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Endophytic fungi are increasingly being recognized for their diverse metabolites that may exhibit antimicrobial properties. In our study, we isolated seven endophytic fungal strains from Calea pinnatifida, which were identified as Hypomontagnella barbarensis, Neopestalotiopsis clavispora, Nigrospora sacchari-officinarum, Annulohypoxylon moriforme, Colletotrichum siamense, and Colletotrichum karstii (with two isolates from the same species). Furthermore, the antimicrobial activity of the extracts was assessed, revealing that the extract from Hypomontagnella barbarensis demonstrated activity against Staphylococcus aureus. Further investigation of secondary metabolites, employing UHPLC-HR-ESI-MS/MS in combination with molecular networking, facilitated annotation of the nine compounds. Of these, five were identified based on matches with the GNPS spectral library, and four were predicted based on the molecular network. Notably, from the extract of Hypomontagnella barbarensis, two pairs of diastereoisomeric acyl-tetronic acid derivatives were isolated and characterized using MS and NMR spectroscopy. This study highlights the potential of endophytic fungi as a valuable source of novel antimicrobial agents. [ABSTRACT FROM AUTHOR]

Stress management is an adaptive advantage for survival in adverse environments. Pathogens face this challenge during host colonization, requiring an appropriate stress response to establish infection. The fungal pathogen Cryptococcus neoformans undergoes thermal, oxidative, and osmotic stresses in the environment and animal host. Signaling systems controlled by Ras1, Hog1, and calcineurin respond to high temperatures and osmotic stress. Cationic stress caused by Na+, K+, and Li+ can be overcome with glycerol, the preferred osmolyte. Deleting the glycerol phosphate phosphatase gene (GPP2) prevents cells from accumulating glycerol due to a block in the last step of its biosynthetic pathway. Gpp2 accumulates in a phosphorylated form in a cna1Δ strain, and a physical interaction between Gpp2 and Cna1 was found; moreover, the gpp2Δ strain undergoes slow growth and has attenuated virulence in animal models of infection. We provide biochemical evidence that growth in 1 M NaCl increases glycerol content in the wild type, whereas gpp2Δ, cna1Δ, and cnb1Δ mutants fail to accumulate it. The deletion of cnb1Δ or cna1Δ renders yeast cells sensitive to cationic stress, and the Gfp-Gpp2 protein assumes an abnormal localization. We suggest a mechanism in which calcineurin controls Gpp2 at the post-translational level, affecting its localization and activity, leading to glycerol biosynthesis. Also, we showed the transcriptional profile of glycerol-deficient mutants and established the cationic stress response mediated by calcineurin; among the biological processes differentially expressed are carbon utilization, translation, transmembrane transport, glutathione metabolism, oxidative stress response, and transcription regulation. To our knowledge, this is the first time that this transcriptional profile has been described. These results have implications for pathogen stress adaptability. [ABSTRACT FROM AUTHOR]

Fungal pathogens are a major cause of death, especially among immunocompromised patients. Therapies against invasive fungal infections are restricted to a few antifungals; therefore, novel therapies are necessary. Nutritional signaling and regulation are important for pathogen establishment in the host. In Cryptococcus neoformans, the causal agent of fungal meningitis, amino acid uptake and biosynthesis are major aspects of nutritional adaptation. Disruptions in these pathways lead to virulence attenuation in an animal model of infection, especially for sulfur uptake and sulfur amino acid biosynthesis. Deletion of Cys3, the main transcription factor that controls these pathways, is the most deleterious gene knockout in vitro and in vivo, making it an important target for further application. Previously, we demonstrated that Cys3 is part of a protein complex, including calcineurin, which is necessary to maintain high Cys3 protein levels during sulfur uptake and sulfur amino acid biosynthesis. In the current study, other aspects of Cys3 regulation are explored. Two lines of evidence suggest that C. neoformans Cys3 does not interact with the F-box WD40 protein annotated as Met30, indicating another protein mediates Cys3 ubiquitin degradation. However, we found another level of Cys3 regulation, which involves protein interactions between Cys3 and ATP sulfurylase (MET3 gene). We show that an atypical leucine zipper at the N-terminus of ATP sulfurylase is essential for physical interaction with Cys3 and calcineurin. Our data suggests that Cys3 and ATP sulfurylase interact to regulate Cys3 transcriptional activity. This work evidences the complexity involved in the regulation of a transcription factor essential for the sulfur metabolism, which is a biological process important to nutritional adaptation, oxidative stress response, nucleic acid stability, and methylation. This information may be useful in designing novel therapies against fungal infections. [ABSTRACT FROM AUTHOR]

