恶性疟原虫动力素相似蛋白1的功能研究
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摘要
疟疾是目前地球上对人类危害最严重的蚊媒寄生原虫病。全世界每年有3至5亿人受到疟原虫感染，二百至三百余万人死于恶性疟疾，其中80%是五岁以下儿童。疟原虫是疟疾的病原体，寄生于人体的疟原虫共有4种，恶性疟原虫是其中最致命的一种。由于恶性疟原虫对现有药物的抗药性不断出现和蔓延，蚊媒对杀虫剂抗药性的产生等因素影响，加上迄今尚无有效的疟疾疫苗出现，致使对疟疾的控制成为世界性的大难题之一。
对疟疾的防治是多种多样的，可以针对不同的发育阶段和传播环节采取不同的措施。目前公认最有发展前景的策略包括：1)能够有效抑制疟原虫的某一特殊生命过程的药物；2)具有预防及治疗作用的疫苗。
恶性疟原虫动力素相似蛋白1(Plasmodium falciparum dynamin like protein1)是本实验筛选出的新虫源基因，前期工作证明其功能具有多样性，可能在内吞通路和分泌通路中具有重要的作用，并且是维持恶性疟原虫生存的重要基因。为了更深入地了解其功能，本文尝试使用基因敲除和特异性抑制剂等技术手段，进行进一步的研究。
首先，构建了用于全长敲除和C端敲除的双交换敲除载体，经电脉冲转染方法，获得了稳定的转染子。随后利用有限稀释法培养了单克隆。遗传实验证明没有得到预期的全长敲除的恶性疟原虫虫株，但是有发生单交换的虫体存在。C末端敲除的稳定转染子由于时间关系，没有及时单克隆化并进行分析，对C末端敲除后的效果尚待观察。
其次，本研究中尝试了P．f dynamin like protein 1蛋白在原核和真核表达系统中全长表达，试用各种诱导条件均未成功。随后改用密码子优化法，将P．fdynamin like protein 1基因密码子全部替换成大肠杆菌(E．coli)偏爱的密码子，实现了P．f dynamin like protein 1全长蛋白的表达，并证实其具有GTPase活性，且不受C末端131个氨基酸缺失的影响。
最后，通过使用dynamin特异性抑制剂dynasore，发现dynasore能够有效地抑制P．fdynamin like protein 1的GTPase活性。将dynasore与培养的恶性疟原虫共孵育，能够抑制恶性疟原虫的生长。结合前期研究结果与已经明确dynamin家族的功能，提出了P．f dynamin like protein 1参与疟原虫血红蛋白内吞的假设。通过生物化学方法显示，dynasore的使用可以明显地减少恶性疟原虫体内血红蛋白的累积以及疟色素的形成，并能极大减少示踪剂FITC标记的葡聚糖在恶性疟原虫中的累积，表明dynasore抑制了P．f dynamin like protein 1的GTPase活性，进而影响了恶性疟原虫对血红蛋白的内吞作用；电镜结果显示，dynasore处理的恶性疟原虫体内累积了大量的囊泡样结构，提示dynamin在内吞通路中有重要作用。本研究表明，P．f dynamin like protein 1参与了恶性疟原虫血红蛋白内吞过程，并发挥了重要作用。为揭示恶性疟原虫的内吞机理提供了新的线索，也为将P．f dynamin like protein 1作为抗疟药靶点提供了理论依据。
Malaria is one of the most infectious diseases to human health in the world.Thereare an estimated 500 million cases and up to 3 million deaths from malaria each year.The mortality levels are greatest in sub-Saharan Africa,where children under 5 years ofage and pregnant women account for ninety percent of all deaths due to malaria.Despitemore than a century of efforts have been taken to eradicate or control malaria,the diseaseremains a major and growing threat to the public health and economic development ofcountries in the tropical and subtropical regions of the world.The emergence and spreadof drug-resistant parasites coupled with the absence of an effective vaccine makesmalaria treatment more complicated,and thus the development of new antimalarial drugsand new vaccine targets is one of the urgent tasks in malaria research.
