Evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii

The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor N omega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway playing a role in zoospore biogenesis. (C) 2009 Elsevier Inc. All rights reserved.

Global Gene Expression Analysis during Germination in the Chytridiomycete Blastocladiella emersonii

Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class. During germination, the zoospore, a motile nongrowing cell, goes through a cascade of morphological changes that culminates with its differentiation into the germling cell, capable of coenocytic vegetative growth. Transcriptome analyses of B. emersonii cells were carried out during germination induced under various environmental conditions. Microarray data analyzing 3,563 distinct B. emersonii genes revealed that 26% of them are differentially expressed during germination in nutrient medium at at least one of the time points investigated. Over 500 genes are upregulated during the time course of germination under those conditions, most being related to cell growth, including genes involved in protein biosynthesis, DNA transcription, energetic metabolism, carbohydrate and oligopeptide transport, and cell cycle control. On the other hand, several transcripts stored in the zoospores are downregulated during germination in nutrient medium, such as genes involved in signal transduction, amino acid transport, and chromosome organization. In addition, germination induced in the presence of nutrients was compared with that triggered either by adenine or potassium ions in inorganic salt solution. Several genes involved in cell growth, induced during germination in nutrient medium, do not show increased expression when B. emersonii zoospores germinate in inorganic solution, suggesting that nutrients exert a positive effect on gene transcription. The transcriptome data also revealed that most genes involved in cell signaling show the same expression pattern irrespective of the initial germination stimulus.

Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste

Soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by Pseudomonas aeruginosa LBI strain. The chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. The chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. The monorhamnolipid RhaC(10)C(10) and the dirhamnolipid Rha(2)C(10)C(10) were the main components of the mixture that showed predominance of 44% and 29%, respectively, after 144-h of cultivation. The biosurfactant was able to form stable emulsions with several hydrocarbons and showed excellent emulsification for soybean oil and chicken fat (100%). The rhamnolipid removed 67% of crude oil present in sand samples and presented antimicrobial activity against Bacillus cereus and Mucor miehei at 64 mu g/mL and inhibition of Neurospora crassa, Staphylococcus aureus, and Micrococcus luteus at 256 mu g/mL. The results demonstrated that the rhamnolipid produced in soybean oil soapstock can be useful in environmental and food industry applications.

Structure and Applications of a Rhamnolipid Surfactant Produced in Soybean Oil Waste

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure of importin-a from a filamentous fungus in complex with a classical nuclear localization signal

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Aspergillus fumigatus calcineurin interacts with a nucleoside diphosphate kinase

The Ca2+-calcineurin pathway affects virulence and morphogenesis in filamentous fungi. Here, we identified 37 CalA-interacting proteins that interact with the catalytic subunit of calcineurin (CalA) in Aspergillus fumigatus, including the nucleoside diphosphate kinase (SwoH). The in vivo interaction between CalA and SwoH was validated by bimolecular fluorescence complementation. A. fumigatus swoH is an essential gene. Therefore, a temperature-sensitive conditional mutant strain with a point mutation in the active site, SwoH(V83F), was constructed, which demonstrated reduced growth and increased sensitivity to elevated temperatures. The SwoH(V83F) mutation did not cause a loss in virulence in the Galleria mellonella infection model. Taken together these results imply that CalA interacts with SwoH. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Comparative EST analysis provides insights into the basal aquatic fungus Blastocladiella emersonii

Abstract Background Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. In this sense, some ancestral characteristics of fungi and animals or fungi and plants could have been retained in this aquatic fungus and lost in members of late-diverging fungal species. To identify in B. emersonii sequences associated with these ancestral characteristics two approaches were followed: (1) a large-scale comparative analysis between putative unigene sequences (uniseqs) from B. emersonii and three databases constructed ad hoc with fungal proteins, animal proteins and plant unigenes deposited in Genbank, and (2) a pairwise comparison between B. emersonii full-length cDNA sequences and their putative orthologues in the ascomycete Neurospora crassa and the basidiomycete Ustilago maydis. Results Comparative analyses of B. emersonii uniseqs with fungi, animal and plant databases through the two approaches mentioned above produced 166 B. emersonii sequences, which were identified as putatively absent from other fungi or not previously described. Through these approaches we found: (1) possible orthologues of genes previously identified as specific to animals and/or plants, and (2) genes conserved in fungi, but with a large difference in divergence rate in B. emersonii. Among these sequences, we observed cDNAs encoding enzymes from coenzyme B12-dependent propionyl-CoA pathway, a metabolic route not previously described in fungi, and validated their expression in Northern blots. Conclusion Using two different approaches involving comparative sequence analyses, we could identify sequences from the early-diverging fungus B. emersonii previously considered specific to animals or plants, and highly divergent sequences from the same fungus relative to other fungi.

