Preliminary Analysis Of Endoplasmic Reticulum-shaping Proteins In Plasmodium Berghei

Severe malaria infection remains a leading cause of mortality in sub-Saharan Africa.The major cause of fatality is cerebral malaria(CM).Early clinical symptoms of CM are very similar to other brain diseases,and once occurred,it often quickly leads to lethality.Pathological mechanism of CM remains elusive,and treatments for CM are very limited.Plasmodium berghei ANKA(Pb)infection in C57BL/6 mice can induce similar symptoms to that of human CM.However,experimental CM(ECM)incidence in mice is variable.Previous studies showed that the induction of ECM was dependent on the amount of inoculum.Therefore,we infected mice with none,103,or 106 parasited red blood cells(pRBCs),and compared ECM incidence.Blood-brain barrier(BBB)examination through Evans Blue stain showed that BBB disruption was present in all of the mice from low-dose infected(LI)group,even when some of them had not displayed clinical signs of CM.The percentage of BBB disruption in high-dose infected(HI)group was ~30%.The LI group has lower parasitemia and higher clinical scores in reduced activities,paraplegia,and spasm.Histopathological tests revealed important features associated with ECM,including brain parenchymal hemorrhages and cerebral edema,consistent with the neurologic symptoms of the mice from LI group.These results indicate that the low-dose inoculation can efficiently induce ECM.We also monitored blood cell types and the metabolic changes in mice from all three groups.LI group had decreased level of white blood cells(WBCs)and platelet when compared with HI and control groups,but monocyte ratio in LI group was increased,blood glucose decreased,and blood lipid increased.FACS analysis for immune-related cells showed that there was no statistical difference between the LI group and the HI group,but the proportion of dendritic cells were decreased,monocytes increased,and activated T lymphocytes increased,when compared to control group.Parasites often used protein secretion to survive in hosts.Plasmodium drastically remodels its own surface when entering the blood stream of the hosts,and at the same time restructures the surface composition of the infected red blood cells.Therefore,interference of parasitic protein secretion is a key strategy for investigating virulence and antigenic determinants.The endoplasmic reticulum(ER)plays a key role in protein secretion.Previous studies showed that ER membrane protein YOP1 help to generate ER tubules,and SEY1 mediates fusion between ER membranes.Mutations of these ER-shaping proteins cause growth and secretion defects.Sequence-based blast revealed homologous ER tubule-shaping proteins in P.berghei.Purified PbYOP1 was able to form tubules in vitro when reconstituted into proteoliposomes.PbYOP1 and PbSEY1 were able to replace their homologs in yeast cells and mammalian cells in the maintenance of ER morphology.To gain functional insights into YOP1 and SEY1-dependent processes,we constructed deletion mutants using malaria transfection.Our results establish a suitable mouse model for CM.It provides important clues for identify pre-clinical markers.By constructing mutant parasites for ER-shaping proteins,we will be able to determine the subset of parasite proteins whose secretion plays a key role in CM development.Finally,we aim to analyze the role of tubular ER network in growth,infection,and sequestration of the Plasmodium parasites.