Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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

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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

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

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 273:32656–32661

  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 111:363–375

  3. Almeida-Campos FR, Noronha FSM, Horta MF (2002) The multitalented pore-forming proteins of intracellular pathogens. Microb Infection 4:741–750

  4. Andrews NW, Portnoy DA (1994) Cytolysins of intracellular pathogens. Trends Microbiol 2:261–263

  5. Bangham AD, Standish MM, Watkins JC (1965) Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol 13:238–252

  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 583:3209–3214

  7. Handman E (1999) Cell Biology of Leishmania. Adv Parasitol 44:1–39

  8. Horta MF (1997) Pore-forming proteins in pathogenic protozoan parasites. Trends Microbiol 5:363–366

  9. Hybiske K, Stephens RS (2008) Exit strategies of intracellular pathogens. Nature Rev Microbiol 6:99–109

  10. Leippe M, Bruhn H, Hecht O, Grötzinger J (2005) Ancient weapons: the three dimensional structure of amoebapore A. Trends Parasitol 21:5–7

  11. Noronha FSM, Cruz JS, Beirão PSL, Horta MF (2000) Macrophage damage by Leishmania amazonensis cytolysin: evidence of pore formation. Infect Immun 68:4578–4584

  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 27:477–482

  13. Noronha FSM, Ramalho-Pinto FJ, Horta MF (1996) Cytolytic activity in the genus Leishmania: involvement of a putative pore-forming protein. Infect Immun 64:3975–3982

  14. Roiko MS, Carruthers VB (2009) New roles for perforins and proteases in apicomplexan egress. Cell Microbiol 11:1444–1452

  15. Wilson ME, Jerônimo SMB, Pearson RD (2005) Immunopathogenesis of infection with the visceralizing Leishmania species. Microb Pathogenesis 38:147–160

Download references

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.

Author information

Correspondence to Maria Fátima Horta.

Additional information

Flávia Regina Almeida-Campos and Thiago Castro-Gomes contributed equally to the experimental work performed for this manuscript.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Keywords

  • DPPC
  • Hemolytic Activity
  • Inactive Form
  • Limited Proteolysis
  • Parasitophorous Vacuole