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Identification of a putative pore-forming hemolysin active at acid pH in Leishmania amazonensis
F S Noronha1, F J Ramalho-Pinto, M F Horta
1Departamento de Bioquímica-Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.
Abstract:
Several organisms, including the protozoa Entamoeba histolytica and Trypanosoma cruzi, have been shown to contain pore-forming proteins (PFP) thought to play a role in the pathogenesis of the diseases they generate. In the present report, we show that promastigotes of Leishmania amazonensis express a hemolysin that appears to cause colloid-osmotic lysis, typical of pore formation. This hemolysin affects red blood cells of different species at 37 degrees C, but not at 0 degrees C, with maximum activity at pH 5.0. The hemolytic activity is heat-labile, but lysis is not affected by protease inhibitors. These results suggest the involvement of a protein with no proteolytic or detergent activity. Hemolysis is inhibited by polyethyleneglycol, suggesting its colloid-osmotic nature. Hemolytic extracts of the parasite contain a polypeptide that reacts with antibodies to perforin from mouse cytotoxic T lymphocytes or to C9 from human complement. In addition, genomic DNA of L. amazonensis contains a fragment that hybridizes to a perforin cDNA probe. The circumstantial evidence suggests that the L. amazonensis hemolytic activity may be mediated by a PFP homologous to perforin and C9.
Insights
Leishmania amazonensis parasites release a hemolysin causing colloid-osmotic lysis of red blood cells. This pore-forming protein (PFP) activity is heat-labile and pH-dependent, suggesting a role in disease pathogenesis.
Area of Science:
- Molecular Biology
- Parasitology
- Immunology
Background:
- Pore-forming proteins (PFPs) are implicated in pathogenesis by various protozoa.
- Leishmania amazonensis is a parasite causing leishmaniasis.
Purpose of the Study:
- To investigate the hemolytic activity of Leishmania amazonensis.
- To determine the nature of the hemolysin and its potential relationship to known PFPs.
Main Methods:
- Assessing hemolysin activity on red blood cells at different temperatures and pH.
- Testing the effect of protease inhibitors and polyethyleneglycol on hemolysis.
- Immunological and genetic analysis of hemolytic extracts using antibodies against perforin and C9, and DNA hybridization.
Main Results:
- Leishmania amazonensis promastigotes exhibit heat-labile hemolytic activity, optimal at pH 5.0 and 37°C, causing colloid-osmotic lysis.
- The hemolysin is not affected by protease inhibitors but is inhibited by polyethyleneglycol.
- Hemolytic extracts contain a polypeptide reactive with anti-perforin/C9 antibodies, and parasite DNA contains a perforin-homologous fragment.
Conclusions:
- Leishmania amazonensis hemolysin likely functions as a pore-forming protein (PFP).
- The PFP may be homologous to mammalian perforin and complement C9.
- This PFP activity could contribute to the pathogenesis of leishmaniasis.