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Eukaryotic Cell, March 2005, p. 556-566, Vol. 4, No. 3
1535-9778/05/$08.00+0     doi:10.1128/EC.4.3.556-566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Intracellular Glycosylphosphatidylinositols Accumulate on Endosomes: Toxicity of Alpha-Toxin to Leishmania major

Zhifeng Zheng,1 Rodney K. Tweten,2 and Kojo Mensa-Wilmot1*

Department of Cellular Biology, The University of Georgia, Athens, Georgia,1 Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma2

Received 22 September 2004/ Accepted 12 November 2004

Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur "free" or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM.

* Corresponding author. Mailing address: Department of Cellular Biology, The University of Georgia, 724 Biological Sciences, Athens, GA 30602. Phone: (706) 542-3355. Fax: (706) 542-4271. E-mail: mensawil{at}cb.uga.edu.

Eukaryotic Cell, March 2005, p. 556-566, Vol. 4, No. 3
1535-9778/05/$08.00+0     doi:10.1128/EC.4.3.556-566.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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