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The microbiological contamination of food causes concern for public health due to the pathogenic action of microorganisms and the increasing of antimicrobial resistance observed in bacterial strains. This work considers the use of fluorescence spectroscopy to study the effects of the presence of microorganisms in the chicken's meat and therefore, how the detection of this presence can help on food safety. The fluorescence of organic boneless and skinless chicken breast pieces contaminated with variable concentrations of E. coli cells inoculated in meat (104, 105, 106, and 107 cells/mL) was measured with excitations at 340 and 410 nm. Raman spectra were obtained to investigate into conformational changes in the collagen structures, from samples kept for 48 h at 25 °C (non-contaminated and contaminated with E. coli, 105 and 107 cells/mL). Protoporphyrin IX fluorescence lifetime was measured in the function of an increased number of E. coli cells. The obtained results of E. coli were compared to the ones of Salmonella and Campylobacter contaminations (106 cells/mL). Some uncommon aspects were found in the spectra of the contaminated meat: enlargement of the collagen band at 400 nm; increase in free reduced nicotinamide adenine dinucleotide fluorescence intensity around 505 nm; and decrease of flavin emission band. A shortening in the porphyrin emission lifetime (from ~ 10 ns for uncontaminated meat to ~ 5 ns) was observed, showing a quenching process for the meat contaminated with E. coli. The presence of coproporphyrin emission band was observed in the samples contaminated with Salmonella. The singularities observed in PpIX fluorescence spectroscopy for E. coli and Salmonella can be used to obtain a quick detection method of pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformans is an opportunist fungal pathogen that causes meningoencephalitis in immunocompromised patients. During infection, this basidiomycete yeast has to adapt to several adverse conditions, especially nutrient availability. The interruption on various amino acid biosynthetic pathways and on amino acid uptake causes reduced viability, inability to cope with various stresses, failure in virulence factors expression and avirulence in animal model of infection. The sulfur amino acid biosynthesis and uptake is an important feature for pathogen survival in vivo and in vitro. Our previous work demonstrates that C. neoformans Cys3 BZip transcription factor controls the gene expression in several steps of the sulfur assimilation and sulfur amino acid biosynthesis. Also, we have shown that Gpp2 phosphatase modulates Cys3 activity. In Saccharomyces cerevisiae Gpp2 is induced in response to hyper osmotic or oxidative stress and during diauxic shift. In this work, we will show that, in C. neoformans , Gpp2 is required to respond to stresses, mainly osmotic stress; also its transcription is induced during exposure to NaCl. Global transcriptional profile of gpp2 Δ by RNAseq shows that CYS3 and other genes in the sulfur assimilation pathway are up regulated, which is consistent with our previous report, in which Gpp2 acts by avoiding Cys3 accumulation and nuclear localization. In addition, several transporters genes, especially amino acid permeases and oxidative stress genes are induced in the gpp2 Δ strain; on the contrary, genes involved in glucose and tricarboxylic acid metabolism are down regulated. gpp2 Δ strain fails to express virulence factors, as melanin, phospholipase, urease and has virulence attenuation in Galleria mellonella. Our data suggest that Gpp2 is an important factor for general pathogen adaptation to various stresses and also to the host, and perhaps it could be an interesting target for therapeutic use. [ABSTRACT FROM AUTHOR]

The serine-threonine kinase AKT/PKB is a critical regulator of various essential cellular processes, and dysregulation of AKT has been implicated in many diseases, including cancer. Despite AKT action is known to function mainly in the cytoplasm, AKT has been reported to translocate to the nucleus. However, very little is known about the mechanism required for the nuclear import of AKT as well as its function in this cellular compartment. In the present study, we characterized the presence of endogenous nuclear AKT in human melanoma cells and addressed the possible role of AKT by exploring its potential association with key interaction nuclear partners. Confocal and Western blot analyses showed that both phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei. Using mass spectrometry in combination with protein-crosslinking and co-immunoprecipitation, we identified a series of putative protein partners of nuclear AKT, including heterogeneous nuclear ribonucleoprotein (hnRNP), cytoskeleton proteins β-actin, γ-actin, β-actin-like 2 and vimentin. Confocal microscopy and biochemical analyses validated β-actin as a new nuclear AKT-interacting partner. Cofilin and active RNA Polymerase II, two proteins that have been described to interact and work in concert with nuclear actin in transcription regulation, were also found associated with nuclear AKT. Overall, the present study uncovered a yet unrecognized nuclear coupling of AKT and provides insights into the involvement of AKT in the interaction network of nuclear actin. [ABSTRACT FROM AUTHOR]

