Biochemical and cellular mechanisms regulating Acanthamoeba castellanii adherence to host cells K. J. SOTO-ARREDONDO 1 , L. L. FLORES-VILLAVICENCIO 1 , J. J. SERRANO-LUNA 2 , M. SHIBAYAMA 3 and M. SABANERO-LÓPEZ 1 * 1 Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Noria Alta S/N Col. Noria Alta, C.P. 36050, Guanajuato, Guanajuato, México 2 Departamento de Biología Celular, Centro de Investigación y Estudios Avanzados, Instituto Politécnico Nacional, Av. Instituto Politecnico Nacional 2508, San Pedro Zacatenco, Gustavo A. Madero, 07360, Ciudad de México, México 3 Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y Estudios Avanzados, Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, San Pedro Zacatenco, Gustavo A. Madero, 07360, Ciudad de México, México (Received 2 August 2013; revised 22 September 2013; accepted 7 October 2013; first published online 26 November 2013) SUMMARY Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process. Key words: Acanthamoeba castellanii, adhesion, cytochalasin, latrunculin, glycoproteins, cytoskeleton. INTRODUCTION Free-living amoebae belonging to the genus Acanthamoeba are among the most prevalent proto- zoa in the environment (Schuster and Visvesvara, 2004). Interest in the study of these protozoa is increasing due to their ability to infect humans and because they can act as a reservoir for other pathogens (Khan, 2003). Acanthamoeba castellanii, a pathogenic species of Acanthamoeba, is the causative agent of amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous acanthamoebiosis (Torno et al. 2000). The pathogenesis of infections by Acanthamoeba is poorly understood. However, it is known that attachment of amoebae to the surface of the host tissue is a crucial step in the establishment of infection, which culminates in the death of the host cell (Serrano-Luna et al. 2006). Similarly, in several protozoan pathogens, such as Entamoeba histolytica, the infection process involves host cell adherence by recognition of glycolipids and/or glycoproteins on the host cell, followed by cellular lysis by secretion of proteases and phagocytosis (Martínez-Palomo et al. 1985; Meza et al. 2006). On the surface of Acanthamoeba trophozoites, a transmembrane pro- tein has been reported to interact with glycoproteins that are rich in mannose residues found on corneal epithelial cells (Yang et al. 1997). However, the adhesion of pathogenic microorganisms is a complex process involving more than one protein in the interactions with the host cell (Karkowska-Kuleta et al. 2009). In Acanthamoeba, the molecules and cellular events governing adherence to the host cell are not completely elucidated. In addition to the expression of surface molecules, actin polymerization plays an important role in various physiological processes, including cell adhesion, through the formation of lamellipodia and filopodia (Pollard and Borisy, 2003). Structurally, Acanthamoeba trophozoites have multiple membrane projections called acantho- podia, which are primarily composed of fibres and * Corresponding author: Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Noria Alta S/N Col. Noria Alta, C.P. 36050, Guanajuato, Guanajuato, México. E-mail: myrna. sabanero@gmail.com 531 Parasitology (2014), 141, 531–541. © Cambridge University Press 2013 doi:10.1017/S0031182013001923