| 其他摘要 | 寄生原生动物(如疟原虫、刚地弓形虫、痢疾阿米巴、阴道毛滴虫、蓝氏贾第虫和隐孢子虫等)是引起常见、甚至恶性的几种寄生虫病的病原体。它们之所以能十分成功地寄生于人类等宿主，重要的原因就在于它们较之自由生活的近亲种类发生了显著的适应各自寄生生活的适应性进化，其中能量代谢途径就是它们发生这种进化的重要方面。本文旨在揭示这些寄生原虫在能量代谢方面所发生的基因/分子水平上的适应性进化现象及其发生的机制和规律，利用比较基因组学的手段对1) 亲缘关系较远但寄生在相似的低氧腔道环境的几种常见寄生原虫，2）同属顶复类但寄生环境不同的几种原虫分别进行了能量代谢途径系统的比较分析研究，得到了以下结果与结论： 1）蓝氏贾滴虫、痢疾阿米巴、阴道毛滴虫和微小隐孢子虫是常见的几种腔道寄生原虫。有研究表明因为寄生环境氧气的缺少，它们的整个线粒体都发生了“退化”或“特化”，不再具有三羧酸循环（糖的有氧分解）和氧化呼吸功能。在此情况下，氧气（哪怕是腔道内的微氧）对这些原虫都是有害的，会造成氧化损伤，它们必须要进化出相应的机制来应对这一压力。基于这些背景和推论，我们通过对KEGG数据库和这些原虫的全基因组数据进行调查，并结合表达数据和文献数据等，对它们参与能量代谢的基因/酶进行了鉴定，重建了它们各自的能量代谢途径，在此基础上对这四种寄生原虫的能量代谢途径进行了系统比较分析。结果发现：这些原虫的三羧酸循环、脂的氧化分解和氧化呼吸链基本都发生了丢失，却都存在丙酮酸（Pyruvate）代谢途径，表现出惊人的相似。对酶的调查进一步发现，这些原虫中大量关键调控酶如PEPCK,PFK和PFO等都催化一样的反应，行使相似的功能，但这些酶的来源却不同。分子系统分析发现它们丙酮酸代谢途径中的如MAL,PPDK,ADHE和ACS等基因都是通过不同的水平基因转移而来的。最重要的是，我们发现这些原虫均是通过丙酮酸代谢途径与氧的“偶联”，产生了一种随氧浓度不同而将丙酮酸代谢成不同终产物的“弹性”代谢机制以适应环境氧气的动态变化。总之，通过我们系统的研究发现，这些腔道寄生原虫在应对微氧环境胁迫时采用了相似的适应策略，而且这种策略是通过不同的原虫在胞器、代谢途径、酶、基因等多个层面上发生不同的适应性进化而实现的，是一种趋同的适应性进化的结果。2）顶复类（Apicomplexans）是一类严格寄生的原虫，隶属于囊泡虫类。顶复类虽然都是寄生种类，但它们的寄生环境却不尽相同。前人的研究表明它们的能量代谢途径也存在明显的多样性。通过对不同寄生环境的顶复类物种之间，以及与它们的自由生活的近亲――纤毛虫或者甲藻之间，进行能量代谢途径的系统比较分析研究，无疑有助于阐明顶复类能量代谢途径在不同的寄生种类间发生了怎样的不同的寄生适应性进化，并还有助于弄清顶复门的寄生性是如何从自由生活进化而来。囊泡虫类群之间和顶复类内部较为清楚的系统发育关系和它们的不少种类已具有基因组数据等为这些问题的探讨提供了前所未有的机遇。我们选取不同寄生部位的四种顶复类寄生原虫：刚地弓形虫、恶性疟原虫、鼠隐孢子虫和微小隐孢子虫为研究对象，并以它们的两种自由生活的近亲：第四双小核草履虫和嗜热四膜虫为对照，通过对KEGG数据库和这些原虫的全基因组数据进行调查，并结合文献数据，系统鉴定并重建了它们各自的能量代谢途径（其中，对于两种隐孢子虫的能量代谢途径的重建，还参考了弓形虫和疟原虫两种寄生原虫的实验验证的数据）。在此基础上对这六种原虫的能量代谢途径进行了系统比较分析。进而，我们对这六个物种每个代谢相关基因进行了分子系统分析。我们的研究结果表明顶复门寄生虫很多酶在自由生活的近亲中基本都存在，在顶复门从自由生活的物种中分化出来后，寄生原虫各自发生了酶的不同的丢失或者改造。例如，隐孢子虫的两个物种都丢失了氧化呼吸链，而且其中的微小隐孢子虫进一步丢失了三羧酸循环，而且它们均发展出了基于丙酮酸的代谢途径；而弓形虫和疟原虫丢失了Malic enzyme, PNO, AOX等酶，疟原虫还进一步丢失了糖异生途径。这就导致了顶复类能量代谢途径多样化。结合它们的寄生环境分析，我们发现这些原虫所处环境的氧气浓度存在差异，比如: 弓形虫生活在氧含量相对较高的哺乳动物细胞内，而隐孢子虫寄生在微氧的小肠内。这就提示我们：氧作为一种关键因子，因它在上述四种寄生原虫的寄生环境中的浓度不同而导致了这些原虫能量代谢的适应性辐射。; Parasitic protozoa (such as the Plasmodium falciparum, Toxoplasma gondii, Entamoeba histolytica, Trichomonas vaginalis, Giardia lamblia and Cryptosporidium parvum etc.) are common malignancy pathogens of parasitic diseases. The reason that these parasites successfully live in their hosts is they can adapt to their parasitic life through adaptive evolution. The energy metabolic pathway is an important aspect of this adaptive evolution. In this paper, we aim to revealing the adaptive evolution and its mechanism and regularity of energy metabolism in these parasites from gene/molecular level, expect to explore an effective prevent and control method to these parasitic diseases. We use comparative genomics method to analyses their energy metabolic pathways, got the following results and conclusions:1) G. lamblia, E. histolytica, T. vaginalis and C. parvum are several lumen parasitic protozoa, of energy metabolic pathways. They all live in microaerobic environment of host, it is said that they short of oxygen in such environment. In fact, the mitochondrion of these parasites have happened "degradation" or even "specialization" and have completely lost the oxidative phosphorylation function. On the other hand, although they lived in microaerobic environment, trace of oxygen will cause damage to these parasites. There have report that they have mechanism to cope with the oxidative stress. Based on this background, we thoroughly searched KEGG database and parasite genome data, combining with experiment data and literature data, identified their energy metabolic pathways respectively, and got a very reliable and complete energy metabolic pathways of these parasitic protozoa. We surprisingly found that their energy metabolic pathways are striking similar. These protozoa have all lost Krebs cycle, lipid oxidation decomposition and oxidative phosphorylation, their ATP generate are mainly rely on glycolysis. And we also find they all have a similar Pyruvate metabolic pathway. Further, we investigate the enzyme of energy metabolism, found that a