title: Disruption of MDCK cell tight junctions by the free-living amoeba Naegleria fowleri.
abstract: Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis. This parasite invades its host by penetrating the olfactory mucosa. However, the mechanism of epithelium penetration is not well understood. In the present study, we evaluated the effect of N. fowleri trophozoites and the non-pathogenic Naegleria gruberi on Madin-Darby canine kidney (MDCK) tight junction proteins, including claudin-1, occludin and ZO-1, as well as on the actin cytoskeleton. Trophozoites from each of the free-living amoeba species were co-cultured with MDCK cells in a 1 : 1 ratio for 1, 3, 6 or 10 h. Light microscopy revealed that N. fowleri caused morphological changes as early as 3 h post-infection in an epithelial MDCK monolayer. Confocal microscopy analysis revealed that after 10 h of co-culture, N. fowleri trophozoites induced epithelial cell damage, which was characterized by changes in the actin apical ring and disruption of the ZO-1 and claudin-1 proteins but not occludin. Western blot assays revealed gradual degradation of ZO-1 and claudin-1 as early as 3 h post-infection. Likewise, there was a drop in transepithelial electrical resistance that resulted in increased epithelial permeability and facilitated the invasion of N. fowleri trophozoites by a paracellular route. In contrast, N. gruberi did not induce alterations in MDCK cells even at 10 h post-infection. Based on these results, we suggest that N. fowleri trophozoites disrupt epithelial monolayers, which could enable their penetration of the olfactory epithelium and subsequent invasion of the central nervous system.
authors.affiliation: Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Mineko
authors.initials: M
authors.lastname: Shibayama
authors.affiliation: Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Moisés
authors.initials: M
authors.lastname: Martínez-Castillo
authors.affiliation: Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Angélica
authors.initials: A
authors.lastname: Silva-Olivares
authors.affiliation: Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Silvia
authors.initials: S
authors.lastname: Galindo-Gómez
authors.affiliation: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Fernando
authors.initials: F
authors.lastname: Navarro-García
authors.affiliation: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Jaime
authors.initials: J
authors.lastname: Escobar-Herrera
authors.affiliation: Department of Biology, University of Guanajuato, 36050 Guanajuato, Mexico.
authors.forename: Myrna
authors.initials: M
authors.lastname: Sabanero
authors.affiliation: Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Víctor
authors.initials: V
authors.lastname: Tsutsumi
authors.affiliation: Department of Cell Biology, Center for Research and Advanced Studies of the National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico.
authors.forename: Jesús
authors.initials: J
authors.lastname: Serrano-Luna
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D018920:Coculture Techniques; D004285:Dogs; D061985:Madin Darby Canine Kidney Cells; D008853:Microscopy; D016848:Naegleria fowleri / Q000472:pathogenicity*; D062725:Tight Junction Proteins / Q000378:metabolism*; D019108:Tight Junctions / Q000382:microbiology*
chemical_list: D000199:Actins; D062725:Tight Junction Proteins