Navegando por Palavras-chave "2D-DIGE"

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    Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex
    (Elsevier B.V., 2015-02-06) Rodrigues, Anderson Messias [UNIFESP]; Kubitschek-Barreira, Paula H.; Fernandes, Geisa Ferreira [UNIFESP]; Almeida, Sandro Rogerio de; Lopes-Bezerra, Leila M.; Camargo, Zoilo Pires de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Universidade do Estado do Rio de Janeiro (UERJ); Universidade de São Paulo (USP)
    Sporotrichosis is a polymorphic disease that affects both humans and animals worldwide. the fungus gains entry into a warm-blooded host through minor trauma to the skin, typically by contaminated vegetation or by scratches and bites from a diseased cat. Cellular and humoral responses triggered upon pathogen introduction play important roles in the development and severity of the disease. We investigated molecules expressed during the host parasite interplay that elicit the humoral response in human sporotrichosis. for antigenic profiling, Sporothrix yeast cell extracts were separated by two-dimensional (2D) gel electrophoresis and probed with pooled sera from individuals with fixed cutaneous and lymphocutaneous sporotrichosis. Thirty-five IgG-seroreactive spots were identified as eight specific proteins by MALDI ToF/MS. Remarkable cross-reactivity among Sporothrix brasiliensis, Sporothrix schenckii, and Sporothrix globosa was noted and antibodies strongly reacted with the 70-kDa protein (gp70), irrespective of clinical manifestation. Gp70 was successfully identified in multiple spots as 3-carboxymuconate cyclase. in addition, 2D-DIGE characterization suggested that the major antigen of sporotrichosis undergoes post-translational modifications involving glycosylation and amino acid substitution, resulting in at least six isoforms and glycoforms that were present in the pathogenic species but absent in the ancestral non-virulent Sporothrix mexicana. Although a primary environmental function related to the benzoate degradation pathway of aromatic polymers has been attributed to orthologs of this molecule, our findings support the hypothesis that gp70 is important for pathogenesis and invasion in human sporotrichosis. We propose a diverse panel of new putative candidate molecules for diagnostic tests and vaccine development.Biological significanceOutbreaks due to Sporothrix spp. have emerged over time, affecting thousands of patients worldwide. A sophisticated host pathogen interplay drives the manifestation and severity of infection, involving immune responses elicited upon traumatic exposure of the skin barrier to the pathogen followed by immune evasion. Using an immunoproteomic approach we characterized proteins of potential significance in pathogenesis and invasion that trigger the humoral response during human sporotrichosis. We found gp70 to be a cross-immunogenic protein shared among pathogenic Sporothrix spp. but absent in the ancestral environmental S. mexicana, supporting the hypothesis that gp70 plays key roles in pathogenicity. for the first time, we demonstrate with 2D-DIGE that post-translational modifications putatively involve glycosylation and amino acid substitution, resulting in at least six isoforms and glycoforms, all of them IgG-reactive. These findings of a convergent humoral response highlight gp70 as an important target serological diagnosis and for vaccine development among phylogenetically related agents of sporotrichosis. (C) 2014 Elsevier B.V. All rights reserved.
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    Xanthomonas citri subsp citri surface proteome by 2D-DIGE: Ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction
    (Elsevier Science Bv, 2017) Carnielli, Carolina Moretto; Artier, Juliana; Franco de Oliveira, Julio Cezar [UNIFESP]; Marques Novo-Mansur, Maria Teresa
    Xanthomonas citri subsp. citri (XAC) is the causative agent of citrus canker, a disease of great economic impact around the world. Understanding the role of proteins on XAC cellular surface can provide new insights on pathogen-plant interaction. Surface proteome was performed in XAC grown in vivo (infectious) and in vitro (non-infectious) conditions, by labeling intact cells followed by cellular lysis and direct 2D-DIGE analysis. Seventy-nine differential spots were analyzed by mass spectrometry. Highest relative abundance for in vivo condition was observed for spots containing Dnal(protein, 60 kDa chaperonin, conserved hypothetical proteins, malate dehydrogenase, phosphomannose isomerase, and ferric enterobactin receptors. Elongation factor Tu, OmpA-related proteins, Oar proteins and some Ton-B dependent receptors were found in spots decreased in vivo. Some proteins identified on XAC's surface in infectious condition and predicted to be cytoplasmic, such as DnaK and 60 KDa chaperonin, have also been previously found at cellular surface in other microorganisms. This is the first study on XAC surface proteome and results point to mediation of molecular chaperones in XAC-citrus interaction. The approach utilized here can be applied to other pathogen-host interaction systems and help to achieve new insights in bacterial pathogenicity toward promising targets of biotechnological interest. Biological significance: This research provides new insights for current knowledge of the Xanthomonas sp. pathogenicity. For the first time the 2D-DIGE approach was applied on intact cells to find surface proteins involved in the pathogen-plant interaction. Results point to the involvement of new surface/outer membrane proteins in the interaction between XAC and its citrus host and can provide potential targets of biotechnological interest for citrus canker control. (C) 2016 Elsevier B.V. All rights reserved.