Detection of immunogenic protein components in excretion/secretion products of Acanthamoeba T5 using polyclonal antibodies

Abstract

Background. 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. Objectices. 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. Methods. 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. Findings. 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. Main conclusion. 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.