中国科学院昆明动物研究所机构知识库

贾第虫是可寄生在包括人在内的几乎所有脊椎动物类群的十二指肠的 单细胞鞭毛虫，在全世界范围内分布。其所引起的疾病--贾第虫病（giardiasis） 被世界卫生组织列为“被忽视的疾病”，可带来严重的经济损失和公众健康问 题。在进化生物学领域，该生物也曾一度成为研究热点。因为，一些学者认 为它是极端原始的真核生物，可以用来研究真核细胞的起源与进化这一重要 的进化生物学问题。但是，另外一些学者认为贾第虫并不原始，它只是一个 普通的高度退化的成功的寄生原虫。那么贾第虫到底处在什么样的进化地位 呢？根据本实验室的长期研究积累，我们初步形成了这样的观点：贾第虫应 该是分化很早的原始真核生物，它的行使细胞基本功能且与寄生生活不相关 的各类特征由于受其原本的功能限制并未发生太大的变化，保留了其原始 性，这些特征能反映贾第虫真实的原始进化地位；而它的与寄生相关的各类 特征应该由于该生物由原来的自由生活转变成寄生生活而发生了次生的寄 生适应性改变，这些特征只能反映它的寄生适应性而非其真实的进化地位。 以前关于贾第虫进化地位的争议很大一部分原因应该正是没有区分开这两 类特征。本论文旨在基于全基因组的分析研究来检验这一关于贾第虫的全新 观点。 基于贾第虫的全基因组数据，本文通过使用同源搜索，结构域分析等生 物信息学手段，鉴定了贾第虫的各类重要结构、功能成分，重建了一些重要 代谢途径，进而通过分子系统发生分析、系统分布调查，并结合部分实验对 贾第虫的原始性与次生寄生适应性进行了全面系统的分析研究。具体的研究 结果与结论如下： 1）鉴于甘油磷脂合成途径在所有真核生物中的保守性，又由于贾第虫 生活在食物消化形成的脂类丰富的十二指肠处，它的甘油磷脂合成途径又有 可能发生相应的适应性改变。本论文首先选取了甘油磷脂合成途径这一具有 典型代表性的代谢途径进行了系统的分析研究。基于基因组数据鉴定了贾第 虫的甘油磷脂合成基因，并构建了其多条甘油磷脂合成途径。经与典型真核 基于全基因组的贾第虫原始性与次生寄生适应性的研究 IV 生物的相比较分析，发现除了 lands cycle 之外，贾第虫的其它甘油磷脂途径 都不完整，尤其是缺少真核生物特异性的酶所催化的步骤。系统发生分析表 明：（I）其中 9 个酶与其他真核生物同源物聚在一起，但都从整个真核生物 的基部分支出来，这提示它们是从真核细胞的共同祖先垂直遗传下来，但保 留了原始性特征。（II）三个酶是从厌氧细菌水平转移过来的，明显为贾第虫 次生性获得的；（Ⅲ）另外一些酶并不处在所有真核生物的基部，而是落入 真核生物类群的内部，且其中一个酶 GiLCAT4 表现出很长的枝长，提示这 些酶在贾第虫中发生了显著的次生性变化。系统分布调查显示：贾第虫中一 些酶的缺失极有可能是因寄生生活在脂类代谢中间产物丰富的肠道环境中， 不需从头合成或可直接获得所需磷脂而导致的丢失；而另外一些缺失的酶极 有可能是因贾第虫的原始性还未进化出来。细胞定位实验表明 GiPSD 定位于 核外围/内质网而不是它的直系同源物 Psd1 所定位的线粒体，这表明伴随着 贾第虫线粒体的退化该酶从原来的线粒体重新定位到核外围/内质网。这些结 果表明：贾第虫的磷脂合成途径原本是非常原始的，但在此原始性的基础上 由于适应寄生生活而发生了一系列的次生适应性改变（如水平基因转移、酶 的丢失及重新细胞定位等）。 2) 为了全面研究贾第虫的原始性与次生适应性问题，本文还对核小体核 心组蛋白，DNA 复制机器，转录机器，DNA 断裂修复系统，腹侧吸盘，糖 酵解途径，糖异生途径，精氨酸脱亚胺酶途径，三羧酸循环等许多方面进行 了分析研究。结果显示这些细胞机器、结构和功能途径它们大部分均具有原 始性和次生适应性两重特性：既具有原始性的基因，又具有次生性来源或发 生了次生性变化的基因。但在行使细胞基本生命活动的核小体组成成分，转 录起始复合物，蛋白翻译机器中存在的基因所构建的大部分分子系统树中， 贾第虫均位于真核生物基部，而在与其寄生生活密切相关的组分如腹侧吸盘 组成成分的基因所构建的大部分系统树中贾第虫均不位于真核生物的基部。 所调查的贾第虫的代谢途径或细胞组分都显示出较典型真核生物来说要简 单得多，系统分布调查发现贾第虫中一部分基因的缺失是由寄生退化造成的 （如三羧酸循环中缺失的基因），一部分基因的缺失有可能是因贾第虫的原 始性还未进化出来之故。 摘 要 V 本文还对目前已测序的五株贾第虫所共有的鉴定出的 92 个水平转移而 来的基因进行了专门的功能富集分析研究。结果表明这些次生性来源的基因 主要富集于贾第虫寄生生活相关的方面，如参与贾第虫抗药性等等。 综上所述，本文基于基因组的全面系统的分析研究表明，贾第虫确实是 一种极为原始的真核生物，在其原始性的基础上因其后来适应次生的寄生生 活而发生了一系列的寄生适应性进化。对贾第虫的这种两重性的的认识和甄 别具有双重价值：其保留了原始性特征的部分可以用来研究真核细胞的起源 与进化问题，而与寄生生活相关的发生了次生适应性变化的部分可以用来研 究寄生适应性进化的规律，以便为该寄生虫的更有效的防治提供基础。因此， 本研究对于结束贾第虫进化地位的争议、重启其进化生物学和寄生适应性方 面的研究具有重要意义。

Giardia spp. is a flagellated protozoan parasite that inhabits the upper small intestine of vertebrates including human beings and other livestocks, it is the aetiological agent of giardiasis. Giardiasis has been called as “negelected disease” by WHO, due to its being leading but neglected cause of infectious gastroenteritis worldwide. This disease has brought about serious economic and public health burden. This organism had ever been a hotsot organism in the field evolutionary biology. Since some scholars consider it as the most primitive eukaryote (and thus extremely important to evolutionary biology and can be used to study the origin of euakryotes from prokaryotes), others treat it just as a highly evolved parasite. Then what’s the real evolutionary position of Giardia? Based on the long term study for Giardia in our lab, we proposed this new viewpoint: it is a very