lot of enzymes, such as PEPCK, PFK and PFO catalyzed same reaction in these parasites, but these enzymes come from different sources. We further constructed phylogenetic tree of MAL, PPDK, ADHE and ACEL genes, find they are acquired by horizontal gene transfer through different donor. Most importantly, We found that these parasites all "coupling" pyruvate metabolism and oxygen, produced a "flexibility" metabolic mechanism that change pyruvate metabolism end products along with different oxygen concentration to adapt to the dynamic oxygen environment. In short, through our systematic study, we found these parasitic protozoa are adopted similar adaptation strategy in dealing with the microaerobic stress, and the adaptation are occurred in organelles, metabolic pathways, enzymes and gene levels. So the metabolic pathway of these parasites is a convergent evolution.2) Apicomplexans are important obligate parasites. Despite they are all parasitic species, their parasitic environment are different. Previous studies have shown that their corresponding energy metabolic pathway also very diversity. The systemic comparison of energy metabolic pathway among different parasitic environment apicomplexans species, and between their free-living close relatives ciliate or dinoflagellates, can help to clarify the apicomplexans energy metabolic pathways adaption to different kind of parasitic environment and what happened in parasitic adaptive evolution process. The relatively clear phylogenetic relationship between alveolate and among apicomplexan taxa and the sequence of their genome data provides unprecedented opportunities to solve these problems. In this study, we select four apicomplexan parasites that living in different parts: T. gondii, P. falciparum, C. muris and C. parvum and along with its two free-living cousins Paramecium tetraurelia and Tetrahymena thermophile as comparison, we thoroughly searched energy metabolic genes in KEGG database and genome data of these parasites, and combining with literature data, system identification and rebuild their respective energy metabolic pathways. To reconstruction of both cryptosporidium species energy metabolic pathway, we chose toxoplasma and plasmodium experiment data as reference. We systemically analyzed energy metabolic pathway of the six protozoans. Furthermore, we constructed phylogenetic tree each metabolic genes of these species. Our results suggest that many enzymes which previous thought only in parasites are generally exist in the free living relatives of apicomplexan. After the apicomplexan separated from the free-living relatives, they have occurred lineage specific loss or modification of metabolic enzymes respectively. Two cryptosporidium species, for example, are lost oxidation respiratory chain, and the C. parvum further lost the Krebs cycle, and they all developed a pyruvate metabolic pathways; T. gondii and P. falciparum lost Malic enzyme, PNO and AOX enzymes, P. falciparum further lost gluconeogenesis. This leads to diversification of energy metabolic pathways in apicomplexan. We analyzed their energy pathway combined with their parasitic environment, found that their parasite environment oxygen concentration is difference, for example: toxoplasma live in relatively high oxygen content in mammalian cells and cryptosporidium in microaerobic environment in the small intestine. As we know that oxygen is the most important factors of energy metabolism, and oxygen will largely influence the biological energy metabolism. Our above results suggest that four parasites energy metabolism is adaptive radiation, and the key factors is the environment oxygen concentration difference which they living. Therefore, this study preliminarily reveals the mechanism and reason of apicomplexan energy metabolic pathways adaptive radiation. |
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