P.falciparum dynamin like protein 1 was a new plasmodium gene identified by ourgroup.Our previous work suggested that P.falciparum dynamin like protein 1 mayparticipate in some important trafficking process in the Plasmodiumfalciparum:theendocytosis and secretion process;It also suggested that P.fDYN 1 may be essential for thesurvival of the parasites during the erythrocytic stage based on RNAi experiment.
Based on the previous work,this study focus on the functional analysis of theP.fDYN1.The results are as follows:
1) Two targeting plasmids were constructed and tansfected into P.falciparum 3D7strain and the two stable transfected parasites were firmly established.PCRbased genetic analysis showed that the whole P.fdynamin like proteinl geneknockout was not success.The truncated knockout experiment was notcompleted but it was still worth to continue.
2) The expression of the whole protein of P.fdynlin Escherichia.coli system and Picha pastoris system was tested but failed.Finally,the whole protein of P.f dynlwas expressed in E.coli based on codon optimization by using the DNAworkssoftware.The whole protein and the truncated protein showed the samecharacters of their GTPase activity in vitro assay.It seems that the C terminal ofthe P.fDYN1 protein was not important for its GTPase activity.
3) A dynamin GTPase inhibitor named dynasore can also inhibit the GTPaseactivity of P.fDYN1 with the concentration of 80μM.
4) Treated the P.falciparum 3D7 strain culture with 80μM dynasore resulted incell growth delay.We supposed that dynasore inhibited the GTPase activity ofthe P.fDYN1,thus the endocytosis of the parasite was inhibited and the nutrientacquisition such as the hemoglobin uptake was arrested.
5) Biochemical and morphological study supported our hypothesis.Theaccumulated hemoglobin and haemozoin in the parasite was remarkablydecreased when the parasite was treated with 80μM dynasore.FITC-dextranendocytosis assay also showed that 80μM dynasore can inhibit the endocytosisof the plasmodium falciparum.The ultrastructure showed that the vesicle likestructure was increased in the parasite when treated with 80μM dynasorecompared with the negative control.
Taken together,we concluded that P.falciparum dynamin like protein 1 participated inthe endocytic trafficking pathway in the malaria parasite Plasmodium falciparum andplayed an important role.These work demonstrated that the P.fDYN 1 had the potential tobe a drug target for malaria chemotherapy.
对疟疾的防治是多种多样的，可以针对不同的发育阶段和传播环节采取不同的措施。目前公认最有发展前景的策略包括：1)能够有效抑制疟原虫的某一特殊生命过程的药物；2)具有预防及治疗作用的疫苗。
恶性疟原虫动力素相似蛋白1(Plasmodium falciparum dynamin like protein1)是本实验筛选出的新虫源基因，前期工作证明其功能具有多样性，可能在内吞通路和分泌通路中具有重要的作用，并且是维持恶性疟原虫生存的重要基因。为了更深入地了解其功能，本文尝试使用基因敲除和特异性抑制剂等技术手段，进行进一步的研究。
首先，构建了用于全长敲除和C端敲除的双交换敲除载体，经电脉冲转染方法，获得了稳定的转染子。随后利用有限稀释法培养了单克隆。遗传实验证明没有得到预期的全长敲除的恶性疟原虫虫株，但是有发生单交换的虫体存在。C末端敲除的稳定转染子由于时间关系，没有及时单克隆化并进行分析，对C末端敲除后的效果尚待观察。
其次，本研究中尝试了P．f dynamin like protein 1蛋白在原核和真核表达系统中全长表达，试用各种诱导条件均未成功。随后改用密码子优化法，将P．fdynamin like protein 1基因密码子全部替换成大肠杆菌(E．coli)偏爱的密码子，实现了P．f dynamin like protein 1全长蛋白的表达，并证实其具有GTPase活性，且不受C末端131个氨基酸缺失的影响。
最后，通过使用dynamin特异性抑制剂dynasore，发现dynasore能够有效地抑制P．fdynamin like protein 1的GTPase活性。将dynasore与培养的恶性疟原虫共孵育，能够抑制恶性疟原虫的生长。结合前期研究结果与已经明确dynamin家族的功能，提出了P．f dynamin like protein 1参与疟原虫血红蛋白内吞的假设。通过生物化学方法显示，dynasore的使用可以明显地减少恶性疟原虫体内血红蛋白的累积以及疟色素的形成，并能极大减少示踪剂FITC标记的葡聚糖在恶性疟原虫中的累积，表明dynasore抑制了P．f dynamin like protein 1的GTPase活性，进而影响了恶性疟原虫对血红蛋白的内吞作用；电镜结果显示，dynasore处理的恶性疟原虫体内累积了大量的囊泡样结构，提示dynamin在内吞通路中有重要作用。本研究表明，P．f dynamin like protein 1参与了恶性疟原虫血红蛋白内吞过程，并发挥了重要作用。为揭示恶性疟原虫的内吞机理提供了新的线索，也为将P．f dynamin like protein 1作为抗疟药靶点提供了理论依据。