ancient eukaryote, the parts which perform essential cellular activities but has nothing to do with parasitic life changed little over time due to the constraint of conserved function; On the contrary, the parts that are tightly related to its parasitic life changed a lot for successful adaptation and can be used for adaptative evolution. Only the unchanged former parts can reflect its true evolutionary position and be used to infer this organism’s evolutionary position while the latter can not. We believed that the controversy over Giardia’s evolutionary position comes from the neglect for the mosaic nature of this organism—the study object in Giardia are related to its parasitic life or not. Our current study is aimed to test this viewpoint.Based on the genome data of Giardia, we identified the cellular components and reconstructed some important metabolic pathways through bioinformatic methods such as homology search, domain analysis and cellular prediction, then phylogenetic analysis for the existing Giardia genes, phylogenetic distribution analysis for some genes that are absent in Giardiawere and experimental cellular localization were carried out. The current results support our initial standpoint. The detailed results and conclusions are as follows:Glycerophospholipid (GPL) synthesis is a conserved essential cellular process, and thus may retain some original features reflecting its evolutionary position, and this process should also have undergone parasitic adaptation to suit Giardia’s dietary lipid-rich environment. Thus, GPL synthesis pathways may be a perfect object to examine the controversy over Giardia. Based on Giardia genome database and the current explicit GPL biosynthesis pathways in model organisms, here, much more reliable and exhaustive GPL biosynthesis enzymes than before were identified and the corresponding pathways were reconstructed in Giardia. Our systematic analysis indicated that compared with common eukaryotes, Giardia’s pathways are very simple/incomplete indeed except its Lands cycle, especially lack those steps catalyzed by eukaryote-specific enzymes; The results of phylogenetic analysis are interesting: Ⅰ) nine Giardia enzymes clustered with their eukaryotic homologs and branched off at the base of the eukaryotic homologs, suggesting they each were inherited from the last eukaryotic common ancestor and maintained their primitive nature. Their simultaneous branch off early strongly indicates the early branching of Giardia; Ⅱ) other three enzymes were proved to be laterally transferred from anaerobic bacteria, these enzymes were secondarily obtained by Giardia; Ⅲ) the other Giardia enzymes fell inside the eukaryotic homologs in the corresponding phylogenetic trees, especially, one gene--GiLCAT4 occupied an extraordinary long branch, indicating their undergone secondary significant changes over time. The phylogenetic distribution of Giardia’s absent enzymes results are also interesting: the absence of some enzymes are probably result of parasitic reduction for example parasitic loss caused by