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.inducible expression ͉ FK506 ͉ glycoconjugates ͉ pathogen ͉ trypanosomatid protozoa

Therapeutic Efficacy of Stable Analogues of Vasoactive Intestinal Peptide against Pathogens

“…For (23). In this inducible system, the synthesis of a key galactofuranose residue within the LPG core is controlled using a conditional "destabilization domain" fused to UDP-galactopyranosylmutase in the WT L. major Friedlin V1 background.…”

Unusual Galactofuranose Modification of a Capsule Polysaccharide in the Pathogenic Yeast Cryptococcus neoformans

“…Kinetoplastids incorporate Galf into a protective glycocalyx that enhances establishment of disease and promotes survival of these parasites within their insect vectors (8). In Leishmania, loss of Galf synthesis or of the key galactofuranosyltransferase required to synthesize surface lipophosphoglycan results in severe defects in survival both in the insect and in the early stages of mammalian infection (15)(16)(17)(18). In the pathogenic fungus Aspergillus fumigatus, the presence of Galf in secreted glycoproteins, glycolipids, and cell wall polysaccharides (7,8) improves cell integrity (19 -21) and reduces adhe-sion to mammalian epithelial and endothelial surfaces by shielding underlying mannan structures (20).…”

Using Genomic Information to Understand Leishmania Biology

“…It was originally described for mammalian cells and it is based on the targeting of FKBP (FK506-Binding Protein) to proteasomal degradation after rapamycin/FK506/Shld1 binding to its destabilizing domain [93]. This system was implemented in L. (L.) major and L. (V.) braziliensis to conditionally express destabilized UDPgalactopyranose mutase (UGM), a fundamental enzyme in lypophosphoglycan (LPG) biosynthesis [94].…”

“…Therefore, reliable strategies such as the destabilization domain system represent promising benefits for molecular biology investigation [94].…”