Novas alternativas terapêuticas para o tratamento da Criptococose: análogos de Resveratrol e microRNAs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência de Lactobacillus rhamnosus na patogenicidade e na expressão de genes de virulência de Candida albicans: estudo in vitro e in vivo

Pós-graduação em Biopatologia Bucal - ICT

Interação Candida albicans COM Acinetobacter baumannii E Klebsiella pneumoniae em biofilmes multiespécies

Pós-graduação em Biopatologia Bucal - ICT

Levantamento de nematoides entomopatogênicos em áreas agrícolas e influência da temperatura e do substrato na sobrevivência, multiplicação e armazenamento

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Levantamento de nematoides entomopatogênicos em áreas agrícolas e influência da temperatura e do substrato na sobrevivência, multiplicação e armazenamento

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

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.

Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes

Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity. (C) 2012 Elsevier Ltd. All rights reserved.

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 μg ml-1 was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions. (C) 2004 Elsevier Ltd. All rights reserved.

Evolutionary origin of Venturia canescens virus-like particles

Insect host-parasitoid interactions provide fascinating examples of evolutionary adaptations in which the parasitoid employs a variety of measures and countermeasures to overcome the immune responses of its host. Maternal factors introduced by the female wasps during egg deposition play an important role in interfering with cellular and humoral components of the host's immune defence. Some of these components actively suppress host immune components and some are believed to confer protection for the developing endoparasitoid by rather passive means. The Venturio conescens/Ephestia kuehniella parrositoid-host system is unique among other systems in that the cellular defence capacity of the host remains virtually intact after parasitization. This system raises some important questions that are discussed in this mini-review: If immune protection of the egg and the emerging larva is achieved by surface properties comprising glycoproteins and virus-like particles (VLPs) produced by the female wasp, why is the prophenoloxidose activating cascade blocked in parasitized caterpillars? Another question is the evolutionary origin of these particles, given that the functional role and structural features of V. canescens VLP proteins are more related to cellular proteins than to viruses.

Recognition and inactivation of LPS by lipophorin particles

Lipophorin is the major lipid carrier in insects, but various observations indicate that lipophorin is also involved in immune reactions. To examine a possible role of lipophorin in defence reactions, we mixed hemolymph plasma from Galleria mellonella with LPS and noticed that lipophorin forms detergent-insoluble aggregates, while most other plasma proteins are not affected. Lipophorin particles isolated by low-density gradient centrifugation retained LPS-induced aggregation properties, which suggested to us that these immune-reactive particles are able to recognise LPS and respond by forming insoluble aggregates. Antibodies against LPS-binding proteins, such as immulectin-2 and beta-1,3-glucan binding protein, cross-reacted with proteins associated with purified lipophorin particles. To examine whether LPS-mediated aggregates inactivate LPS, we added LPS-lipophorin mixtures to purified lipophorin particles and monitored aggregate formation. Under these conditions lipophorin did not form insoluble aggregates, which indicates that lipophorin particles sequester LPS into non-toxic aggregates. (c) 2005 Elsevier Ltd. All rights reserved.

LtpD is a novel legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD_471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD_471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide- binding L. pneumophila effector that has a role in intracellular bacterial replication. © 2013, American Society for Microbiology.

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7%) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7% - 49.5% identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin and spanins) and shows 29-98% homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60°C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest the AP3 phage is a promising potent agent against bacteria belonging to most common B. cenocepacia IIIA lineage strains.

Desarrollo de formulaciones bioplaguicidas a base de Bauveria bassiana (Bals & Vuils) con materiales sólidos y líquidos.

El objetivo de este trabajo fue evaluar en diferentes periodos de almacenamiento la calidad, en términos de viabilidad y concentración de conidias, en formulaciones bioplaguicidas a base de Beauveria bassiana, elaboradas con diferentes materiales inertes. Los materiales inertes (tratamientos) evaluados fueron: harina de trigo, arcilla blanca, arcilla verde, sulfato de calcio, aceite de soya, aceite de maní y el testigo fue B. bassiana en sustrato (no formulado). La concentración del hongo, utilizada para las formulaciones fue 8.5 x 109 conidias por gramo. Se utilizó B. bassiana cepa 114, obtenida del cepario del laboratorio de hongos entomopatógenos de la UNA. La reproducción del hongo se hizo mediante el método de producción semi-industrial, utilizando arroz grano entero como sustrato. La caracterización se hizo mediante características macroscópicas y ritmo de crecimiento del hongo en los medios de cultivo: PDA, SDA y EMA. Luego de elaboradas las formulaciones, fueron almacenadas en condiciones de laboratorio (24 oC -28ºC, HR60%) durante seis meses se determinó la concentración y viabilidad de conidias. Para la evaluación de la concentración de conidias se prepararon diluciones seriadas y el conteo de conidias se realizó en una cámara con rayado Neubauer. La evaluación de viabilidad, se hizo en medio Agar-agua 2%. La evaluación de la concentración de conidias así como la evaluación de la viabilidad se hizo cada 15 días durante los primeros tres meses y luego cada 30 días durante los últimos tres meses. El efecto de los materiales inertes sobre la efectividad biológica de B. bassiana fue evaluado en un bioensayo con la especie Galleria mellonella la que se obtuvo del laboratorio de cría de la UNA, usando una suspensión del hongo a una concentración de 1.6 x 108 por ml. Se realizó Análisis de Varianza y separación de medias según Tukey, por cada fecha de monitoreo. Se concluye que el mejor material para formular B. bassiana es harina de trigo, ya que fue el material inerte que presentó menor disminución en la concentración y viabilidad de conidias durante el periodo evaluado. La mayor velocidad promedio de crecimiento se observó en el medio de cultivo PDA. En general, los materiales inertes no afectaron significativamente la efectividad biológica de Beauveria bassiana en larvas de Galleria mellonella.

Impacto da utilização de ácido gálhico, ácido ferúlico e eugenol em modo contínuo na suscetibilidade de Pseudomonas aeruginosa

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016