title: Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins.
abstract: Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence.
authors.affiliation: Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Luciana
authors.initials: L
authors.lastname: Madeira da Silva
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Owens
authors.forename: Silvane M F
authors.initials: SM
authors.lastname: Murta
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Beverley
mesh_terms: D000818:Animals; D015536:Down-Regulation; D006028:Glycosphingolipids / Q000096:biosynthesis*; D019751:Intramolecular Transferases / Q000378:metabolism; D018320:Leishmania major / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000096:biosynthesis*
chemical_list: D006028:Glycosphingolipids; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; C008290:lipophosphonoglycan; D022021:Tacrolimus Binding Proteins; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase; D020123:Sirolimus