Advertisement

Parasitology Research

, Volume 112, Issue 9, pp 3305–3314 | Cite as

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

  • Flávia Regina Almeida-Campos
  • Thiago Castro-Gomes
  • Alice Machado-Silva
  • Jamil Silvano de Oliveira
  • Marcelo Matos Santoro
  • Frédéric Frézard
  • Maria Fátima HortaEmail author
Original Paper

Abstract

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.

Keywords

DPPC Hemolytic Activity Inactive Form Limited Proteolysis Parasitophorous Vacuole 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

CHAPSO

3[(3-Cholamidopropyl) dimethyl-ammonio]-2-hydroxy-propanesulfonate

DPPC

Dipalmitoylphosphatidyl choline

(g-HCl)

Guanidine-HCl

HEPES

N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid

H50

Inverse of the dilution that caused 50 % of hemolysis

HuE

Human erythrocytes

mExt

Promastigotes membrane detergent-soluble extract

PFP

Pore-forming protein(s)

PBS

Phosphate-buffered saline

Notes

Acknowledgments

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.

References

  1. Abrami L, Fivaz M, Decroly E, Seidah NG, Jean F, Thomas G, Leppla SH, Buckley JT, van der Goot FG (1998) The pore-forming toxin proaerolysin is activated by furin. J Biol Chem 273:32656–32661PubMedCrossRefGoogle Scholar
  2. Almeida-Campos FR, Horta MF (2000) Proteolytic activation of leishporin: evidence that Leishmania amazonensis and Leishmania guyanensis have distinct inactive forms. Mol Biochem Parasitol 111:363–375PubMedCrossRefGoogle Scholar
  3. Almeida-Campos FR, Noronha FSM, Horta MF (2002) The multitalented pore-forming proteins of intracellular pathogens. Microb Infection 4:741–750CrossRefGoogle Scholar
  4. Andrews NW, Portnoy DA (1994) Cytolysins of intracellular pathogens. Trends Microbiol 2:261–263PubMedCrossRefGoogle Scholar
  5. Bangham AD, Standish MM, Watkins JC (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13:238–252PubMedCrossRefGoogle Scholar
  6. Castro-Gomes T, Almeida-Campos FR, Calzavara-Silva CE, da Silva RA, Frézard F, Horta MF (2009) Membrane binding requirements for the cytolytic activity of Leishmania amazonensis leishporin. FEBS Lett 583:3209–3214PubMedCrossRefGoogle Scholar
  7. Handman E (1999) Cell Biology of Leishmania. Adv Parasitol 44:1–39PubMedCrossRefGoogle Scholar
  8. Horta MF (1997) Pore-forming proteins in pathogenic protozoan parasites. Trends Microbiol 5:363–366PubMedCrossRefGoogle Scholar
  9. Hybiske K, Stephens RS (2008) Exit strategies of intracellular pathogens. Nature Rev Microbiol 6:99–109CrossRefGoogle Scholar
  10. Leippe M, Bruhn H, Hecht O, Grötzinger J (2005) Ancient weapons: the three dimensional structure of amoebapore A. Trends Parasitol 21:5–7PubMedCrossRefGoogle Scholar
  11. Noronha FSM, Cruz JS, Beirão PSL, Horta MF (2000) Macrophage damage by Leishmania amazonensis cytolysin: evidence of pore formation. Infect Immun 68:4578–4584PubMedCrossRefGoogle Scholar
  12. Noronha FSM, Ramalho-Pinto FJ, Horta MF (1994) Identification of a putative pore-forming hemolysin active at acid pH in Leishmania amazonensis. Braz J Med Biol Res 27:477–482PubMedGoogle Scholar
  13. Noronha FSM, Ramalho-Pinto FJ, Horta MF (1996) Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein. Infect Immun 64:3975–3982PubMedGoogle Scholar
  14. Roiko MS, Carruthers VB (2009) New roles for perforins and proteases in apicomplexan egress. Cell Microbiol 11:1444–1452PubMedCrossRefGoogle Scholar
  15. Wilson ME, Jerônimo SMB, Pearson RD (2005) Immunopathogenesis of infection with the visceralizing Leishmania species. Microb Pathogenesis 38:147–160CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Flávia Regina Almeida-Campos
    • 1
    • 3
  • Thiago Castro-Gomes
    • 1
    • 4
  • Alice Machado-Silva
    • 1
    • 5
  • Jamil Silvano de Oliveira
    • 1
  • Marcelo Matos Santoro
    • 1
  • Frédéric Frézard
    • 2
  • Maria Fátima Horta
    • 1
    Email author
  1. 1.Departamento de Bioquímica e Imunologia, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  2. 2.Departamento de Fisiologia e Biofísica, Instituto de Ciências BiológicasUniversidade Federal de Minas GeraisBelo HorizonteBrazil
  3. 3.Departamento de Ciências Fisiológicas, Instituto de Ciências BiológicasUniversidade Federal do AmazonasManausBrazil
  4. 4.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA
  5. 5.Departamento de Química, Instituto de Ciências ExatasUniversidade Federal de Minas GeraisBelo HorizonteBrazil

Personalised recommendations