Enhanced activity of strobilurin and fludioxonil by using berberine and phenolic compounds to target fungal antioxidative stress response.

AIMS: Identify natural products that effectively target antioxidative signal transduction/stress response systems [i.e., mitogen-activated protein kinase (MAPK) pathway, mitochondrial superoxide dismutase (Mn-SOD)] of fungi. Enhance activity of strobilurin or fludioxonil with discovered compounds. METHODS AND RESULTS: Enhancement of antifungal activity of strobilurins, inhibitors of complex III of the mitochondrial respiratory chain, was tested using berberine hemisulfate and different phenolic compounds. The Saccharomyces cerevisiae sod2Delta, a deletion mutant lacking Mn-SOD gene, was highly sensitive to berberine and veratraldehyde. Functional complementation analysis verified these compounds target Mn-SOD. Activity of strobilurin (25-50 micromol l(-1)) was elevated on most aspergilli and Penicillium expansum by co-application with berberine or veratraldehyde (2-4 mmol l(-1)). These compounds also prevented Aspergillus fumigatus MAPK mutants (sakADelta and mpkCDelta) from escaping toxicity of fludioxonil (a phenylpyrrole fungicide potentiated by the MAPK pathway), a typical phenotype of fungal MAPK mutants. CONCLUSIONS: Strobilurin activity or prevention of fungal escape from fludioxonil toxicity can be enhanced by co-application of certain alkaloids or phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural products can be used to target cellular stress response systems in fungal pathogens and serve as alternatives/additives to commercial antifungal agents.

Targeting antioxidative signal transduction and stress response system: control of pathogenic Aspergillus with phenolics that inhibit mitochondrial function.

AIMS: The aim of this study was to show whether antioxidative response systems are potentially useful molecular targets for control of Aspergillus fumigatus and Aspergillus flavus. Selected phenolic agents are used in target-gene-based bioassays to determine their impact on mitochondrial respiration. METHODS AND RESULTS: Vanillyl acetone, vanillic acid, vanillin, cinnamic acid, veratraldehyde, m-coumaric acid (phenolic agents to which Saccharomyces cerevisiae sod2delta mutant showed sensitivity), carboxin (inhibits complex II of the mitochondrial respiratory chain), strobilurins/antimycin A (inhibits complex III of the mitochondrial respiratory chain) and fludioxonil/fenpiclonil [antifungals potentiated by mitogen-activated protein kinase (MAPK)] were examined in A. fumigatus, A. flavus and S. cerevisiae. Individual or combined application of phenolics with inhibitors of mitochondrial respiration showed some of the phenolics effectively inhibited fungal growth. Target-gene bioassays were performed using a sakAdelta (MAPK deletion) strain of A. fumigatus and a complementation analysis using the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) of A. flavus in the ortholog mutant, sod2delta, of S. cerevisiae. The results demonstrated that mitochondrial antioxidative stress system plays important roles in fungal response to antifungal agents tested. CONCLUSIONS: Antioxidative response systems of fungi can be an efficient molecular target of phenolics for pathogen control. Combined application of phenolics with inhibitors of mitochondrial respiration can effectively suppress the growth of fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural compounds that do not pose any significant medical or environmental risks could serve as useful alternatives or additives to conventional antifungals. Identifying the antioxidative response systems in other pathogens could improve methods for fungal control.

Mitogen activated protein kinases of Aspergillus fumigatus.

In microbial eukaryotes, mitogen activated protein kinase (MAPK) pathways play a pivotal role in regulating cellular physiology. In fungi MAPK pathways have established functions in mating-pheromone responses, maintaining cell wall integrity, responding to changes in osmolarity and nutrient sensing. We have been studying MAPK functions in the human pathogenic fungus Aspergillus fumigatus. The genome of A. fumigatus has four MAPK genes, sakA/hogA, mpkA, mpkB and mpkC. Deletion of the sakA gene produces a strain that does not correctly regulate conidial germination, sense environmental nitrogen or responds to hypertonic stress. The function of the remaining MAPK genes is still under investigation, but by analogy to work in other filamentous fungi, we speculate as to their possible functions in A. fumigatus.

