| Objective: An estimated 20 million cases of malaria occur worldwide and cause 400,000 deaths globally every year with approximately 70% of fatal malaria cases occurring in children less than five years old.The most severe complication,cerebral malaria,presents a significant problem for patients during Plasmodium infection and is attributed to most of the fatal malaria cases.Many elements of Plasmodium,including the malaria pigment,GPIs,GPI anchors,and nucleic acids can trigger a proinflammatory response in the host.Glycosylphosphatidylinositols(GPIs)are complex glycolipids.One of the most apparent functions of GPIs is to anchor surface proteins to the cell membrane.GPIs expressed throughout the life cycle of the malaria parasite have been studied functionally.The effects of GPIs and GPI anchors in many kinds of parasites have been attributed to their ability to induce TNF-? and other proinflammatory cytokines,which contribute to the disease pathology.During the development in mosquito,GPIs of plasmodium can induce NOS expression in immune and endothelial cells,NOS expression could result in apoptosis of follicular and fecundity reduction.These effects are mediated in part by mimicry of insulin signaling by GPIs.Before GPIs and GPI anchors are transported to the cell surface,the GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum(ER)through a transamidation reaction.The GPI transamidase is a key enzyme of this posttranslational modification.The GPI transamidase consists of five different subunits and is conserved among different species.The subunits of Saccharomyces cerevisiae(S.cerevisiae)GPI-T consist of Gaa1 p,Gpi8p,Gpi17 p Gpi16p,and Gab1 p,which form two sub-complexes(one containing Gpi8 p,Gpi16p,and Gaa1 p,and the other Gab1 p and Gpi17p).The three-component complex is the core and is conserved in many species.Gpi16 p may mediate protein presentation to Gpi8 p.Gpi16p may also regulate Gaa1 p and Gpi8 p expressionMethods : 1.Bioinformatics.The pbph genomic sequences used in this study were retrieved from Plasmo DB,(http://www.plasmodb.org).Putative signal peptide and functional domains were predicated using the SMART online server(http://smart.emblheidelberg.de/).Multiple sequence alignments were performed using the Clustal W multiple sequence alignment program.2.Generation of transgenic parasites.KO and ha-tagged parasite lines were generation using the double crossover homologous recombination technology.Describedadd ha tag at the C-terminus of Pb GPI16 gene or replace all the protein-coding sequence of the Pb GPI16 gene with an hdhfr expression cassette conferring resistance to pyrimethamine.3.Pb GPI16 expression stages detection.pbgpi16-ha parasite line was used for Pb GPI16 expression stages detection,slides were incubated with monoclonal anti-HA antibody to identify Pb GPI16-HA fusion proteins,the expression stages detection including ring,trophozoite,schizont,male and female gametocyte,male and female gamete,ookinete and sporozoite.4.Phenotypic analysis of ?pbgpi16.Five mice were each injected with ?pbgpi16-infected red blood cells(i RBCs),while the other five were injected with WT Pb A-i RBCs.Parasitemia was monitored daily by Giemsa-stained blood smears.On day 3 after infection,gametocytemia and the gametocyte sex ratio were determined,exflagellation of male gametocytes was quantified under a phase contrast microscope.ookinete formation was determined using mouse anti-Pbs21 antibody,mosquito feeding experiment was performed,Ten days after feeding,up to 25 mosquitoes were dissected in each group.Oocysts were counted to determine the prevalence(number of infected mosquitoes)and intensity of infection(number of oocysts per positive midgut).Sporozoite counts were carried out at day 21 of infection.To characterize the function of Pb GPI16 in all life cycles of Pb A,10000 sporozoites of ?pbgpi16 or WT Pb A were injected intravenously,Mice were screened for blood stage infections.5.Histopathology and immunohistochemistry.Infection was initiated by intraperitoneal injection into each mouse.Parasitemia was monitored by light microscopy examination of Giemsa-stained thin smears from tail blood,and mortality was monitored daily.The BBB blood-brain barrier(BBB)integrity was evaluated when neurological signs of ECM.When the WT Pb A-infected mice showed neurological symptoms.The brains were removed,stained with hematoxylin and eosin(HE)to examine microvascular obstruction and endothelial cell damage.Immunohistochemical staining was performed to detect VCAM-1,ICAM-1,and CD36 expression.6.Measurement of cytokines.Total RNA was extracted from the brains and spleens,The expression levels of VCAM-1,ICAM-1,CD36,CXCL9,CXCL10,and CXCR3 in the brain and TNF-?,IFN-?,TGF-?,IL-1?,IL-6,IL-10,and IL-12 in the spleen were determined by q RT-PCR.The levels of TNF-?,IFN-?,IL-1?,IL-6,IL-12,TGF-?,and IL-10 were measured in serum using MSD.the cytokines of splenocyte cytokines were measured using commercial enzyme-linked immunosorbent assay(ELISA).7.Flow Cytometry.The number of CD8~+ cells in the brain and Th1 cells,dendritic cells(DCs)and regulatory T cells(Tregs)was measured using flow cytometry analysis.8.NF-?B detection in brain and spleen.Mice were euthanized on day 6 and splenocytes and brain mononuclear cells were isolated.The nuclear proteins were isolated.Nuclear NF-?B was detected by western