Transcriptional and Post-Translational Roles of Calcineurin in Cationic Stress and Glycerol Biosynthesis in Cryptococcus neoformans.

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.

ATP sulfurylase atypical leucine zipper interacts with Cys3 and calcineurin A in the regulation of sulfur amino acid biosynthesis in Cryptococcus neoformans.

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.

Characterization of chicken meat contaminated with Salmonella by fluorescence spectroscopy.

Contaminated poultry products as eggs and meat are the primary vehicles of Salmonella infection. Conventional methods for microorganisms detections involve multiple steps, and despite its accuracy, these assays are time-consuming. Biosensing methods have shown great potential for the rapid detection of foodborne pathogens. Some of the biosensors are based on fluorescence. Various fluorophores such as collagen, elastin, NAD(P)H, and porphyrins can be used to evaluate possible chemical changes in meat. In this manuscript, the fluorescence properties of chicken meat contaminated with Salmonella enterica (ATCC 14028) cell suspensions (500; 5000; 50,000 and 500,000 cells/mL) were obtained and compared with non-contaminated control, for meat kept at 25 °C for 24 and 48 h. The effects of ambient light were also considered. Our results indicated that free NAD(P)H and coproporphyrin emission bands present in contaminated meat, increased over time, and can provide access to valuable information for the detection of Salmonella in chicken meat.

Biological functions of the autophagy-related proteins Atg4 and Atg8 in Cryptococcus neoformans.

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.

First report of cis-1,4-polyisoprene degradation by Gordonia paraffinivorans.

The use of rubber has increased over the years, leading to a series of environmental problems due to its indefinite decomposition time. Bioremediation employing microorganisms have drawn an increasing interest and originated several studies of microbial rubber degradation. Genome sequencing and in silico analysis demonstrated that G. paraffinivorans MTZ041 isolate encodes the lcp gene (Latex Clearing Protein), responsible for expressing an enzyme that performs the first step in the assimilation of synthetic and natural rubber. Growth curves and scanning electron microscopy (SEM) were conducted for MTZ041 in natural (NR) and synthetic rubber (IR) as sole carbon source during 11 weeks. After 80 days, robust growth was observed and SEM analysis revealed the presence of bacilli and the formation of biofilm-like structures on natural and synthetic rubber. This is the first report of a G. paraffinivorans rubber degrader. Given the complexity of this substrate and the relative small number of microorganisms with this ability, the description and characterization of MTZ041 is of great importance on bioremediation processes of rubber products.

AKT/protein kinase B associates with ß-actin in the nucleus of melanoma cells.

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 ß-actin, Î³-actin, ß-actin-like 2 and vimentin. Confocal microscopy and biochemical analyses validated ß-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.

A Wor1-Like Transcription Factor Is Essential for Virulence of Cryptococcus neoformans.

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).

A simple and effective method to synthesize fluorescent nanoparticles using tryptophan and light and their lethal effect against bacteria.

A simple, environmentally friendly and cost-effective method was used to synthesize silver nanoparticles using tryptophan and light. To prepare the nanoparticles, the following components were used: deionized water, silver nitrate, light and tryptophan. The effects of the tryptophan concentration and light exposure time on the formation of tryptophan silver nanoparticles (Tnnps) were studied. The synthesized Tnnps were characterized using transmission electron microscopy (TEM), absorption and fluorescence spectroscopy and zeta potential measurements. The synthesized Tnnps were nearly spherical, with sizes of approximately 17 nm. In addition, the antibacterial activity of Tnnps was determined by monitoring the growth curves of strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Serratia marcescens, and Enterococcus faecalis using the microdilution test. The Minimum Inhibitory Concentration (MIC) for 4 of 5 tested bacteria was determined to be between 20.0 and 17.5 µg/mL for 48 h and between 22.5 and 20.0 µg/mL for 72 h.

Amylolytic microorganism from são paulo zoo composting: isolation, identification, and amylase production.

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.