Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Depledge DP, MacLean LM, Hodgkinson MR, Smith BA, Jackson AP, Ma S, Uliana SR, Smith DF (2010) Leishmania-specific surface antigens show sub-genus sequence variation and immune recognition. PLoS Negl Trop Dis 4(9), e829. doi:10.1371/journal.pntd.0000829
Denny PW, Gokool S, Russell DG, Field MC, Smith DF (2000) Acylation-dependent protein export in Leishmania. J Biol Chem 275(15):11017–11025
Alce TM, Gokool S, McGhie D, Stager S, Smith DF (1999) Expression of hydrophilic surface proteins in infective stages of Leishmania donovani. Mol Biochem Parasitol 102(1):191–196
McKean PG, Trenholme KR, Rangarajan D, Keen JK, Smith DF (1997) Diversity in repeat-containing surface proteins of Leishmania major. Mol Biochem Parasitol 86(2):225–235
Rangarajan D, Gokool S, McCrossan MV, Smith DF (1995) The gene B protein localises to the surface of Leishmania major parasites in the absence of metacyclic stage lipophosphoglycan. J Cell Sci 108(Pt 11):3359–3366
Flinn HM, Rangarajan D, Smith DF (1994) Expression of a hydrophilic surface protein in infective stages of Leishmania major. Mol Biochem Parasitol 65(2):259–270
Sadlova J, Price HP, Smith BA, Votypka J, Volf P, Smith DF (2010) The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi. Cell Microbiol 12(12):1765–1779. doi:10.1111/j.1462-5822.2010.01507.x
Jensen AT, Gasim S, Moller T, Ismail A, Gaafar A, Kemp M, el Hassan AM, Kharazmi A, Alce TM, Smith DF, Theander TG (1999) Serodiagnosis of Leishmania donovani infections: assessment of enzyme-linked immunosorbent assays using recombinant L. donovani gene B protein (GBP) and a peptide sequence of L. donovani GBP. Trans R Soc Trop Med Hyg 93(2):157–160
Jensen AT, Gaafar A, Ismail A, Christensen CB, Kemp M, Hassan AM, Kharazmi A, Theander TG (1996) Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein. Am J Trop Med Hyg 55(5):490–495
Stager S, Alexander J, Kirby AC, Botto M, Rooijen NV, Smith DF, Brombacher F, Kaye PM (2003) Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses. Nat Med 9(10):1287–1292. doi:10.1038/nm933
Stager S, Smith DF, Kaye PM (2000) Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. J Immunol 165(12):7064–7071
Maroof A, Brown N, Smith B, Hodgkinson MR, Maxwell A, Losch FO, Fritz U, Walden P, Lacey CN, Smith DF, Aebischer T, Kaye PM (2012) Therapeutic vaccination with recombinant adenovirus reduces splenic parasite burden in experimental visceral leishmaniasis. J Infect Dis 205(5):853–863. doi:10.1093/infdis/jir842
Stegmayer C, Kehlenbach A, Tournaviti S, Wegehingel S, Zehe C, Denny P, Smith DF, Schwappach B, Nickel W (2005) Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant. J Cell Sci 118(Pt 3):517–527. doi:10.1242/jcs.01645
Tournaviti S, Pietro ES, Terjung S, Schafmeier T, Wegehingel S, Ritzerfeld J, Schulz J, Smith DF, Pepperkok R, Nickel W (2009) Reversible phosphorylation as a molecular switch to regulate plasma membrane targeting of acylated SH4 domain proteins. Traffic 10(8):1047–1060. doi:10.1111/j.1600-0854.2009.00921.x
Besteiro S, Williams RA, Morrison LS, Coombs GH, Mottram JC (2006) Endosome sorting and autophagy are essential for differentiation and virulence of Leishmania major. J Biol Chem 281(16):11384–11396. doi:10.1074/jbc.M512307200
Williams RA, Tetley L, Mottram JC, Coombs GH (2006) Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana. Mol Microbiol 61(3):655–674. doi:10.1111/j.1365-2958.2006.05274.x
Maclean LM, O'Toole PJ, Stark M, Marrison J, Seelenmeyer C, Nickel W, Smith DF (2012) Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion. Cell Microbiol 14(5):740–761. doi:10.1111/j.1462-5822.2012.01756.x
Duran JM, Anjard C, Stefan C, Loomis WF, Malhotra V (2010) Unconventional secretion of Acb1 is mediated by autophagosomes. J Cell Biol 188(4):527–536. doi:10.1083/jcb.200911154
Manjithaya R, Anjard C, Loomis WF, Subramani S (2010) Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation. J Cell Biol 188(4):537–546. doi:10.1083/jcb.200911149
Price HP, MacLean L, Marrison J, O'Toole PJ, Smith DF (2010) Validation of a new method for immobilising kinetoplastid parasites for live cell imaging. Mol Biochem Parasitol 169(1):66–69. doi:10.1016/j.molbiopara.2009.09.008
Engstler M, Pfohl T, Herminghaus S, Boshart M, Wiegertjes G, Heddergott N, Overath P (2007) Hydrodynamic flow-mediated protein sorting on the cell surface of trypanosomes. Cell 131(3):505–515. doi:10.1016/j.cell.2007.08.046
Grunfelder CG, Engstler M, Weise F, Schwarz H, Stierhof YD, Morgan GW, Field MC, Overath P (2003) Endocytosis of a glycosylphosphatidylinositol-anchored protein via clathrin-coated vesicles, sorting by default in endosomes, and exocytosis via RAB11-positive carriers. Mol Biol Cell 14(5):2029–2040. doi:10.1091/mbc.E02-10-0640
Sacks DL, da Silva RP (1987) The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid. J Immunol 139(9):3099–3106
Acknowledgements
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
MacLean, L., Price, H., O’Toole, P. (2016). Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods. In: Pompa, A., De Marchis, F. (eds) Unconventional Protein Secretion. Methods in Molecular Biology, vol 1459. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3804-9_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3804-9_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3802-5
Online ISBN: 978-1-4939-3804-9
eBook Packages: Springer Protocols