Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods
Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a “nonclassically” secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.
Key wordsLive cell imaging Leishmania Nonclassical protein secretion FRAP (fluorescence recovery after photobleaching) HASPB (hydrophilic acylated surface protein B) Acylation
We thank Jeremy Mottram (University of Glasgow) for the gift of RFP-ATG8 plasmid, and Barbara Smith, Michael Hodgkinson, and Ian Morrison for technical assistance. We also thank Deborah Smith for her support. This work was funded by Wellcome Trust programme grant 077503 awarded to Deborah F. Smith (University of York).
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