Mutation in a calpain-like protease affects the posttranslational mannosylation of phosphatases in Aspergillus nidulans.

In this communication, we show that the palB7 mutation drastically reduced the mannose and N-acetylgalactosamine content of the pacA-encoded acid phosphatase secreted by the fungus Aspergillus nidulans at pH 5.0, compared to a control strain. By using mRNA differential display reverse transcription and polymerase chain reaction, we isolated two cDNAs from the control pabaA1 strain that were not detected in the palB7 mutant strain that encode a mannosyl transferase and a NADH-ubiquinone oxidoreductase. Thus, a defect in the posttranslational mannosylation of proteins could be the consequence of mutations in the palB gene, which encodes for a nuclear calpain-like protease that may have specific functions in the processing of transcription factor(s) similar to its homolog, RIM13, in Saccharomyces cereviseae.

Aspergillus nidulans is frequently resistant to amphotericin B.

The high failure rate of amphotericin B-based therapy in patients with Aspergillus nidulans infections may not be entirely a result of host factors as suggested previously. Innate resistance of A. nidulans to polyenes may contribute to the poor response in patients.

Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essential in vivo function(s).

The single class I myosin (MYOA) of Aspergillus nidulans is essential for hyphal growth. It is generally assumed that the functions of all myosins depend on their actin-activated MgATPase activity. Here we show that MYOA mutants with no more than 1% of the actin-activated MgATPase activity of wild-type MYOA in vitro and no detectable in vitro motility activity can support fungal cell growth, albeit with a delay in germination time and a reduction in hyphal elongation. From these and other data, we conclude that the essential role(s) of myosin I in A. nidulans is probably structural, requiring little, if any, actin-activated MgATPase or motor activity, which have long been considered the defining characteristics of the myosin family.

Resistance to itraconazole in Aspergillus nidulans and Aspergillus fumigatus is conferred by extra copies of the A. nidulans P-450 14alpha-demethylase gene, pdmA.

Triazoles selectively inhibit the cytochrome P-450-dependent C-14 lanosterol alpha-demethylase (P-450 14 alpha DM), a key enzyme in ergosterol biosynthesis in fungi. To investigate mechanisms of triazole resistance in a mould, we used Aspergillus nidulans, a genetically amenable model fungus closely related to more pathogenic members of the genus. We selected for genes that would give resistance to itraconazole following transformation with a high copy genomic library of A. nidulans. In all the resistant colonies that we isolated, resistance was conferred by extra copies of the A. nidulans P-450 14 alpha DM gene, pdmA. We determined that in A. nidulans, extra copies of pdmA increase the MIC for itraconazole 36 times over wild-type controls. Similarly, transformation of an Aspergillus fumigatus strain with pITZR1 resulted in increased resistance to itraconazole. Our results indicate that triazole resistance in clinical isolates of moulds may result from amplification or overexpression of the P-450 14 alpha DM and demonstrate the utility of A. nidulans as a promising model fungus for the analysis of drug resistance and susceptibility in the pathogenic fungus A. fumigatus.

The molecular mechanisms of conidial germination.

The asexual spore, or conidium, is critical in the life cycle of many fungi because it is the primary means for dispersion and serves as a 'safe house' for the fungal genome in adverse environmental conditions. This review discusses the physiological process of germination, conidial adhesion and initiation of protein synthesis and also the regulatory pathways used to activate conidial germination. These include Ca(2+)/calmodulin-mediated signaling, the cyclic AMP/protein kinase A and the ras/mitogen-activated protein kinase pathways. Insights into the process of conidial germination will increase our understanding of the mechanisms of dormancy and sensing of environmental stimuli, and permit identification of novel therapeutic targets for the treatment of spore-borne fungal infections in plants and animals.

The Aspergillus nidulans pyrG89 mutation alters glycosylation of secreted acid phosphatase.

