Abstract：
BACKGROUND: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host\'s immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity.
METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB).
RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB.
CONCLUSIONS: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.
METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB).
RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB.
CONCLUSIONS: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.
摘要：
背景：自由生活的原生动物棘阿米巴可引起严重的角膜炎，称为棘阿米巴角膜炎（AK）和肉芽肿性阿米巴脑炎（GAE）。棘阿米巴的发病机制包括生物体与宿主免疫系统之间复杂的相互作用。对良好注释的基因组组装的下游分析以及蛋白质组分析可以解开几个生物学过程，并有助于鉴定与致病性有关的潜在基因。
方法：基于下一代测序数据分析，基因包括溶血磷脂酶,磷脂酶,S8/S53肽酶,羧酸酯酶，选择甘露糖结合蛋白作为可能的致病靶标，并通过常规PCR在总共30株棘阿米巴分离物中进行验证。随后进行实时PCR，以评估使用角膜炎（CHA5）诱导的角膜炎和阿米巴性脑炎动物模型中的相对基因表达。脑炎（CHA24）和非致病性环境分离株（CHA36）。此外,对角膜炎进行液相色谱-质谱(LC-MS/MS),脑炎,聚六亚甲基双胍（PHMB）处理前后的非致病性环境分离株。
结果：常规PCR证明溶血磷脂酶扩增成功，磷脂酶,S8/S53肽酶,羧酸酯酶，临床和环境分离株中的甘露糖结合蛋白基因。表达分析显示磷脂酶，溶血磷脂酶,在动物模型的AK期间，角膜炎分离株（CHA5）中甘露糖结合基因显着上调。在阿米巴脑炎模型的情况下，磷脂酶,溶血磷脂酶,S8/S53肽酶,与角膜炎分离物相比，脑炎分离物中的羧酸酯酶显着上调。蛋白质组数据揭示了在用PHMB处理前和后处理中致病性与非致病性分离株中的差异蛋白质表达。
结论：基因表达数据表明溶血磷脂酶，磷脂酶,S8/S53肽酶,羧酸酯酶，甘露糖结合蛋白（MBP）可能在棘阿米巴发病的接触依赖性和独立机制中发挥作用。此外,3种分离物的蛋白质组学分析揭示了对寄生虫生长至关重要的差异蛋白表达，生存,和毒力。我们的结果为选择可能的致病靶标提供了基线数据，这些靶标可用于将来设计敲除实验。
方法：基于下一代测序数据分析，基因包括溶血磷脂酶,磷脂酶,S8/S53肽酶,羧酸酯酶，选择甘露糖结合蛋白作为可能的致病靶标，并通过常规PCR在总共30株棘阿米巴分离物中进行验证。随后进行实时PCR，以评估使用角膜炎（CHA5）诱导的角膜炎和阿米巴性脑炎动物模型中的相对基因表达。脑炎（CHA24）和非致病性环境分离株（CHA36）。此外,对角膜炎进行液相色谱-质谱(LC-MS/MS),脑炎,聚六亚甲基双胍（PHMB）处理前后的非致病性环境分离株。
结果：常规PCR证明溶血磷脂酶扩增成功，磷脂酶,S8/S53肽酶,羧酸酯酶，临床和环境分离株中的甘露糖结合蛋白基因。表达分析显示磷脂酶，溶血磷脂酶,在动物模型的AK期间，角膜炎分离株（CHA5）中甘露糖结合基因显着上调。在阿米巴脑炎模型的情况下，磷脂酶,溶血磷脂酶,S8/S53肽酶,与角膜炎分离物相比，脑炎分离物中的羧酸酯酶显着上调。蛋白质组数据揭示了在用PHMB处理前和后处理中致病性与非致病性分离株中的差异蛋白质表达。
结论：基因表达数据表明溶血磷脂酶，磷脂酶,S8/S53肽酶,羧酸酯酶，甘露糖结合蛋白（MBP）可能在棘阿米巴发病的接触依赖性和独立机制中发挥作用。此外,3种分离物的蛋白质组学分析揭示了对寄生虫生长至关重要的差异蛋白表达，生存,和毒力。我们的结果为选择可能的致病靶标提供了基线数据，这些靶标可用于将来设计敲除实验。
