Tools for regulated gene expression in Entamoeba invadens

 描述(申请人提供)：溶组织内阿米巴是一种原生动物寄生虫，是一种重要的人类病原体。由溶组织埃希氏菌引起的疾病包括痢疾和肝脓肿，这种生物是全球范围内主要的寄生性死亡原因。内阿米巴的生命周期包括滋养体和包囊之间的阶段相互转换。重要的是，转变为包囊使病原体传播到新的宿主，而向滋养体的发展允许有机体在宿主中引起侵袭性疾病。因此，阶段相互转换对于疾病的传播和发病是必不可少的。然而，尽管阶段转换是阿米巴生物学的核心，但人们对阶段转换知之甚少。许多因素导致了数据的缺乏，其中包括不能再现组织裂殖吸虫的发育周期。取而代之的是，与溶组织内阿米巴具有相同致病机制的爬行动物阿米巴入侵阿米巴被用作研究内阿米巴发育的模式系统。在侵袭性肠杆菌中，包囊化和激发化都可以在体外高效地重现。然而，直到最近，糟糕的基因组注释和缺乏基因操作的方法阻碍了对这一系统的充分利用。最近对入侵埃希氏菌基因组的重新测序，阶段转换转录组的出版，以及构成基因表达方法的发展，开辟了新的发现途径。我们的目标是在最近这些成功的基础上，通过建立新的工具来研究入侵E.inadens，包括(I)调节基因表达，(Ii)基因沉默，和(Iii)负选择和单细胞标记的发展。这些方法将使研究内阿米巴阶段转化的新实验策略成为可能，并极大地帮助整个内阿米巴群落。解剖发育级联对于理解这种重要的人类病原体的生物学至关重要。

 DESCRIPTION (provided by applicant): Entamoeba histolytica, a protozoan parasite, is an important human pathogen. Diseases caused by E. histolytica include dysentery and liver abscesses, and this organism is a leading parasitic cause of death on a global scale. The life cycle of Entamoeba involves stage inter-conversion between trophozoites and cysts. Importantly, the conversion to cysts allows the pathogen to disseminate to new hosts while development to trophozoites allows the organism to cause invasive disease in the host. Thus, stage interconversion is essential for disease transmission and pathogenesis. However, despite being central to amebic biology, stage conversion is poorly understood. Many factors have contributed to the paucity of data including the inability to reproduce the developmental cycle in E. histolytica. Instead, the reptilian ameba E. invadens, which has the same disease pathogenesis as E. histolytica, has been used as a model system to study Entamoeba development. In E. invadens, both encystation and excystation can be recapitulated with high efficiency in vitro. However, until recently a poorly annotated genome and lack of methods for genetic manipulation have prevented full exploitation of this system. The recent re-sequencing of the E. invadens genome, publication of the transcriptome of stage conversion, and development of methods for constitutive gene expression open up new avenues for discovery. We aim to build upon these recent successes by establishing new tools for the study of E. invadens including (i) regulated gene expression, (ii) gene silencing, and (iii) development of negative selection and single cell markers. These methods will make possible new experimental strategies for studying Entamoeba stage conversion, and greatly aid the entire Entamoeba community. Dissecting the developmental cascade is crucial to understanding the biology of this important human pathogen.