mediated, PKA‐dependent phosphorylation. Further evidence for cross‐talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar–apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA‐dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.

Highly active, citrate inhibition resistant form of Aspergillus niger 6-phosphofructo-1-kinase encoded by a modified pfkA gene

In Aspergillus niger cells spontaneous posttranslational modification of 6-phosphofructo-1-kinase (PFK1) occurs. In a two step process the native enzyme (85kDa) is first cleaved to an inactive fragment (49kDa) that regains its activity after phosphorylation of the protein. The shorter PFK1 fragment exhibits changed kinetics, such as resistance to citrate inhibition. In order to avoid spontaneous complex posttranslational modification, modified gene was prepared encoding an active shorter PFK1 fragment. Since no appropriate microbial strains with disrupted native pfkA genes were available, Aspergillus niger strain with reduced likelihood for spontaneous posttranslational modification of PFK1 has been chosen for in vivo tests. First, the appropriate length of a truncated gene was defined after a number of enzymes encoded by genes of different lengths had been tested. After adding sodium azide to the medium, phosphorylation was induced in the transformed hyphae to activate the shorter fragments which were subsequently screened for changed PFK1 kinetics. In the second step the responsible threonine residue was replaced with glutamic acid to elude the need for phosphorylation. An active shorter PFK1 fragment, resistant to citrate inhibition and activated to a higher level by fructose-2,6-bisphosphate with respect to the native enzyme was encoded directly from the modified gene.

Antiarrhythmic drug amiodarone displays antifungal activity, induces irregular calcium response and intracellular acidification of Aspergillus niger – Amiodarone targets calcium and pH homeostasis of A. niger

The rapidly developing resistance of fungi to antifungal drugs is a serious health problem. Todays drugs mainly target cell membrane composition and synthesis. Moreover, some of them have serious side effects. New antifungal drugs targeting different molecular pathways are necessary. Amiodarone, an FDA approved antiarrhythmic drug displays antifungal activity. It targets calcium and pH homeostasis. In concentrations above 25 μM, it inhibits the growth of the filamentous fungi Aspergillus niger. It triggers a biphasic calcium response accompanied by a high [Ca(2+)](c) resting level and an intracellular acidification from 7.5 to 6.0, both of which are concentration dependent. Both extracellular calcium and calcium from intracellular organelles are sources of the transient second cytosolic calcium peak, whose amplitude is 0.12 μM for cells treated with 0.1mM amiodarone. In P-type ATPase deficient A. niger strains pmrAΔ and pmcAΔ, the [Ca(2+)](c) resting level after amiodarone treatment is at least twice as high as that of the wild type, which correlates with fungal viability and hypersensitivity to amiodarone. A combination of amiodarone and amphotericin B is additive in terms of cell viability and cytosolic calcium influx. In contrast, a combination of azole drugs and amiodarone has a synergistic effect on the viability of fungi.

Protein kinase A signaling and calcium ions are major players in PAF mediated toxicity against Aspergillus niger

The Penicillium chrysogenum antifungal protein PAF is toxic against potentially pathogenic Ascomycetes. We used the highly sensitive aequorin‐expressing model Aspergillus niger to identify a defined change in cytoplasmic free Ca2+ dynamics in response to PAF. This Ca2+ signature depended on an intact positively charged lysine‐rich PAF motif. By combining Ca2+ measurements in A. niger mutants with deregulated cAMP/protein kinase A (PKA) signaling, we proved the interconnection of Ca2+ perturbation and cAMP/PKA signaling in the mechanistic function of PAF. A deep understanding of the mode of action of PAF is an invaluable prerequisite for its future application as new antifungal drug.

Stress mediated changes in expression of the pkaC gene, encoding the catalytic subunit of cAMP-dependent protein kinase, in Aspergillus niger.

The transcriptional regulation of the pkaC gene, encoding the catalytic subunit of cAMP-dependent protein kinase from Aspergillus niger was analysed under different environmental conditions. Quantitative determination of pkaC transcript showed a significant decrease in concentration of specific mRNA immediately after a temperature, hypoosmotic and hyperosmotic shock followed by stimulated synthesis. The amount of pkaC mRNA as well as PKA enzymatic activity steadily decreased during the initial phase of growth in 15 % sucrose medium while a slight increase was observed at the time of a change in morphology from bulbous cells to filamentous growth. Transcriptional alternation might be mediated by multiple putative stress elements in the promoter region of pkaC gene.