Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components

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

Lignases Production and Partial Purification of Mnp from Brazilian Fungal Isolate in Submerged Fermentation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Signaling via cAMP in Fungi: Interconnections with Mitogen-Activated Protein Kinase Pathways

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen- activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes.

Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Background: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. Objective: To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of beta-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Methods: Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. Results: PBMCs from patients with APECED syndrome had reduced TNF-alpha responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-a release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane. Conclusion: AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome. (J Allergy Clin Immunol 2012;129:464-72.)

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

The fungal metabolite eugenitin as additive for Aspergillus niveus glucoamylase activation

Endophytic microorganisms live inside tissues of host plants apparently do not causing warning to them, and area promising source of bioactive molecules as antimicrobial and antitumoral drugs. In this work, we report the isolation of eugenitin from cultures of the endophyte Mycoleptodiscus indicus and its potential as additive for Aspergillus niveus glucoamylase activation. The glucoamylase hydrolytic activity increased twofold using 5 mM of eugenitin and this activation could be explained by the binding mode of eugenitin with the three-dimensional structure of glucoamylase. The in silica prediction of ligand binding sites revealed at least 9 possible interaction sites able to accommodate eugenitin on glucoamylase from Hypocrea jecorina. Besides, we evaluated the effect of pH and temperature on activity and stability, as well as in the hydrolysis of different substrates and kinetic parameters either in presence or absence of eugenitin. The results displayed by eugenitin as additive to glucoamylase activation are promising and provide novel perspectives for applications of fungal metabolites. (C) 2011 Elsevier B.V. All rights reserved.

Endo-xylanase GH11 activation by the fungal metabolite eugenitin

Eugenitin, a chromone derivative and a metabolite of the endophyte Mycoleptodiscus indicus, at 5 mM activated a recombinant GH11 endo-xylanase by 40 %. The in silico prediction of ligand-binding sites on the three-dimensional structure of the endo-xylanase revealed that eugenitin interacts mainly by a hydrogen bond with a serine residue and a stacking interaction of the heterocyclic aromatic ring system with a tryptophan residue. Eugenitin improved the GH11 endo-xylanase activity on different substrates, modified the optimal pH and temperature activities and slightly affected the kinetic parameters of the enzyme.

Paracoccidoides brasiliensis 30 kDa adhesin: identification as a 14-3-3 protein, cloning and subcellular localization in infection models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.

Association of lectin pathway proteins with intra-abdominal Candida infection in high-risk surgical intensive-care unit patients. A prospective cohort study within the fungal infection network of Switzerland

OBJECTIVES Human studies on the role of mannose-binding lectin (MBL) in patients with invasive candidiasis have yielded conflicting results. We investigated the influence of MBL and other lectin pathway proteins on Candida colonization and intra-abdominal candidiasis (IAC) in a cohort of high-risk patients. METHODS Prospective observational cohort study of 89 high-risk intensive-care unit (ICU) patients. Levels of lectin pathway proteins at study entry and six MBL2 single-nucleotide polymorphisms were analyzed by sandwich-type immunoassays and genotyping, respectively, and correlated with development of heavy Candida colonization (corrected colonization index (CCI) ≥0.4) and occurrence of IAC during a 4-week period. RESULTS Within 4 weeks after inclusion a CCI ≥0.4 and IAC was observed in 47% and 38% of patients respectively. Neither serum levels of MBL, ficolin-1, -2, -3, MASP-2 or collectin liver 1 nor MBL2 genotypes were associated with a CCI ≥0.4. Similarly, none of the analyzed proteins was found to be associated with IAC with the exception of lower MBL levels (HR 0.74, p = 0.02) at study entry. However, there was no association of MBL deficiency (<0.5 μg/ml), MBL2 haplo- or genotypes with IAC. CONCLUSION Lectin pathway protein levels and MBL2 genotype investigated in this study were not associated with heavy Candida colonization or IAC in a cohort of high-risk ICU patients.

Functional analysis of the Galpha protein, GNA -3, in the development of Neurospora crassa using biochemical and genetic studies

Extracellular signals regulate fungal development and, to sense and respond to these cues, fungi evolved signal transduction pathways similar to those in mammalian systems. In fungi, heterotrimeric G proteins, composed of α, β, and γ subunits, transduce many signals, such as pheromones and nutrients, intracellularly to alter adenylyl cyclase and MAPK cascades activity. ^ Previously, the Gα proteins GNA-1 and GNA-2 were characterized in regulating development in the fungus Neurospora crassa. R. A. Baasiri isolated a third Gα, gna-3, and P. S. Rowley generated Δgna-3 mutants. GNA-3 belongs to a fungal Gα family that regulates cAMP metabolism and virulence. The Δ gna-3 sexual cycle is defective in homozygous crosses, producing inviable spores. Δgna-3 mutants have reduced aerial hyphae formation and derepressed asexual sporulation (conidiation), causing accumulation of asexual spores (conidia). These defects are similar to an adenylyl cyclase mutant, cr-1; cAMP supplementation suppressed Δ gna-3 and cr-1. Inappropriate conidiation and expression of a conidiation gene, con-10, were higher in Δ gna-3 than cr-1 submerged cultures; peptone suppressed conidiation. Adenylyl cyclase activity and expression demonstrated that GNA-3 regulates enzyme levels. ^ A Δgna-1 cr-1 was analyzed with F. D. Ivey to differentiate GNA-1 roles in cAMP-dependent and -independent pathways. Δ gna-1 cr-1 defects were worse than cr-1 and refractory to cAMP, suggesting that GNA-1 is necessary for sensing extracellular CAMP. Submerged culture conidiation was highest in Δgna-1 cr-1, and only high cell density Δgna-1 cultures conidiated, which correlated with con-10 levels. Transcription of a putative heat shock cognate protein was highest in Δgna-1 cr-1. ^ Functional relationships between the three Gαs was analyzed by constructing Δgna-1 Δgna-2 Δ gna-3, Δgna-1 Δgna-3, and Δgna-2 Δgna-3 strains. Δ gna-2 Δgna-3 strains exhibited intensified Δ gna-3 phenotypes; Δgna-1 Δgna-2 Δgna-3 and Δgna-1 Δ gna-3 strains were identical to Δgna-1 cr-1 on plates and were non-responsive to cAMP. The highest levels of conidiation and con-10 were detected in submerged cultures of Δ gna-1 Δgna-2 Δgna-3 and Δgna-1 Δgna-3 mutants, which was partially suppressed by peptone supplementation. Stimulation of adenylyl cyclase is completely deficient in Δgna-1 Δ gna-2 Δgna-3 and Δgna-1 Δ gna-3 strains. Δgna-3 and Δ gna-1 Δgna-3 aerial hyphae and conidiation defects were suppressed by mutation of a PKA regulatory subunit. ^

Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK

It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995) Science 270, 1988–1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Derepressed Hyphal Growth and Reduced Virulence in a VH1 Family-related Protein Phosphatase Mutant of the Human Pathogen Candida albicans

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.