Low Concentrations of Hydrogen Peroxide or Nitrite Induced of Paracoccidioides brasiliensis Cell Proliferation in a Ras-Dependent Manner

Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. in lower eukaryotes, Byr2 kinase represents a Ras target. the present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM) or NO2 (0.1-0.25 mu M) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. in conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenacao Aperfeicoamento de Pessoal de Nivel SuperiorUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol CTCMol, Dept Bioquim Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol CTCMol, Dept Bioquim Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, BrazilFAPESP: 2011/14392-2CNPq: 477764/2009-6Web of Scienc

Cell proliferation of <i>P. brasiliensis</i> stimulated by low concentrations of H₂O₂ or nitrite is suppressed in the presence of FPT III inhibitor.

<p>(<b>A</b>) Pb18 yeast cells were seeded in a 6-well culture plate subjected to a 24-h period of starvation with F12 medium and pretreated with 250 µM FPT III and stimulated with 0.1 mM H₂O₂ or 0.25 µM nitrite at pH 5.6 for 5 h at 37°C. Cells were plated in BHI medium at 37°C for 10 days (n = 6 at each point) and CFU were counted. (<b>B</b>) Same as in (<b>A</b>), but cells (1.5×10⁵) were cultured in the YPD medium (n = 4 each point) and after 4 days the fungal growth was determined by counting in a Neubauer chamber. The graphs show the mean CFU or number of cells ± SD for each sample. The results represent three independent experiments. Statistically significant samples are indicated (<i>p</i><0.01 or 0.05).</p

Low concentrations of H₂O₂ or nitrite promoted cell proliferation in <i>P. brasiliensis</i>.

<p>Pb18 yeast cells were seeded in a 6-well culture plate subjected to a 24-h period of starvation with F12 medium and treated with different concentrations of H₂O₂ (<b>A</b>) or nitrite (<b>B</b>) at pH 5.6 for 5 h at 37°C. Treated cells were plated in BHI and incubated at 37°C for 7–10 days (n = 6 at each point). The graph shows the means ± SD of total CFU before and after treatment with H₂O₂ or nitrite for each concentration. Statistically significant samples are indicated (<i>p</i><0.05). The results are representative of three independent experiments.</p

Low concentrations of H₂O₂ or nitrite induce Ras activation.

<p>(<b>A</b>) Pb18 yeast cells were cultivated in modified YPD for 5 days at 37°C, subjected to a 24-h period of starvation with F12 medium and incubated with 0.1 mM H₂O₂ or 0.25 µM NO₂ at pH 5.6 for different timepoints at 37°C. (<b>B</b>) Pb18 yeast cells were cultivated in modified YPD for 5 days at 37°C, subjected to a 24-h period of starvation with F12 medium and cultured with or without 250 µM FPT III inhibitor followed by treatment with 0.1 mM H₂O₂ or 0.25 µM NO₂ at pH 5.6 for 1 h at 37°C. After yeast cells lysis (<b>A</b>) and (<b>B</b>), Ras activation was determined using the GST-RBD(Byr2) fusion protein, which binds with high affinity to the GTP-associated form of Ras. Ras-GTP (active) and total Ras (50 µg total protein) were assayed by western blots probed with anti-Ras antibody. Relative densitometric values of bands are shown in the bar graphs.</p

Plasmid construction and production of the RBD(Byr2)-GST probe.

<p>(<b>A</b>) Schematic representation of Byr2 showing the localization of conserved domains, highlighting in red the localization of the Ras Binding Domain (RBD). RBD sequences from <i>P. brasiliensis</i> (Pb – EEH46080), <i>Blastomyces dermatitidis</i> (Bd – EGE86103), <i>H. capsulatum</i> (Hc – EGC48892), <i>Coccidioides immitis</i> (Ci – XP 001242119), <i>S. cerevisiae</i> (Sc – AAB67571), and RBD-Raf-1 of <i>Homo sapiens</i> (Hs – AGC09606) were aligned with ClustalW (module MegAlign, DNAstar Inc). Conserved sequences are boxed, the residues directly involved in the interaction with activated Ras (Ras-GTP) are indicated in red boxes and key amino acids involved in the interaction with Ras-GTP are shown in yellow. (<b>B</b>) Coomassie blue-stained 10% SDS-PAGE gels showing total bacterial extracts from recombinant bacteria expressing RBD(Byr2)-GST (arrow) stimulated (3 h) or not (0 h) with 1 mM IPTG. (<b>C</b>) Ten microliters of total (T), soluble (S) and insoluble (I) fractions were assayed in Western blots probed with anti-GST antibody. The migration of molecular mass standards (MW) is shown on the left. (<b>D</b>) Pb18 total extract (1 mg) was bound either to GTP (1 mM) or GDP (1 mM) and incubated with RBD(Byr2)-GST fusion protein linked to glutathione-Sepharose. Ras-GTP and total Ras (50 µg total protein) eluted with SDS-PAGE sample buffer were loaded in an SDS-PAGE gel. Ras was detected by immunoblotting with anti-Ras antibody.</p

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data