Multiple mitochondrial introgression events and heteroplasmy in trypanosoma cruzi revealed by maxicircle MLST and next generation sequencing.
Multiple mitochondrial introgression events and heteroplasmy in trypanosoma cruzi revealed by maxicircle MLST and next generation sequencing.
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DOI：
10.1371/journal.pntd.0001584
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发表时间：
2012
影响因子：
3.8
通讯作者：
Miles MA
中科院分区：
文献类型：
--
作者：
Messenger LA;Llewellyn MS;Bhattacharyya T;Franzén O;Lewis MD;Ramírez JD;Carrasco HJ;Andersson B;Miles MA
线粒体DNA由于其相对较快的进化速度而成为一种有价值的分类标记。在恰加斯病的病原体克氏锥虫中，线粒体基因组具有独特的结构组织，由20 - 50个最大圆环（~ 20 kb）和数千个小圆环（0.5-10 kb）组成。克氏锥虫是一种早期分化的原生生物，具有显著的遗传异质性，被认为是由6个离散分型单元（dtu）组成的复合体。大多数感染者终身无症状，而30-35%的人出现可能致命的心脏和/或消化综合征。然而，特定临床结果与克氏锥虫基因型之间的关系仍然难以捉摸。全基因组序列的可用性推动了高分辨率基因分型技术的进步，并重新激发了探索各种dtu内存在的多样性的兴趣。为了描述dtu内部的多样性，我们基于10个基因片段开发了一个高分辨率的maxiccircle multi - locus sequence typing （mtMLST）方案。采用mtMLST方案、GPI、mini-外显子和25个微卫星位点对32个TcI分离株进行基因分型。核和线粒体数据的比较表明，不同地理种群和主要dtu之间的系统发育历史明显不一致。与此同时，我们利用Illumina测序TcI参考菌株Sylvio X10/1的maxiccle基因组产生的读取深度数据，为克氏锥虫线粒体异质性（单个细胞中的异质线粒体基因组）提供了第一个证据。mtMLST提供了一种强大的亚dtu水平基因分型方法。这一策略将有助于解决克氏锥虫表型变异的尝试，并结合密集的时空采样来解决流行病学上重要的假设。对核线粒体系统发育不一致的一般和特殊发生率的观察表明，遗传重组在地理上广泛存在，并继续影响着TcI的自然种群结构，这一结论挑战了克氏体的传统克隆范式。恰加斯病是由原生动物寄生虫克氏锥虫引起的，是拉丁美洲一个重要的公共卫生问题。虽然分子技术可以区分主要的克氏锥虫遗传谱系，但很少有足够的分辨率来描述密切相关菌株之间的多样性。三个线粒体基因组的在线可用性使我们能够设计一个多位点序列分型（mtMLST）方案来利用这些快速进化的标记。我们将mtMLST与目前的核分型工具进行比较，使用属于最古老和最广泛发生的谱系TcI的分离株。一般认为克氏锥虫是无性繁殖的。然而，在本研究中，线粒体和核系统发育树之间的不同分支模式揭示了不同地理种群和主要谱系之间的多重遗传交换发生率。我们还检查了来自TcI基因组菌株的Illumina测序数据，该数据揭示了单个寄生虫（异质性）中多个不同的线粒体基因组，然而，这些线粒体基因组的差异不足以代表分型错误的主要来源。我们强烈推荐这种结合核和线粒体基因分型的方法来揭示克氏锥虫的隐性多样性和遗传交换。该mtMLST提供的分辨率水平将极大地有助于阐明寄生虫基因型、临床结果和疾病分布之间复杂的相互作用。
Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20–50 maxicircles (∼20 kb) and thousands of minicircles (0.5–10 kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30–35% develop potentially fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the diversity present within the various DTUs. To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme based on ten gene fragments. A panel of 32 TcI isolates was genotyped using the mtMLST scheme, GPI, mini-exon and 25 microsatellite loci. Comparison of nuclear and mitochondrial data revealed clearly incongruent phylogenetic histories among different geographical populations as well as major DTUs. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain Sylvio X10/1, to provide the first evidence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi. mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi. Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is an important public health problem in Latin America. While molecular techniques can differentiate the major T. cruzi genetic lineages, few have sufficient resolution to describe diversity among closely related strains. The online availability of three mitochondrial genomes allowed us to design a multilocus sequence typing (mtMLST) scheme to exploit these rapidly evolving markers. We compared mtMLST with current nuclear typing tools using isolates belonging to the oldest and most widely occurring lineage TcI. T. cruzi is generally believed to reproduce clonally. However, in this study, distinct branching patterns between mitochondrial and nuclear phylogenetic trees revealed multiple incidences of genetic exchange within different geographical populations and major lineages. We also examined Illumina sequencing data from the TcI genome strain which revealed multiple different mitochondrial genomes within an individual parasite (heteroplasmy) that were, however, not sufficiently divergent to represent a major source of typing error. We strongly recommend this combined nuclear and mitochondrial genotyping methodology to reveal cryptic diversity and genetic exchange in T. cruzi. The level of resolution that this mtMLST provides should greatly assist attempts to elucidate the complex interactions between parasite genotype, clinical outcome and disease distribution.
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影响因子：
64.8
作者：
Gaunt, MW;Yeo, M;Miles, MA
通讯作者：
Miles, MA
影响因子：
2.1
作者：
Barnabé, C;Yaeger, R;Tibayrenc, M
通讯作者：
Tibayrenc, M
影响因子：
3.2
作者：
Gibson, Wendy;Peacock, Lori;Bailey, Mick
通讯作者：
Bailey, Mick
影响因子：
3.2
作者：
Herrera, Claudia;Bargues, M. Dolores;Guhl, Felipe
通讯作者：
Guhl, Felipe
影响因子：
3.3
作者：
Añez, N;Crisante, G;Teixeira, MMG
通讯作者：
Teixeira, MMG