Stress conditions in the host induce persister cells and influence biofilm formation by Staphylococcus epidermidis RP62A

INTRODUCTION: Studies have demonstrated that pathogens react to the harsh conditions in human tissues by inducing mechanisms that promote survival. METHODS: Persistence and biofilm-forming ability were evaluated during stress conditions that mimic those in the host. RESULTS: Carbon-source availability had a positive effect on Staphylococcus epidermidis RP62A adhesion during hypoxia, accompanied by a decrease in pH. In contrast, iron limitation led to decreased surface-adherent biomass, accompanied by an increase medium acidification and lactate levels. Interestingly, iron starvation and hypoxia induced persister cells in planktonic culture. CONCLUSIONS: These findings highlight the role of host stress in the virulence of S. epidermidis

Educomunicação e suas áreas de intervenção: Novos paradigmas para o diálogo intercultural

oai:omp.abpeducom.org.br:publicationFormat/1O material aqui divulgado representa, em essência, a contribuição do VII Encontro Brasileiro de Educomunicação ao V Global MIL Week, da UNESCO, ocorrido na ECA/USP, entre 3 e 5 de novembro de 2016. Estamos diante de um conjunto de 104 papers executivos, com uma média de entre 7 e 10 páginas, cada um. Com este rico e abundante material, chegamos ao sétimo e-book publicado pela ABPEducom, em seus seis primeiros anos de existência. A especificidade desta obra é a de trazer as “Áreas de Intervenção” do campo da Educomunicação, colocando-as a serviço de uma meta essencial ao agir educomunicativo: o diálogo intercultural, trabalhado na linha do tema geral do evento internacional: Media and Information Literacy: New Paradigms for Intercultural Dialogue

Expressão de GFP em Bacillus thuringiensis S76 uma estirpe selvagem ativa para Lepidoptera

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2009.B. thuringiensis é um importante entomopatógeno amplamente estudado e empregado como agente de controle biológico, ainda assim, a ecologia desse microrganismo é pouco explorada. Apesar da crescente utilização na agricultura desse agente, as interações com plantas são pouco conhecidas. Com o intuito de construir uma ferramenta eficaz para o rastreamento dessa bactéria, foi desenvolvida a estirpe S76GFP+ a partir de uma estirpe brasileira selvagem de B. thuringiensis. O vetor de expressão pAD43-25 contendo o gene gfpmut3a funcional, além do vetor parental, pAD123, contendo o mesmo gene, porém sem promotor foram transferidos para a estirpe S76, permitindo no primeiro caso a expressão constitutiva do gene durante o crescimento vegetativo. Adicionalmente, como controle, ambos vetores foram transferidos para uma estirpe acristalífera (Cry-). Células verdes brilhantes foram detectadas por microscopia de fluorescência, em ambas hospedeiras transformadas com o vetor pAD43-25, a partir de duas horas após a inoculação em meio líquido e que puderam ser observadas pelo restante do tempo de cultivo até o fim da esporulação. A estirpe S76GFP+ apresentou um discreto acréscimo no tempo de geração, embora não tenham sido observadas alterações morfológicas das colônias e células. Análises dos perfis protéico e plasmidial indicaram, respectivamente, a manutenção da expressão das proteínas Cry e do elenco dos plasmídeos residentes. Do nosso conhecimento, não apenas é o primeiro relato de visualização da emissão de fluorescência pela expressão de GFP em B. thuringiensis. Igualmente, é o primeiro relato de expressão de GFP em uma estirpe de B. thuringiensis selvagem. _______________________________________________________________________________ ABSTRACTAlthough extensively studied by its recognized potential as bioinseticide, B. thuringiensis ecology is poorly understood, and despite its increasing use, little is known regarding interactions between this microorganism and plants. Thus, a tractable marker for detection of this bacterium under specific environment and physiological conditions is a valuable tool. A plasmid (pAD43-25) bearing a functional gfp gene and the parental vector, bearing the promotorless gfp gene, were introduced in the Brazilian wild-type strain B. thuringiensis kurstaki S76, allowing, in the first case, the constitutive synthesis of GFP during vegetative growth. Additionally, both vectors were transferred to a Cry- B. thuringiensis host. Green bright cells could be detected, by fluorescence microscopy and in both hosts, since 2h after inoculation in liquid medium and could be seen throughout remaining cultivation time, until complete sporulation was accomplished. Strain S76GFP+ seems to grow slower than the remaining recombinants and parental cells, whereas no perceptible change in cell or colony morphologies was observed. Protein profile and plasmidial DNA analyses indicate, respectively, that this recombinant maintained Cry proteins expression and resident plasmid outline. To our knowledge, it is the first time fluorescence is detected in a B. thuringiensis strain, as well as, that GFP is expressed in a wild-type B. thuringiensis