The glycosylation level of the pacA-encoded acid phosphatase secreted by Aspergillus nidulans was reduced in strains pabaA1 pyroA4and pabaA1 pyroA4 pyrG89, compared to strains carrying these mutations singly. The molecular mass of the enzyme secreted by the triple mutant grown at pH 5.0 was 105 and 45 kDa as determined by exclusion chromatography and SDS-PAGE, respectively. In contrast, the pabaA1 strain secreted acid phosphatases of 119 and 62 kDa. The enzyme also had an altered electrophoretic mobility and glycosylation had a protective effect against its heat inactivation. Thus, this combination of mutants alters glycosylation of the enzyme, leading to changes in their structural properties. In spite of this, no deviation was observed in the apparent optimum pH and Michaelis kinetics for enzymatic hydrolysis of p-nitrophenyl phosphate or alpha-naphthyl phosphate.

Localization of wild type and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins.

We have examined the distribution of MYOA, the class I myosin protein of the filamentous fungus Aspergillus nidulans, as a GFP fusion protein. Wild type GFP-MYOA expressed from the myoA promoter is able to rescue a conditional myoA null mutant. Growth of a strain expressing GFP-MYOA as the only class I myosin was approximately 50% that of a control strain, demonstrating that the fusion protein retains substantial myosin function. The distribution of the wild type GFP-MYOA fusion is enriched in growing hyphal tips and at sites of septum formation. In addition, we find that GFP-MYOA is also found in patches at the cell cortex. We have also investigated the effects of deletion or truncation mutations in the tail domain on MYOA localization. Mutant GFP-MYOA fusions that lacked either the C-terminal SH3 or a portion of the C-terminal proline-rich domain had subcellular distributions like wild type MYOA, consistent with their ability to complement a myoA null mutant. In contrast, mutants lacking all of the C-terminal proline-rich domain or the TH-1-like domain were mainly localized diffusely throughout the cytoplasm, but could less frequently be found in patches, and were unable to complement a myoA null mutant. The GFP-MYOA DeltaIQ mutant was localized into large bright fluorescent patches in the cytoplasm. This mutant protein was subsequently found to be insoluble.

Polarity-defective mutants of Aspergillus nidulans.

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the alpha subunit of yeast mitochondrial phenylalanyl--tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed.

The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers.

Numerous disparate studies in plants, filamentous fungi, yeast, Archaea, and bacteria have identified one of the most highly conserved proteins (SNZ family) for which no function was previously defined. Members have been implicated in the stress response of plants and yeast and resistance to singlet oxygen toxicity in the plant pathogen Cercospora. However, it is found in some anaerobic bacteria and is absent in some aerobic bacteria. We have cloned the Aspergillus nidulans homologue (pyroA) of this highly conserved gene and define this gene family as encoding an enzyme specifically required for pyridoxine biosynthesis. This realization has enabled us to define a second pathway for pyridoxine biosynthesis. Some bacteria utilize the pdx pyridoxine biosynthetic pathway defined in Escherichia coli and others utilize the pyroA pathway. However, Eukarya and Archaea exclusively use the pyroA pathway. We also found that pyridoxine is destroyed in the presence of singlet oxygen, helping to explain the connection to singlet oxygen sensitivity defined in Cercospora. These data bring clarity to the previously confusing data on this gene family. However, a new conundrum now exists; why have highly related bacteria evolved with different pathways for pyridoxine biosynthesis?

Structural requirements for in vivo myosin I function in Aspergillus nidulans.

We have investigated the minimal requirements of the tail region for myosin I function in vivo using the filamentous fungus Aspergillus nidulans. The CL3 strain (McGoldrick, C. A., Gruver, C., and May, G. S. (1995) J. Cell Biol. 128, 577-587) was transformed with a variety of myoA constructs containing mutations in the IQ, TH-1-like, SH3, and proline-rich domains by frameshift or in-frame deletions of the tail domains. The resulting strains contained wild type myoA driven by the alcA promoter and a mutant myoA driven by its endogenous promoter. This strategy allowed for selective expression of the wild type and/or mutant form of MYOA by the choice of growth medium. Proper septation and hyphal branching were found to be dependent on the interaction of the IQ motifs with calmodulin, as well as, the presence of its proline-rich domain. Additionally, a single proline-rich motif was sufficient for nearly wild type MYOA function. Most surprisingly, the SH3 domain was not essential for MYOA function. These studies expand our previous knowledge of the function of MYOA to include roles in hyphal morphogenesis, septal wall formation, and cell polarity, laying the groundwork for more detailed investigations on the function of the various tail domains in MYOA.

