Peptidases and gp63-like proteins in Herpetomonas megaseliae: possible involvement in the adhesion to the invertebrate host |
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Authors: | Nogueira de Melo Ana C d'Avila-Levy Claudia M Dias Felipe A Armada Jorge Luís A Silva Heriberto D Lopes Angela H C S Santos André L S Branquinha Marta H Vermelho Alane B |
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Affiliation: | Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária, Ilha do Fund?o, Rio de Janeiro, RJ 21941-590, Brazil. |
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Abstract: | The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues. |
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Keywords: | Herpetomonas megaselia Megaselia scalaris Aedes aegypti Insect trypanosomatid Peptidase gp63 Cellular interaction |
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