A genome-wide over-expression screen identifies genes involved in phagocytosis in the human protozoan parasite, Entamoeba histolytica.
A genome-wide over-expression screen identifies genes involved in phagocytosis in the human protozoan parasite, Entamoeba histolytica.
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DOI：
10.1371/journal.pone.0043025
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Temesvari LA
中科院分区：
文献类型：
--
作者：
King AV;Welter BH;Koushik AB;Gordon LN;Temesvari LA
功能基因组学和正向遗传学试图将功能分配给基因组中所有已知的基因。溶组织内阿米巴是一种原生动物寄生虫，其正向遗传学方法尚未广泛应用。它是阿米巴痢疾和肝脓肿的病原体，感染在无法阻止其粪口传播的发展中国家普遍存在。它是造成全球相当大的发病率和死亡率的原因。鉴于溶组织芽孢杆菌基因组已被测序，应该有可能应用基因组方法来发现基因功能。我们使用全基因组过表达筛选来发现调节溶组织芽胞杆菌重要毒力功能的基因，即吞噬作用。我们建立了一个episomal E. histolytica cDNA过表达文库，将收集的质粒转染到滋养体中，并应用高通量筛选在过表达细胞群体中鉴定吞噬突变体。筛选是基于人体红细胞对代谢毒素结核菌素的吞噬作用。从暴露于含结核菌素的红细胞（吞噬突变体）中存活的滋养体中分离出表达质粒，并对其cdna进行测序。我们分离了编码profilin的基因，profilin是一种特性良好的细胞骨架调节蛋白，已知在吞噬作用中起作用。这证明了我们方法的有效性。此外，我们将吞噬作用分配给一些以前不知道以这种方式起作用的基因。据我们所知，这是第一个应用于这种病原体的全基因组正向遗传学筛查。该研究证明了前向遗传学在揭示溶组织芽孢杆菌毒力调节基因方面的力量。此外，该研究验证了溶组织芽孢杆菌cDNA过表达文库作为功能基因组学的一个有价值的工具。
Functional genomics and forward genetics seek to assign function to all known genes in a genome. Entamoeba histolytica is a protozoan parasite for which forward genetics approaches have not been extensively applied. It is the causative agent of amoebic dysentery and liver abscess, and infection is prevalent in developing countries that cannot prevent its fecal-oral spread. It is responsible for considerable global morbidity and mortality. Given that the E. histolytica genome has been sequenced, it should be possible to apply genomic approaches to discover gene function. We used a genome-wide over-expression screen to uncover genes regulating an important virulence function of E. histolytica, namely phagocytosis. We developed an episomal E. histolytica cDNA over-expression library, transfected the collection of plasmids into trophozoites, and applied a high-throughput screen to identify phagocytosis mutants in the population of over-expressing cells. The screen was based on the phagocytic uptake of human red blood cells loaded with the metabolic toxin, tubercidin. Expression plasmids were isolated from trophozoites that survived exposure to tubercidin-charged erythrocytes (phagocytosis mutants), and the cDNAs were sequenced. We isolated the gene encoding profilin, a well-characterized cytoskeleton-regulating protein with a known role in phagocytosis. This supports the validity of our approach. Furthermore, we assigned a phagocytic role to several genes not previously known to function in this manner. To our knowledge, this is the first genome-wide forward genetics screen to be applied to this pathogen. The study demonstrates the power of forward genetics in revealing genes regulating virulence in E. histolytica. In addition, the study validates an E. histolytica cDNA over-expression library as a valuable tool for functional genomics.
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