Analysis of the secretomes of paracoccidioides mycelia and yeast cells

Submitted by Jaqueline Silva ([email protected]) on 2018-05-15T22:20:11Z No. of bitstreams: 2 Artigo - Simone Schneider Weber - 2012.PDF: 498981 bytes, checksum: 5e5cc2b69e9d37e0442d54c04f1be937 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Approved for entry into archive by Luciana Ferreira ([email protected]) on 2018-05-16T12:06:48Z (GMT) No. of bitstreams: 2 Artigo - Simone Schneider Weber - 2012.PDF: 498981 bytes, checksum: 5e5cc2b69e9d37e0442d54c04f1be937 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2018-05-16T12:06:48Z (GMT). No. of bitstreams: 2 Artigo - Simone Schneider Weber - 2012.PDF: 498981 bytes, checksum: 5e5cc2b69e9d37e0442d54c04f1be937 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012Paracoccidioides, a complex of several phylogenetic species, is the causative agent of paracoccidioidomycosis. The ability of pathogenic fungi to develop a multifaceted response to the wide variety of stressors found in the host environment is important for virulence and pathogenesis. Extracellular proteins represent key mediators of the host-parasite interaction. To analyze the expression profile of the proteins secreted by Paracoccidioides, Pb01 mycelia and yeast cells, we used a proteomics approach combining two-dimensional electrophoresis with matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF MS/MS). From three biological replicates, 356 and 388 spots were detected, in mycelium and yeast cell secretomes, respectively. In this study, 160 non-redundant proteins/isoforms were indentified, including 30 and 24 proteins preferentially secreted in mycelia and yeast cells, respectively. In silico analyses revealed that 65% of the identified proteins/isoforms were secreted primarily via non-conventional pathways. We also investigated the influence of protein export inhibition in the phagocytosis of Paracoccidioides by macrophages. The addition of Brefeldin A to the culture medium significantly decreased the production of secreted proteins by both Paracoccidioides and internalized yeast cells by macrophages. In contrast, the addition of concentrated culture supernatant to the co-cultivation significantly increased the number of internalized yeast cells by macrophages. Importantly, the proteins detected in the fungal secretome were also identified within macrophages. These results indicate that Paracoccidioides extracellular proteins are important for the fungal interaction with the host

<i>Paracoccidiodes, Pb</i>01 yeast cells secreted proteins in macrophages by nanoLC-MS/MS.

1<p>NCBI database general information number (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p

Graphic summation of proteomics analysis.

<p><b>A-</b> The comparative analysis using Image master 2D Platinum software displays the analyzed spots and the preferentially expressed spots in mycelia and yeast secretomes, while the mass spectrometry analyses displays the identified differentially expressed spots. <b>B-</b> The Venn diagram shows the number of identified proteins via MS/MS. The preferentially secreted extracellular proteins include those with statistically significant alteration in the mycelia and yeast secretome, while the constitutive proteins refer to those with similar expression patterns.</p

Proteins detected in the secretome of yeast cells and mycelia of <i>Paracoccidiodes</i> via 2D-gel analysis.

<p>Protein profile generated after the separation of the secreted fraction of proteins by yeast cells (<b>A</b>) and mycelia (<b>B</b>) using 2D-eletrophoresis (first dimension: IEF pH range 3 –11 non-linear, second dimension: 12% (w/v) SDS-PAGE) and visualized using Coomassie brilliant blue staining. The 2-D gel images of three biological replications of each phase were compared to identify the differential expression levels of proteins using Image master 2D Platinum software. The protein spots that were identified via MS/MS are numbered and listed in Table S1. The pH gradient is shown above the gel, and the molecular mass protein standards (kDa) are indicated to the left of the gels.</p

Constitutive proteins secreted by <i>Paracoccidioides Pb</i>01 yeast and mycelia.

1<p>NCBI database general information number (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>).</p>2<p>Number of identified isoforms of protein in <i>Paracoccidioides, Pb01</i> secretome.</p>3<p>The average of amount of values of abundances of all identified isoforms used to statistical test.</p>4<p>ANOVA – statistically significant differences are considered with <i>p</i><0.05 (*).</p>5<p>Secretion prediction according to Signal P 3.0 server, the number corresponds to signal peptide probability (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>).</p>6<p>Secretion prediction according to Secretome P 2.0 server, the number corresponds to neural network that exceeded a value of 0.5 (NN-score <b>≥</b>0.50) (<a href="http://www.cbs.dtu.dk/services/SecretomeP/" target="_blank">http://www.cbs.dtu.dk/services/SecretomeP/</a>).</p

Validation of the extracellular protein extraction method.

<p><b>A-</b> The viability of <i>Paracoccidioides</i> yeast cells incubated in Fava Netto's liquid medium (dark gray square) and the incubation of yeast cells in Fava Netto's liquid medium containing 6 µg/mL Brefeldin A (light gray square). Viability was assessed using trypan blue staining. The error bars represent the standard deviation of three biological replicates. <b>B-</b> The growth of <i>Paracoccidioides</i> yeast cells in liquid medium in either the absence (dark line) or presence of 6 µg/mL Brefeldin A (light gray line). Culture growth was evaluated by quantifying the number of yeast cells per mL. The error bars represent the standard deviation of three biological replicates. <b>C-</b> PCR sensitivity for the formamidase gene was assessed using <i>Paracoccidiodes Pb01</i> genomic DNA (at five dilutions) as a template (50 ng to 1 pg). Lanes: 1 −50 ng; 2 −5 ng; 3 −50 pg; 4 −5 pg; 5 −1 pg; 6 - negative control (without genomic DNA). The formamidase PCR amplicons were assessed via 1% agarose gel electrophoresis and stained with ethidium bromide. <b>D-</b> The yeast and mycelia cell-free supernatant samples (2 µL) were assessed for the presence of <i>Paracoccidiodes</i> DNA via PCR using oligonucleotides specific for the formamidase gene.</p