Mitosis in wild-type and beta-tubulin mutant strains of Aspergillus nidulans.

We review and illustrate the wild-type mitotic cycle of Aspergillus nidulans and report the sequence alterations in six mutant alleles of the A. nidulans benA, beta-tubulin, gene. These alleles confer heat sensitivity and resistance to the antifungal, antimicrotubule compound benomyl, and they have been very important in the study of mitosis and microtubule function in A. nidulans. The mutations are novel and fall at amino acids 50, 134, and 257. We have examined the phenotypes conferred by the mutations at restrictive temperatures. None blocks the assembly of microtubules. One allele, benA33, blocks anaphase A and partially inhibits the disassembly of cytoplasmic microtubules in mitosis. We also often observe abnormal spindle morphologies in strains carrying benA33. Another allele, benA31, causes arrest in mitosis with short mitotic spindles and, thus, appears to inhibit spindle elongation.

Isolation of the Aspergillus nidulans sudD gene and its human homologue.

We have been studying the heat-sensitive bimD6 mutation of Aspergillus nidulans. At a restrictive temperature, the chromosomes of bimD6 mutant strains fail to attach properly to the spindle microtubules, and the mutant also displays a high rate of chromosome loss. We previously cloned the sudA gene, an extragenic suppressor of the heat-sensitive bimD6 mutation and showed that it coded for a DA-box or SMC protein. SMC proteins have been demonstrated to function in chromosome condensation, segregation and global gene regulation. We have now cloned the sudD gene, another of the extragenic suppressor genes of the bimD6 mutation. The predicted SUDD protein is the founding member of a widely expressed protein family. Similar proteins are found in sequence databases for Saccharomyces cerevisiae, Caenorhabditis elegans, mammals and four species of archaebacteria. We have also cloned and sequenced a human cDNA that encodes the human homologue of SUDD and mapped the gene to 18q11.2. The predicted SUDD proteins from A. nidulans, Homo sapiens and S. cerevisiae all share a variety of features. The predicted proteins are approximately 60000Da in mass and have a serine-plus-threonine content of about 11%. The evolutionary conservation of the proteins suggests an ancient origin and conserved function for these proteins.

Constitutive activation of endocytosis by mutation of myoA, the myosin I gene of Aspergillus nidulans.

Class I myosins function in cell motility, intracellular vesicle trafficking and endocytosis. Recently, it was shown that class I myosins are phosphorylated by a member of the p21-activated kinase (PAK) family. PAK phosphorylates a conserved serine or threonine residue in the myosin heavy chain. Phosphorylation at this site is required for maximal activation of the actin-activated Mg2+-ATPase activity in vitro. This serine or threonine residue is conserved in all known class I myosins of microbial origin and in the human and mouse class VI myosins. We have investigated the in vivo significance of this phosphorylation by mutating serine 371 of the class I myosin heavy chain gene myoA of Aspergillus nidulans. Mutation to glutamic acid, which mimics phosphorylation and therefore activation of the myosin, results in an accumulation of membranes in growing hyphae. This accumulation of membranes results from an activation of endocytosis. In contrast, mutation of serine 371 to alanine had no discernible effect on endocytosis. These studies are the first to demonstrate the in vivo significance of a regulatory phosphorylation on a class I myosin. Furthermore, our results suggest that MYOA has two functions, one dependent and one independent of phosphorylation.

Motoring along the hyphae: molecular motors and the fungal cytoskeleton.

This review discusses molecular motors that use the microfilament and microtubule cytoskeletal systems in filamentous fungi. There has been an explosion in our knowledge of kinesins over the past year, because of the integration of genetic and biochemical data. The recognition of possible interactions between septation genes and cytokinesis has also advanced our understanding of microfilament-based cytoskeletal systems. We review recent findings on microfilament motors, including conventional and unconventional myosins, and the microtubule motors of the kinesin family and cytoplasmic dynein. The roles that these molecules play in hyphal morphogenesis and organelle transport provide an insight into cytoskeletal-based transport systems.