Identification and characterization of putative Aeromonas spp. T3SS effectors
Identification and characterization of putative Aeromonas spp. T3SS effectors
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DOI：
10.1371/journal.pone.0214035
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发表时间：
2019-06-04
期刊：
影响因子：
3.7
通讯作者：
Gogarten, Johann Peter
中科院分区：
文献类型：
--
作者：
Rangel, Luiz Thiberio;Marden, Jeremiah;Gogarten, Johann Peter
细菌致病性的遗传决定因素在物种和菌株之间是高度可变的。然而，一个通常与烈性革兰氏阴性菌（包括许多气单胞菌属）相关的因素，是3型分泌系统（T3SS），用于将效应蛋白注入靶真核细胞。在这项研究中，我们开发了一种生物信息学管道来识别T3SS效应蛋白，将这种方法应用于从环境，互惠或病原环境中分离的105株气单胞菌菌株的基因组，并通过其在酵母中的异源表达来评估所识别的效应物的细胞毒性。开发的管道使用两步方法，其中候选气单胞菌基因家族最初使用隐马尔可夫模型（HMM）对毒力因子数据库（VFDB）进行特征搜索，然后与阳性和阴性对照数据集进行严格比较，大大减少了假阳性的数量。该方法鉴定了21个气单胞菌T3SS可能的效应子家族，其中8个代表已知或表征的效应子，而其余13个先前未在气单胞菌中描述。我们通过评估酿酒酵母BY4741中代表性效应物的细胞毒性实验验证了我们的计算机研究结果，21种测定蛋白质中有15种在酵母中引起细胞毒性作用。本研究的结果证明了我们的方法的实用性，将新型计算机模拟搜索方法与体内实验验证相结合，并且将在未来旨在识别和鉴定其他属的细菌效应蛋白的研究中有用。
The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate Aeromonas gene families are initially selected using Hidden Markov Model (HMM) profile searches against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. This approach identified 21 Aeromonas T3SS likely effector families, of which 8 represent known or characterized effectors, while the remaining 13 have not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.