Biochemical and cell biological analysis of a virulence enhancing protein from Leishmania major; its impact on macrophage activity and gene expression
Final Report Abstract
A functional cloning approach using an avirulent Leishmania major strain as acceptor and donor identified a 46 kD, L. major-specific virulence-enhancing protein (P46) which upon over expression in the parasites increased infectivity in vitro, pathogenicity in vivo, and a widened range of experimental hosts. The goal of this project was to determine the biochemical characteristics of P46, its export pathway, its function inside mammalian macrophages, and its expression in different L. major isolates of varying pathogenicity. In addition, by the use of reverse genetics, the overall impact of P46 on L. major virulence was to be determined. We found that oligomers of P46 are not readily formed by recombinantly expressed protein, nor by GFP fusion protein. P46, like many virulence-enhancing proteins of Leishmania is exported to the host cell via exosomes, membrane vesicles carrying much of the exoproteome of the parasite. While the exact mode of action inside macrophages remains unsolved, we could generate L. major P46 null mutants which indeed display a loss of virulence in vivo and a loss of infectivity in vitro. A surprising finding was that P46 is subject to regional sequence variation, correlating with varying animal reservoirs found in the regions of endemicity, ranging from West Africa to Central Asia. Passage experiments indeed confirmed that certain P46 sequence variations are strongly favoured in mice, suggesting a role for P46 in host adaption.
Publications
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(2015). Geographical sequence variation in the Leishmania major virulence factor P46. Infection, genetics and evolution 30, 195-205
Bifeld, E., Chrobak, M., Zander, D., Schleicher, U., Schonian, G., and Clos, J.
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The genetics of Leishmania virulence. Medical Microbiology and Immunology, December 2015, Volume 204, Issue 6, pp 619–634
Bifeld, E., and Clos, J.
