Shedding of TRAP by a Rhomboid Protease from the Malaria Sporozoite Surface Is Essential for Gliding Motility and Sporozoite Infectivity

Abstract

Plasmodium sporozoites, the infective stage of the malaria parasite, move by gliding motility, a unique form of locomotion required for tissue migration and host cell invasion. TRAP, a transmembrane protein with extracellular adhesive domains and a cytoplasmic tail linked to the actomyosin motor, is central to this process. Forward movement is achieved when TRAP, bound to matrix or host cell receptors, is translocated posteriorly. It has been hypothesized that these adhesive interactions must ultimately be disengaged for continuous forward movement to occur. TRAP has a canonical rhomboid-cleavage site within its transmembrane domain and mutations were introduced into this sequence to elucidate the function of TRAP cleavage and determine the nature of the responsible protease. Rhomboid cleavage site mutants were defective in TRAP shedding and displayed slow, staccato motility and reduced infectivity. Moreover, they had a more dramatic reduction in infectivity after intradermal inoculation compared to intravenous inoculation, suggesting that robust gliding is critical for dermal exit. The intermediate phenotype of the rhomboid cleavage site mutants suggested residual, albeit inefficient cleavage by another protease. We therefore generated a mutant in which both the rhomboid-cleavage site and the alternate cleavage site were altered. This mutant was non-motile and non-infectious, demonstrating that TRAP removal from the sporozoite surface functions to break adhesive connections between the parasite and extracellular matrix or host cell receptors, which in turn is essential for motility and invasion.

Author Summary

Malaria infection begins with the bite of an infected mosquito which inoculates sporozoites into the skin. Sporozoites then go to the liver where they invade hepatocytes and replicate, ultimately leading to the blood stage of infection. Sporozoites are motile and actively invade hepatocytes using a unique form of motility called gliding motility. The mechanism by which the parasite moves forward is somewhat similar to a treadmill and the sporozoite protein TRAP, is key to this process. Its extracellular portion binds to host proteins while its intracellular portion binds to the parasite's motor. As the motor moves the protein rearwards, the sporozoite moves forward. It follows that the extracellular adhesive interactions of TRAP must ultimately be disengaged for forward movement to occur. We have generated mutant sporozoites that can only partially disengage these parasite-host adhesive interactions and find that these sporozoites have a halting, constipated movement. Following this, we generated a mutant that cannot disengage these interactions at all and these sporozoites are nonmotile and noninfectious. Lastly we found that a parasite rhomboid protease, ROM4, is on the surface of the sporozoite and thus may be responsible for TRAP cleavage and shedding from the sporozoite surface. Overall, our results demonstrate that robust gliding motility requires the disengagement of adhesive interactions.