Retana-Moreira Lissette, Abrahams-Sandí Elizabeth, Ruiz-Campos Marco, Alvarado-Ocampo Johan, Castro Julián, Lorenzo-Morales Jacob, Sáenz-Arce Giovanni, Osuna Antonio
Universidad de Costa Rica, Facultad de Microbiología, Departamento de Parasitología, San José, Costa Rica.
Universidad de Costa Rica, Centro de Investigación en Enfermedades Tropicales, San José, Costa Rica.
Mem Inst Oswaldo Cruz. 2025 Jun 9;120:e240190. doi: 10.1590/0074-02760240190. eCollection 2025.
Acanthamoeba is a free-living amoeba widely distributed, responsible for keratitis and granulomatous amoebic encephalitis. The presence of virulence factors in its excretion/secretion products has been demonstrated. Characterisation of these products, including the determination of immunogenic protein components using polyclonal antibodies, could be the basis for the development of new diagnostic tools and help to understand aspects related to its pathogenesis.
To identify immunogenic protein components in Acanthamoeba conditioned medium (ACM) and extracellular vesicles (EVs) using polyclonal anti-Acanthamoeba antibodies produced in the laboratory and to evaluate the effect of these antibodies in adhesion and cytopathic effect.
Excretion/secretion products were obtained after the axenic culture of a potentially pathogenic environmental Acanthamoeba T5 isolate. The presence of immunogenic components in lysates of trophozoites, ACM and EVs was determined using polyclonal anti-Acanthamoeba antibodies produced in Wistar rats. Proteomic analyses to identify the immunogenic protein components in ACM and EVs were included. Experiments to evaluate the effect of polyclonal anti-Acanthamoeba antibodies in adhesion and cytopathic effect in vitro were also performed in Vero cells.
Protein recognition by anti-Acanthamoeba antibodies in lysates, ACM and EVs was demonstrated, and these components were identified using proteomics. Decreases in adhesion and cytopathic effect after the preincubation of trophozoites with antibodies, prior to the contact with cells, were observed.
The development of polyclonal antibodies, capable of recognising proteins related to pathogenesis in ACM and EVs, and with significant effects in adhesion, provides an important tool for the search for new therapeutic and diagnostic targets in infections caused by Acanthamoeba.
棘阿米巴是一种广泛分布的自由生活阿米巴，可导致角膜炎和肉芽肿性阿米巴脑炎。已证实其排泄/分泌产物中存在毒力因子。对这些产物进行表征，包括使用多克隆抗体确定免疫原性蛋白成分，可为开发新的诊断工具奠定基础，并有助于了解与其发病机制相关的方面。
使用实验室制备的抗棘阿米巴多克隆抗体，鉴定棘阿米巴条件培养基（ACM）和细胞外囊泡（EVs）中的免疫原性蛋白成分，并评估这些抗体对黏附及细胞病变效应（CPE）的影响。
对一株具有潜在致病性的环境棘阿米巴T5分离株进行无菌培养后，获得其排泄/分泌产物。使用在Wistar大鼠体内产生的抗棘阿米巴多克隆抗体，确定滋养体裂解物、ACM和EVs中免疫原性成分的存在情况。纳入蛋白质组学分析以鉴定ACM和EVs中的免疫原性蛋白成分。还在Vero细胞中进行了实验，以评估抗棘阿米巴多克隆抗体对体外黏附及细胞病变效应的影响。
证实抗棘阿米巴抗体可识别裂解物、ACM和EVs中的蛋白质，并通过蛋白质组学鉴定了这些成分。观察到在滋养体与细胞接触前用抗体预孵育后，黏附及细胞病变效应降低。
能够识别ACM和EVs中与发病机制相关蛋白质且对黏附有显著影响的多克隆抗体的开发，为寻找棘阿米巴感染的新治疗和诊断靶点提供了重要工具。