Malaria is one of the most infectious diseases to human health in the world.Thereare an estimated 500 million cases and up to 3 million deaths from malaria each year.The mortality levels are greatest in sub-Saharan Africa,where children under 5 years ofage and pregnant women account for ninety percent of all deaths due to malaria.Despitemore than a century of efforts have been taken to eradicate or control malaria,the diseaseremains a major and growing threat to the public health and economic development ofcountries in the tropical and subtropical regions of the world.The emergence and spreadof drug-resistant parasites coupled with the absence of an effective vaccine makesmalaria treatment more complicated,and thus the development of new antimalarial drugsand new vaccine targets is one of the urgent tasks in malaria research.
P.falciparum dynamin like protein 1 was a new plasmodium gene identified by ourgroup.Our previous work suggested that P.falciparum dynamin like protein 1 mayparticipate in some important trafficking process in the Plasmodiumfalciparum:theendocytosis and secretion process;It also suggested that P.fDYN 1 may be essential for thesurvival of the parasites during the erythrocytic stage based on RNAi experiment.
Based on the previous work,this study focus on the functional analysis of theP.fDYN1.The results are as follows:
1) Two targeting plasmids were constructed and tansfected into P.falciparum 3D7strain and the two stable transfected parasites were firmly established.PCRbased genetic analysis showed that the whole P.fdynamin like proteinl geneknockout was not success.The truncated knockout experiment was notcompleted but it was still worth to continue.
2) The expression of the whole protein of P.fdynlin Escherichia.coli system and Picha pastoris system was tested but failed.Finally,the whole protein of P.f dynlwas expressed in E.coli based on codon optimization by using the DNAworkssoftware.The whole protein and the truncated protein showed the samecharacters of their GTPase activity in vitro assay.It seems that the C terminal ofthe P.fDYN1 protein was not important for its GTPase activity.
3) A dynamin GTPase inhibitor named dynasore can also inhibit the GTPaseactivity of P.fDYN1 with the concentration of 80μM.
4) Treated the P.falciparum 3D7 strain culture with 80μM dynasore resulted incell growth delay.We supposed that dynasore inhibited the GTPase activity ofthe P.fDYN1,thus the endocytosis of the parasite was inhibited and the nutrientacquisition such as the hemoglobin uptake was arrested.
5) Biochemical and morphological study supported our hypothesis.Theaccumulated hemoglobin and haemozoin in the parasite was remarkablydecreased when the parasite was treated with 80μM dynasore.FITC-dextranendocytosis assay also showed that 80μM dynasore can inhibit the endocytosisof the plasmodium falciparum.The ultrastructure showed that the vesicle likestructure was increased in the parasite when treated with 80μM dynasorecompared with the negative control.
Taken together,we concluded that P.falciparum dynamin like protein 1 participated inthe endocytic trafficking pathway in the malaria parasite Plasmodium falciparum andplayed an important role.These work demonstrated that the P.fDYN 1 had the potential tobe a drug target for malaria chemotherapy.
引文
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