Cryptococcosis is an nvasive ungal nfection (IFI) caused by Cryptococcus neoformans, mainly in immunocompromised patients. Therapeutic failure due to pathogen drug resistance, treatment inconstancy and few antifungal options is a problem. The study of amino acid biosynthesis and uptake represents an opportunity to explore possible development of novel antifungals. C. neoformans has 10 amino acids permeases, two of them (Aap3 and Aap7) not expressed at the conditions tested, and five were studied previously (Aap2, Aap4, Aap5, Mup1 and Mup3). Our previous results showed that Aap4 and Aap5 are major permeases with overlapping functions. The aap4Δ/aap5Δ double mutant fails to grow in amino acids as sole nitrogen source and is avirulent in animal model. Here, we deleted the remaining amino acid permeases (AAP1, AAP6, AAP8) that showed gene expression modulation by nutritional condition and created a double mutant (aap1Δ/aap2Δ). We studied the virulence attributes of these mutants and explored the regulatory mechanism behind amino acid uptake in C. neoformans. The aap1Δ/aap2Δ strain had reduced growth at 37°C in L-amino acids, reduced capsule production and was hypovirulent in the Galleria mellonella animal model. Our data, along with previous studies, (i) complement the analysis for all 10 amino acid permeases mutants, (ii) corroborate the idea that these transporters behave as global permeases, (iii) are required during heat and nutritional stress, and (iv) are important for virulence. Our study also indicates a new possible link between Ras1 signaling and amino acids uptake. [ABSTRACT FROM AUTHOR]

Gti1/Pac2 transcription factors occur exclusively in fungi and their roles vary according to species, including regulating morphological transition and virulence, mating and secondary metabolism. Many of these functions are important for fungal pathogenesis. We therefore hypothesized that one of the two proteins of this family in Cryptococcus neoformans , a major pathogen of humans, would also control virulence-associated cellular processes. Elimination of this protein in C. neoformans results in reduced polysaccharide capsule expression and defective cytokinesis and growth at 37°C. The mutant loses virulence in a mouse model of cryptococcal infection and retains only partial virulence in the Galleria mellonella alternative model at 30°C. We performed RNA-Seq experiments on the mutant and found abolished transcription of genes that, in combination, are known to account for all the observed phenotypes. The protein has been named Required for cytokinesis and virulence 1 (Rcv1). [ABSTRACT FROM AUTHOR]

In order to survive and cause disease, microbial pathogens must be able to proliferate at the temperature of their infected host. We identified novel microbial features associated with thermotolerance in the opportunistic fungal pathogen Cryptococcus neoformans using a random insertional mutagenesis strategy, screening for mutants with defective growth at 37°C. Among several thermosensitive mutants, we identified one bearing a disruption in a gene predicted to encode the Ape4 aspartyl aminopeptidase protein. Ape4 metalloproteases in other fungi, including Saccharomyces cerevisiae, are activated by nitrogen starvation, and they are required for autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway. However, none have been previously associated with altered growth at elevated temperatures. We demonstrated that the C. neoformans ape4 mutant does not grow at 37°C, and it also has defects in the expression of important virulence factors such as phospholipase production and capsule formation. C. neoformans Ape4 activity was required for this facultative intracellular pathogen to survive within macrophages, as well as for virulence in an animal model of cryptococcal infection. Similar to S. cerevisiae Ape4, the C. neoformans GFP-Ape4 fusion protein co-localized with intracytoplasmic vesicles during nitrogen depletion. APE4 expression was also induced by the combination of nutrient and thermal stress. Together these results suggest that autophagy is an important cellular process for this microbial pathogen to survive within the environment of the infected host. [ABSTRACT FROM AUTHOR]

Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor. [ABSTRACT FROM AUTHOR]

Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (itrogen atabolism epression, NCR) and carbon sources (arbon atabolism epression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (lobal mino cid ontrol, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro. [ABSTRACT FROM AUTHOR]

The chemical composition and antimicrobial activity of essential oils obtained from three Brazilian plant species-leaves and branches of Eremanthus erythropappus (Asteraceae), leaves of Plectranthus barbatus, and leaves of P. amboinicus (Lamiaceae)-were determined. Analysis by GC/MS and determination of Kovats indexes both indicated δ-elemene (leaves-42.61% and branches-23.41%) as well as (-)-α-bisabolol (leaves-24.80% and stem bark-66.16%) as major constituents of E. erythropappus essential oils. The main components of leaves of P. barbatus were identified as (Z)-caryophyllene (17.98%), germacrene D (17.35%), and viridiflorol (14.13%); whereas those of leaves of P. amboinicus were characterized as p-cymene (12.01%), γ-terpinene (14.74%), carvacrol (37.70%), and (Z)-caryophyllene (14.07%). The antimicrobial activity against yeasts and bacteria was assessed in broth microdilution assays to determine the minimum inhibitory concentration (MIC) necessary to inhibit microbial growth. In addition, the crude oil of branches of E. erythropappus was subjected to chromatographic separation procedures to afford purified (-)-α-bisabolol. This compound displayed biological activity against pathogenic yeasts, thus suggesting that the antimicrobial effect observed with crude oils of E. erythropappus leaves and branches may be related to the occurrence of (-)-α-bisabolol as their main component. Our results showed that crude oils of Brazilian plants, specifically E. erythropappus, P. barbatus, and P. amboinicus and its components, could be used as a tool for the developing novel and more efficacious antimicrobial agents. [ABSTRACT FROM AUTHOR]

Bioactivity-guided fractionation of an antimicrobial active extract from twigs of Baccharis retusa C. DC. (Asteraceae) yielded the flavanone 5,4'-dihydroxy-7-methoxyflavanone (sakuranetin) as responsible for the detected activity. The structure of the bioactive compound was established on the basis of spectroscopic data analysis, including NMR and MS. Additionally, the structure of a new crystal form of sakuranetin was confirmed by X-ray diffratometry. The minimum inhibitory concentrations (MIC) of isolated compound were determined against pathogenic yeast belonging to the genus Candida (six species), Cryptococcus (two species/four serotypes) and S. cerevisiae BY 4742 (S288c background) and ranged from 0.32 to 0.63 μg/μL. Our results showed that sakuranetin, which structure was fully characterized, could be used as a tool for the design of novel and more efficacious antifungal agents. [ABSTRACT FROM AUTHOR]

This study investigates the impact of seasonal variation on the chemical composition of essential oils from the leaves of Porcelia macrocarpa (Annonaceae) obtained over the course of one year (January-December 2011) and the chemical composition of the essential oils obtained from the ripe fruits of the same plant. Furthermore, the essential oils of the leaves were investigated with respect to their antimicrobial activity. The essential oils of the leaves contain a mixture of monoterpenes, one diterpene and several sesquiterpenes. The main components were identified as the sesquiterpenes germacrene D (29%-50%) and bicyclogermacrene (24%-37%). No significant variation was observed for the composition of the essential oil of the leaves over the course ofthe year, except for the month of November, when the ripe fruit were collected. In this month, substantially decreased concentrations of germacrene D (28.8 ± 0.8%) and bicyclogermacrene (23.9 ± 0.6%) were measured and the emergence of spathulenol (10.4 ± 0.2%) was observed. The essential oils extracted from the ripe fruit revealed the presence of a variety of monoterpenes, sesquiterpenes and hydrocarbons. The main constituents of these oils were neryl (8.8 ± 0.2%) and geranyl (27.3 ± 0.7%) formates, y-muurolene (10.3 ± 0.9%) and dendrolasin (8.23 ± 0.06%). The antimicrobial activity of the essential oil obtained from the leaves of P. macrocarpa towards a range of bacterial and yeast strains was examined. In order to determine the minimum inhibitory concentration (MIC) of essential oils obtained from the January collection of the leaves, broth microdilution assays were carried out, which showed a significant antimicrobial activity towards Cryptococcus neoformans serotypes A and D as well as C. gattii serotypes B and C. [ABSTRACT FROM AUTHOR]

: Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the São Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders. [ABSTRACT FROM AUTHOR]

The chemical composition and antimicrobial activity of essential oils obtained from leaves of two Myrtaceae species-Eugenia uniflora L. and Plinia trunciflora (O. Berg) Kausel-were determined. Analysis by GC/MS as well as determination of Kovatz indexes indicated atractylone (26.78%) and curzerene (17.96%) as major constituents of E. uniflora oil and α-cadinol (19.15%), apiole (11.15%) and cubenol (5.43%) as main components in P. trunciflora oil. Both essential oils were tested for antimicrobial activity against yeasts and bacteria. E. uniflora and P. trunciflora essential oils were active towards two Gram-positive bacteria, Streptococcus equi and Staphylococcus epidermis. In addition, biological activity of both essential oils was detected for pathogenic yeasts of the genus Candida and Cryptococcus. E. uniflora was active towards all yeast tested and exhibited interesting minimal inhibitory concentrations (0.11 to 3.75 mg/mL) across a broad spectrum of activity. [ABSTRACT FROM AUTHOR]

Composting is a way of transforming the organic waste into fertilizer, minimizing the use of inorganic compounds that may contaminate the environment. This transformation is the result of the microorganism action, converting complex carbon sources into energy. Enzymes that are exported by the microorganisms to the surrounding environment mediate this process. The aiming of the present work is to prospect the compost produced by the organic composting unit (OCU) of the Fundação Parque Zoológico de São Paulo (FPZSP) to find novel starch hydrolyzing organisms (SHO) that secrete large amounts of amylases under harsh conditions, such as high temperature. We found five bacterial isolates that have amylolytic activity induced by soluble starch and 39°C temperature of growth. These bacterial strains were identified by MALDI-TOF (Matrix-assisted laser desorption/ionization-Time of Flight) analysis, a rapid and efficient methodology for microbe identification in large scale. Our results present amylolytic strains that belong to diverse taxonomic groups (Solibacillus silvestris, Arthrobacter arilaitensis, Isoptericola variabilis, and Acinetobacter calcoaceticus); some of them have never been associated with this kind of hydrolytic activity before. The information regarding enzyme induction will be important to optimize the production by the bacterial isolates, which may be a great value for biotechnological applications. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63-75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12αp. SteAp and Ste12αp lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C[sub 2]/H[sub 2]-Zn[sup +2] finger domains not present in the other Ste12 proteins. A ΔsteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry. [ABSTRACT FROM AUTHOR]

Cryptococcosis is a fungal disease caused by C. neoformans. To adapt and survive in diverse ecological niches, including the animal host, this opportunistic pathogen relies on its ability to uptake nutrients, such as carbon, nitrogen, iron, phosphate, sulfur, and amino acids. Genetic circuits play a role in the response to environmental changes, modulating gene expression and adjusting the microbial metabolism to the nutrients available for the best energy usage and survival. We studied the sulfur amino acid biosynthesis and its implications on C. neoformans biology and virulence. CNAG_04798 encodes a BZip protein and was annotated as CYS3, which has been considered an essential gene. However, we demonstrated that CYS3 is not essential, in fact, its knockout led to sulfur amino acids auxotroph. Western blots and fluorescence microscopy indicated that GFP-Cys3, which is expressed from a constitutive promoter, localizes to the nucleus in rich medium (YEPD); the addition of methionine and cysteine as sole nitrogen source (SD–N + Met/Cys) led to reduced nuclear localization and protein degradation. By proteomics, we identified and confirmed physical interaction among Gpp2, Cna1, Cnb1 and GFP-Cys3. Deletion of the calcineurin and GPP2 genes in a GFP-Cys3 background demonstrated that calcineurin is required to maintain Cys3 high protein levels in YEPD and that deletion of GPP2 causes GFP-Cys3 to persist in the presence of sulfur amino acids. Global transcriptional profile of mutant and wild type by RNAseq revealed that Cys3 controls all branches of the sulfur amino acid biosynthesis, and sulfur starvation leads to induction of several amino acid biosynthetic routes. In addition, we found that Cys3 is required for virulence in Galleria mellonella animal model. [ABSTRACT FROM AUTHOR]