UBC Theses and Dissertations
Identification of a novel secretion system in Leishmania : composition, mechanisms, and immune modulating properties Silverman, Judith Maxwell
Human infection with protozoa of the genus Leishmania results in a spectrum of disease manifestations collectively termed the leishmaniases. These diseases involve a chronic infection of macrophages which display a deactivated phenotype. Various proteins secreted by leishmania are known to interact with host signaling molecules, bringing about activation of negative feedback loops. Some of these have been shown to block interferon-γ signaling and to inhibit macrophage microbicidal functions. Unlike the well characterized secretion mechanisms used by bacterial pathogens, the mechanism(s) by which leishmania and other eukaryotic pathogens secrete proteins into host cells has remained elusive. The overall aim of this project was to gain a more thorough understanding of host immune-modulation by leishmania, based on the hypothesis that much of these effects are mediated by secreted proteins. The project goals were to: 1) comprehensively identify the proteins secreted by leishmania, 2) determine the mechanism by which proteins are secreted from leishmania into host cells, and 3) determine the functional properties of leishmania secreted compounds. To achieve these goals, a global proteomic analysis of leishmania secreted proteins was carried out using quantitative mass spectrometry. This identified 358 bona fide leishmania secreted proteins many of which were orthologs of proteins considered to be markers of mammalian exosomes. Subsequent experiments confirmed that leishmania secrete exosomes into conditioned media. Comparative proteomics showed that exosomes account for at least 50% of protein secretion by leishmania. Furthermore, the results showed that the cargo profile of leishmania exosomes is influenced by changes in temperature and pH, similar to those experienced by promastigotes after host invasion. Microscopy of leishmania infected cells confirmed the novel finding that leishmania use exosomes to deliver proteins into host cells. Additional studies demonstrated that leishmania exosomes have immunosuppressive properties which, in a cargo dependent manner, modulate the responses of monocytes, dendritic cells, and T lymphocytes. These findings suggest that leishmania utilize exosomes in long-range cellular communication and immune-modulation. In conclusion, this research has significantly advanced the current knowledge of leishmania biology, through the identification of novel secreted molecules, discovery of a secretion system, and description of the immune-modulating effects of leishmania exosomes.
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