the abundance of digestive lipid intermediates in the intestine, while the absence of other enzymes are likely their‘having-not-yet-evolved’ due to Giardia’s early branching. The cellular localization experiments showed the location of GiPsd in perinuclear/ER region instead of the degenerated mitochondrion – mitosome, its different localization compared to its other orthologs’ mitochondrion localization indicates its possible secondarily subcellular re-localization from mitochondrion to perinuclear/ER region, in company with the parasitic reduction of Giardia’s mitochondrion. Overall, we revealed that Giardia GPL biosynthesis pathways turn out to be evolutionarily primitive ones, but with many secondary parasitic adaptation ‘patches’ including gene loss, rapid evolution, product relocation, and horizontal gene transfer. The results also indicate that modern Giardia is of both ‘primary primitivity’ and ‘secondary parasitic adaptability’.To explore the ‘primary primitivity’ and ‘secondary parasitic adaptability’ nature of Giardia thoroughly, we studied more representative metabolic pathways and cellular machinery. Our study objects include nucleosome, DNA replication machinery, mRNA gene transcription machinery, mRNA gene processing machinery, DNA repairing system, ventral attachment disk, the glycolysis pathway, the gluconeogenesis pathway, the arginine deiminase pathway, the tricarboxylic acid cycle and so on. The results indicate the metabolic pathways or cellular machinery each themselves are of both‘primary primitivity’ and ‘secondary parasitic adaptability’: possessing both primitive enzymes and secondary changed enzymes. But a general pattern can be found: while Giardia all branched off early on most phylogenetic trees reconstructed from genes of nucleosome, DNA replication machinery and mRNA gene transcription machinery which perform essential cellular functions, it branched later on most phylogenetic trees reconstructed from genes of parasitic life related parts such as ventral attachment disk. The investigated parts in Giardia are simple compared to their counterparts in other eukaryotes. The phylogentic distribution analysis indicate that some absence are secondary loss while some others are likely the original absence due to Giardia’s early branching.we also obtained 92 laterally transferred genes form the shared genes of the five sequenced Giardia organisms. Functional enrichment analysis shows that most of them are related to Giardia’s parasitic life for example drug resistance genes. Overall, the current work supports ourviewpoint: Giardia is really an early-diverging eukaryote with many remnant primitive features, and on the basis of this primitivity, it has acquired many secondary parasitic adaptive features. Revelation of its mosaic nature make Giardia a dually valuable model: the primitive features can be used for studying the origin and evolution of eukaryotes and the parasitic ones can be utilized for studying the law of adaptative evolution to provide theoretical basis of more efficient control of this organism.