Function and Trafficking of Flagellar Membrane Proteins in Leishmania mexicana

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Leishmania and other kinetoplastid parasites cause devastating diseases that afflict millions of people. All kinetoplastid parasites contain a whip-like organelle, the flagellum, in all life cycle stages. Studies over decades have underscored the vital and diverse roles that flagella play in the biology of these parasites, including motility, attachment to the gut of insect vectors, sensing the environment, mediating mating, interacting with internal membranes of mammalian host cells, evading the host immune response, etc. As such, flagella are essential for both parasite colonization of and transmission through the insect vector and for survival and virulence inside the vertebrate host. While internal components of the flagellum, such as the axoneme, have been studied in greater detail, the surface of the flagellar membrane (FM) is poorly understood, despite the fact that the FM mediates most of the above biological functions. To better understand the FM and its role in parasitism, this proposal will investigate: Aim 1) how specific FM proteins traffic selectively to this discrete component of the surface membrane; Aim 2) what the biological functions are for several selected FM proteins. The project is founded on initial studies on the KHARON protein, that mediates FM trafficking of a flagellar glucose transporter GT1, a trafficking machine that is essential for viability of intracellular disease- causing amastigote stages. A molecular mechanism for KHARON in delivering GT1 to Intraflagellar Transport particles for trafficking into the flagellum will be tested. Since most FM proteins do not depend upon KHARON for FM trafficking, other FM trafficking machines that interact with those FM proteins will be identified by tandem affinity purification/mass spectrometry. These novel FM trafficking machines will then be investigated, by gene knockout approaches, for critical functions in both the insect vector and the mammalian infective stages of the life cycle. Three selected FM proteins with known or likely biochemical functions will then be investigated, by gene knockout, for functions in the insect vector and mammalian host stages. These studies will reveal how specific molecular components of the FM mediate critical functions for the parasite in both the insect vector and the mammalian host.