blot.9.Fecundity measurement.Groups of 50–100 mosquitoes 4–6 days old,were offered a bloodmeal with ?pbgpi16 infected mice,WT Pb A infected mice and uninfected mice.After 3 days,fecundity was measured as the number of eggs laid over the following 2 days,as well as the number of mature eggs counted after dissection 10.Quantitative PCR for apoptosis related genes.Five engorged mosquitoes were removed 24 h post-bloodfeeding,Total RNA was extracted from each group ofmosquitoes.The expression levels of As NOS and As INR of the mosquitoes were determined by q RT-PCR.11.Statistical analyses.Data are presented as the mean ± standard error of the mean(SEM).Survival analysis was performed using the Kaplan-Meier log-rank test.Statistical significance was determined using the Student's t-test or one-way ANOVA(SPSS 17.0).A P-value < 0.05 was considered significant.Results: 1.Pb GPI16 is highly conserved among Plasmodium species and expressed in asexual stages and asexual stages.Pb GPI16 contains a signal peptide at N-terminal and a GPI transamidase subunit16(Gpi16)domain.Multiple sequence alignments using a Clustl W showed that this protein is highly conserved among Plasmodium species.The localization of Pb GPI16 in the asexual and sexual stages of P.berghei was investigated by IFA.Pb GPI16 almost expressed in all asexual stages and sexual including ring,trophozoite,schizont,male and female gametocyte,male and female gamete,ookinete and sporozoite.2.Pb GPI16 deficiency reduce the ability of erythrocyte invasion and influence the development in mosquito.Deletion of the pbgpi16 gene in P.berghei showed lower parasitemia,gametocytemia and gametocyte sex ratio had no significant difference between ?pbgpi16 parasites and wild-type parasites as well,the ?pbgpi16 parasites displayed a normal exflagellation,but ookinete conversion in vitro was severely affected,the result showed that ?pbgpi16 parasites formed ookinetes at number that was significantly lower than wild-type parasites.In mosquito feeding assays,A striking reduction of oocysts number was observed in ?pbgpi16 parasites compared with wild-type parasites.At day 21 after mosquito feeding,in mosquitoes infected with ?pbgpi16 parasites,sporozoites numbers in salivary glands were significantly reduced,both ?pbgpi16 parasites group and wild-type parasites group with sporozoites in salivary glands were infectious to mice.3.ECM pathology is reduced in ?pbgpi16-infected mice.The majority of WT Pb A-infected C57BL/6 mice exhibited neurological symptoms around day 6 and death occurred between days 5 and 12.In contrast,less than 30% of mice died of ECM in the ?pbgpi16-infected group,and death occurred beginning on day 12.The BBB experiment was performed to evaluate BBB leakage,BBB leakage was significantly lower in ?pbgpi16-infected mice compared to WT Pb A-infected mice.The number of vessels containing leukocytes in the ?pbgpi16-infected mice was significantly lower than that in the WT Pb A-infected mice based on the HE staining results.The corresponding VCAM-1,ICAM-1,and CD36 staining on the endothelium of the microvessels was less intense in ?pbgpi16-infected mice than the WT Pb A-infected mice.The VCAM-1,ICAM-1,and CD36 m RNA expression levels and the levels of the chemokines CXCL9,CXCL10,and CXCR3 in brains,which are associated with ECM,were also relatively decreased.4.Proinflammatory response is downregulated in ?pbgpi16-infected mice.TNF-?,IFN-?,IL-1?,IL-6,and IL-12 expression levels were evaluated.m RNA expression levels of these cytokines in spleens were significantly lower in ?pbgpi16-infected mice compare with WT Pb A-infected mice as well as the content in and splenocyte culture supernatants.The absolute number of Th1 cells in the spleen and CD8+ T cells in the brain were significantly reduced compared to WT Pb A-infected mice.the expression of TLR4 and MHCII on DCs was drastically reduced in ?pbgpi16-infected mice compared to WT Pb A-infected mice.the number of Tregs were increased in the ?pbgpi16-infected group as well as anti-inflammatory cytokines TGF-? and IL-10.5.?pbgpi16 disables NF-?B activation in mice.Western blotting results showed that NF-?B(p65)in the nucleus was significantly downregulated in the brains and spleens of ?pbgpi16-infected mice six days post-infection indicated that NF-?B(p65)translocated from the cytosol to the nucleus and activated downstream gene transcription in the WT Pb A-infected,but not ?pbgpi16-infected,mice.6.Pb GPI16 deletion inhibit ovarian apoptosis of mosquito.The fecundity of mosquito in ?pbgpi16 feeding group was higher than those in WT Pb A feeding group.During plasmodium infection,the GPIs of the parasite are insulin-mimetic and that insulin signaling can induce NOS expression,As NOS induction through pathways involving As NOS and As INR.In mosquito of ?pbgpi16 feeding group,the expression levels of As NOS was lower than those in WT Pb A feeding group.Conclution: 1: Pb GPI16 is highly conserved among Plasmodium species and expressed in asexual stages and asexual stages.2: Pb GPI16 deficiency reduce the ability of erythrocyte invasion and influence the development in mosquito.3: Pb GPI16 of P.berghei is essential for experimental cerebral malaria in C57BL/6 mice 4: Pb GPI16 deficiency P.berghei could not trigger the ovarian apoptosis of mosquito... |
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