EXPLORATION OF THE CONTRIBUTION OF PLASMODIUM GRASP TO MULTIPLE PARASITIC STAGE DEVELOPMENT, WITH SPECIAL FOCUS ON METAMORPHOSIS IN THE LIVER
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Date
2020-04-27
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Johns Hopkins University
Abstract
The Plasmodium parasite undergoes dramatic morphological and metabolic transformations as it adapts to diverse environments within a mosquito vector and a vertebrate host. During liver infection, the morphological changes of sporozoites (insect forms) includes the elimination of micronemes, organelles previously required for hepatocyte penetration. Preliminary experimental evidence suggests that Plasmodium utilizes an autophagic pathway that shares components with the unconventional protein secretion (UPS) pathway for microneme clearance from liver forms. The UPS pathway is implicated in the sequestration and expulsion of damaged proteins or organelles, bypassing the endoplasmic reticulum, instead requiring ATG8, and the Golgi reassembly and stacking protein (GRASP), a process called exophagy. In exophagy, GRASP has been identified as a molecular effector, tethering autophagic vesicles to the cell plasma membrane for cargo release. A GRASP ortholog is present in Plasmodium but its physiological role remains elusive. To investigate the relevance of Plasmodium GRASP to malaria infection, we have engineered a knockout Plasmodium mutant, named PbGRASP-KO (Plasmodium berghei GRASP Knockout). The objective of this study is to explore the role of P. berghei GRASP through all stages of the parasite’s lifecycle, and particularly the possible involvement of GRASP in exophagy of micronemes during liver stage development. If PbGRASP-KO parasites are unable to clear micronemes during the transformation of sporozoites to liver stage trophozoites, this will subsequently impact parasite multiplication in the liver. Comparing the development of P. berghei WT and PbGRASP-KO in the mosquito, liver, and blood stages, we show that PbGRASP-KO displays no growth defects in the mosquito and blood stages. However, after sporozoite inoculation of the PbGRASP-KO into mice, a spike in blood stage parasitemia was observed for several days. Interestingly, morphological examination of blood stage parasites after sporozoite inoculation shows a greater number of ring stage parasites than WT. Through fluorescence and electron microscopy of infected hepatocytes, we observe that the PbGRASP-KO was able to clear micronemes effectively, despite a more pronounced accumulation of micronemes beneath the parasite plasma membrane, and PbGRASP-KO underwent normal schizogony. We conclude that GRASP may have a role outside of the liver stage, perhaps during initial blood stage development.
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Keywords
Plasmodium, GRASP, Autophagy