Molecular coevolution within a Drosophila clock gene

The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract. Similar repeats are found in the frq and wc2 clock genes of Neurospora crassa and in the mammalian per homologues, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster, whose repetitive regions differ in length by about 150 amino acids. The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock’s temperature compensation.

COS1, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen Candida albicans

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene, COS1, from the opportunistic pathogen Candida albicans by using a PCR-based screening strategy. The sequence of COS1 indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus Neurospora crassa. COS1 is also identical to a gene called CaNIK1 identified in C. albicans by low stringency hybridization using CaSLN1 as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998) Microbiol. 44, 425–432]. We assess the function of COS1/CaNIK1 by constructing a diploid deletion mutant. Mutants lacking both copies of COS1 appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the Δnik-1 mutant, the Δcos1/Δcos1 mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both Δnik-1 and Δcos1/Δcos1 mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.

Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

Phosphate transporters from the higher plant Arabidopsis thaliana.

Two cDNAs (AtPT1 and AtPT2) encoding plant phosphate transporters have been isolated from a library prepared with mRNA extracted from phosphate-starved Arabidopsis thaliana roots, The encoded polypeptides are 78% identical to each other and show high degree of amino acid sequence similarity with high-affinity phosphate transporters of Saccharomyces cerevisiae, Neurospora crassa, and the mycorrhizal fungus Glomus versiforme. The AtPT1 and AtPT2 polypeptides are integral membrane proteins predicted to contain 12 membrane-spanning domains separated into two groups of six by a large charged hydrophilic region. Upon expression, both AtPT1 and AtPT2 were able to complement the pho84 mutant phenotype of yeast strain NS219 lacking the high-affinity phosphate transport activity. AtPT1 and AtPT2 are representatives of two distinct, small gene families in A. thaliana. The transcripts of both genes are expressed in roots and are not detectable in leaves. The steady-state level of their mRNAs increases in response to phosphate starvation.

Omics for Investigating Chitosan as an Antifungal and Gene Modulator

Chitosan is a biopolymer with a wide range of applications. The use of chitosan in clinical medicine to control infections by fungal pathogens such as Candida spp. is one of its most promising applications in view of the reduced number of antifungals available. Chitosan increases intracellular oxidative stress, then permeabilizes the plasma membrane of sensitive filamentous fungus Neurospora crassa and yeast. Transcriptomics reveals plasma membrane homeostasis and oxidative metabolism genes as key players in the response of fungi to chitosan. A lipase and a monosaccharide transporter, both inner plasma membrane proteins, and a glutathione transferase are main chitosan targets in N. crassa. Biocontrol fungi such as Pochonia chlamydosporia have a low content of polyunsaturated free fatty acids in their plasma membranes and are resistant to chitosan. Genome sequencing of P. chlamydosporia reveals a wide gene machinery to degrade and assimilate chitosan. Chitosan increases P. chlamydosporia sporulation and enhances parasitism of plant parasitic nematodes by the fungus. Omics studies allow understanding the mode of action of chitosan and help its development as an antifungal and gene modulator.

Pollen and non-pollen palynomorphs counting data from Borsteler Moor (Germany)

In order to reconstruct regional vegetation changes and local conditions during the fen-bog transition in the Borsteler Moor (northwestern Germany), a sediment core covering the period between 7.1 and 4.5 cal kyrs BP was palynologically in vestigated. The pollen diagram demonstrates the dominance of oak forests and a gradual replacement of trees by raised bog vegetation with the wetter conditions in the Late Atlantic. At ~ 6 cal kyrs BP, the non-pollen palynomorphs (NPP) demonstrate the succession from mesotrophic conditions, clearly indicated by a number of fungal spore types, to oligotrophic conditions, indicated by Sphagnum spores, Bryophytomyces sphagni, and testate amoebae Amphitrema, Assulina and Arcella, etc. Four relatively dry phases during the transition from fen to bog are clearly indicated by the dominance of Calluna and associated fungi as well as by the increase of microcharcoal. Several new NPP types are described and known NPP types are identified. All NPP are discussed in the context of their palaeoecological indicator values.

The Cratylia Mollis Seed Lectin Induces Membrane Permeability Transition In Isolated Rat Liver Mitochondria And A Cyclosporine A-insensitive Permeability Transition In Trypanosoma Cruzi Mitochondria.

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria.