Plasmodium sporozoites move by gliding motility, a form of motility that is powered by a subpellicular actin-myosin motor. A central protein in this process is TRAP, a type I transmembrane protein that links the motor to the extracellular substrate. It has been hypothesized that TRAP is translocated posteriorly via the force of the motor and at the posterior end of the sporozoite, it is likely shed from the parasite's surface via proteolytic cleavage, a process that would enable the sporozoite to disengage adhesive interactions and allow for forward movement. Proteolytic processing of TRAP has not been well characterized and we set out to determine the function of TRAP cleavage as well as the nature of the protease responsible for TRAP shedding. Pulse-chase metabolic labeling experiments showed that TRAP is cleaved in its carboxy-terminus, releasing the entire extracellular domain from the sporozoite surface. Processing is inhibited by a subset of serine protease inhibitors. It has been hypothesized that TRAP processing is mediated by a class of intramembranous serine proteases called rhomboids, because its transmembrane domain contains a conserved rhomboid substrate motif. To determine whether this is the case and to better understand the function of TRAP shedding from the sporozoite surface, we generated mutants in which the putative rhomboid substrate motif of TRAP was altered. Sporozoites expressing TRAP with these mutations cleave TRAP much less efficiently and the cleaved fragment is smaller, indicating that some cleavage is occurring at another site. Our data suggested that the alternate site was located juxtamembraneously and to address this issue, we generated double mutants in which the juxtamembrane region of TRAP was deleted along with disruption of the rhomboid substrate motif. These double mutant sporozoites were completely unable to process TRAP. While the rhomboid-cleavage site mutants exhibit abnormal gliding motility and have significantly decreased infectivity for target organs, the double mutants are non-motile and non infectious in both mosquito and mammalian hosts. These data demonstrate that cleavage of TRAP is required for sporozoite motility and invasion of both mosquito salivary glands and mammalian hepatocytes.
|Commitee:||Blackman, Micheal, Darwin, Heran, Eichinger, Daniel, Ghiso, Jorge|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 70/12, Dissertation Abstracts International|
|Subjects:||Cellular biology, Parasitology|
|Keywords:||Gliding motility, Plasmodium, Rhomboids, Sporozoites, TRAP|
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