We have previously shown that various species of Leishmania produce a lytic activity, which, in Leishmania amazonensis, is mediated by a pore-forming cytolysin, called leishporin. It is toxic for macrophages in vitro and optimally active at pH 5.0 to 5.5 and at 37 °C, suggesting that it might be active inside phagolysosomes. Leishporin from both L. amazonensis (a-leishporin) and Leishmania guyanensis (g-leishporin) can be activated by proteases, suggesting either a limited proteolysis of an inactive precursor or a proteolytic degradation of a non-covalently linked inhibitor. Here, we show that both a- and g-leishporin are also activated in dissociating conditions, indicating the second hypothesis as the correct one. In fact, we further demonstrated that inactive leishporin is non-covalently associated with an inhibitor, possibly more than one oligopeptide that, when removed, renders leishporin hemolytically active. This activation was shown to be the result of both the improvement of leishporin’s ability to bind to phospholipids and the emergence of its pore-forming ability. In vitro results demonstrate that leishporin can be released by the parasites, as they evolve in axenic cultures, in an inactive form that can be activated. These results are compatible with our hypothesis that leishporin can be activated in the protease-rich, low pH, and dissociating environment of parasitophorous vacuoles, leading to disruption of both vacuoles and plasma membranes with the release of amastigotes.
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- H50 :
Inverse of the dilution that caused 50 % of hemolysis
Promastigotes membrane detergent-soluble extract
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We thank Elimar Faria’s technical assistance. Financial support: UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, and Programa de Apoio a Núcleos de Excelência. FRAC and TCG were supported by Coordenadoria de Aperfeiçoamento de Pessoal do Ensino Superior. MFH and FF are Conselho Nacional de Desenvolvimento Científico e Tecnológico research fellows.
Flávia Regina Almeida-Campos and Thiago Castro-Gomes contributed equally to the experimental work performed for this manuscript.
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Almeida-Campos, F.R., Castro-Gomes, T., Machado-Silva, A. et al. Activation of Leishmania spp. leishporin: evidence that dissociation of an inhibitor not only improves its lipid-binding efficiency but also endows it with the ability to form pores. Parasitol Res 112, 3305–3314 (2013). https://doi.org/10.1007/s00436-013-3510-4
- Hemolytic Activity
- Inactive Form
- Limited Proteolysis
- Parasitophorous Vacuole