de la Cruz Olga Hernández, Muñiz-Lino Marcos, Guillén Nancy, Weber Christian, Marchat Laurence A, López-Rosas Itzel, Ruíz-García Erika, Astudillo-de la Vega Horacio, Fuentes-Mera Lizeth, Álvarez-Sánchez Elizbeth, Mendoza-Hernández Guillermo, López-Camarillo César
Genomics Sciences Program, Autonomous University of Mexico City, Mexico City 03100, Mexico.
Unit of Cellular Biology of Parasitism, Pasteur Institute, 75724 Paris, France; INSERM U786, Paris, France.
J Proteomics. 2014 Dec 5;111:46-58. doi: 10.1016/j.jprot.2014.03.041. Epub 2014 Apr 8.
Actin cytoskeleton is an essential structure involved in cell migration and invasion in parasites. In Entamoeba histolytica, the protozoan parasite causing human amoebiasis, the mechanisms underlying the expression of migration-related genes are poorly understood. Here, we investigated the biological effects of ectopic overexpression of EhPC4 (positive coactivator 4) in cell migration of E. histolytica trophozoites. Using differential in gel two-dimensional electrophoresis, 33 modulated proteins were detected in EhPC4-overexpressing cells. By electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis, 16 of these proteins were identified. Interestingly, four up-regulated proteins involved in cytoskeleton organization and cell migration were identified. Particularly, we found the up-regulation of a 16-kDa actin-binding protein (EhABP16) which is a putative member of the cofilin/tropomyosin family involved in actin polymerization. EhPC4 overexpression induced a significant increase in migration of trophozoites and in the destruction of human SW480 colon cells. Consistently, silencing of gene expression by RNA interference of EhABP16 significantly impairs cell migration. These changes were associated to alterations in the organization of actin cytoskeleton, and suppression of uropod-like structure formation in EhABP16-deficient cells. In summary, we have uncovered novel proteins modulated by EhPC4, including EhABP16, with a potential role in cell migration, cytopathogenicity and virulence in E. histolytica.
The human pathogen Entamoeba histolytica infects around 50million people worldwide resulting in 40,000-100,000 deaths annually. Cell motility is a complex trait that is critical for parasites adaptation, spread and invasion processes into host tissues; it has been associated with virulence. In this study, we used a differential proteomic approach to demonstrate that E. histolytica EhPC4 induces changes in the expression of actin cytoskeleton proteins, including EhABP16, promoting a significant increase in cell motility and destruction of intestinal human cells. Particularly, we demonstrated for the first time that abrogation of EhABP16 impairs cell migration by altering the actin cytoskeleton dynamics and uropod-like structure formation in trophozoites. These data contribute to the understanding of molecular mechanisms that regulate virulence properties in this neglected protozoan parasite.
肌动蛋白细胞骨架是参与寄生虫细胞迁移和侵袭的重要结构。在引起人类阿米巴病的原生动物寄生虫溶组织内阿米巴中，与迁移相关基因表达的潜在机制尚不清楚。在此，我们研究了异位过表达EhPC4（正向共激活因子4）对溶组织内阿米巴滋养体细胞迁移的生物学效应。使用差异凝胶二维电泳，在过表达EhPC4的细胞中检测到33种调节蛋白。通过电喷雾电离串联质谱（ESI-MS/MS）分析，鉴定出其中16种蛋白。有趣的是，鉴定出4种参与细胞骨架组织和细胞迁移的上调蛋白。特别地，我们发现一种16 kDa肌动蛋白结合蛋白（EhABP16）上调，它是参与肌动蛋白聚合的丝切蛋白/原肌球蛋白家族的推定成员。EhPC4过表达导致滋养体迁移以及对人SW480结肠细胞的破坏显著增加。一致地，通过RNA干扰沉默EhABP16的基因表达显著损害细胞迁移。这些变化与肌动蛋白细胞骨架组织的改变以及EhABP16缺陷细胞中尾足样结构形成的抑制有关。总之，我们发现了受EhPC4调节的新蛋白，包括EhABP16，它们在溶组织内阿米巴的细胞迁移、细胞致病性和毒力方面具有潜在作用。
人类病原体溶组织内阿米巴在全球感染约5000万人，每年导致40000 - 100000人死亡。细胞运动性是一种复杂的特性，对寄生虫适应、扩散和侵入宿主组织的过程至关重要；它与毒力相关。在本研究中，我们使用差异蛋白质组学方法证明溶组织内阿米巴EhPC4诱导肌动蛋白细胞骨架蛋白表达的变化，包括EhABP16，促进细胞运动性显著增加以及对肠道人类细胞的破坏。特别地，我们首次证明，通过改变滋养体中的肌动蛋白细胞骨架动力学和尾足样结构形成，缺失EhABP16会损害细胞迁移。这些数据有助于理解调节这种被忽视的原生动物寄生虫毒力特性的分子机制。
