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.

Naegleria fowleri after 50 years: is it a neglected pathogen?

“…Investigations of the early stages of PAM demonstrated that 1 h after instillation of the amoebae, they interacted with the mucus present in the nasal cavity and that, at 6 h post-inoculation, the trophozoites were surrounded by an acute inflammatory reaction, mainly consisting of neutrophils (Cervantes-Sandoval et al, 2008a); however, this innate response appeared to be insufficient to eliminate N. fowleri. Then, at 12 h post-inoculation, the amoebae attached to and penetrated the olfactory neuroepithelium (Rojas-Hern andez et al, 2004;Cervantes-Sandoval et al, 2008a;Shibayama et al, 2013). Furthermore, at 30 h postinfection, trophozoites were found in the cribriform plate, and at 48-72 h post-infection, amoebae reached the OBs without causing an inflammatory reaction (Jarolim et al, 2000;Rojas-Hern andez et al, 2004).…”

“…When an infection of the protozoan occurs, it can damage tissues after the invasion to the CNS. A broad battery of mechanisms, such as poreforming proteins, proteases and adhesion-mediating glycoproteins, among others, are involved in the pathogenic mechanisms through which N. fowleri acts (Aldape et al, 1994;Herbst et al, 2002;Serrano-Luna et al, 2007;Shibayama et al, 2013) (Fig. 1a).…”

Pathogens Penetrating the Central Nervous System: Infection Pathways and the Cellular and Molecular Mechanisms of Invasion

“…Penetration of the olfactory epithelium in mice occurred without cellular disruption or damage (390), and microscopy studies have shown that N. fowleri may be ingested by sustentacular cells within the epithelium or may migrate between the sustentacular cells (393). A recent study demonstrated that N. fowleri degraded the epithelial tight junction proteins claudin-1 and zonula occludens-1 in vitro, highlighting a potential paracellular mechanism of penetration (394).…”

An In Vitro Model of the Blood–Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells

“…; Khan and Siddiqui ; Shibayama et al. ). To evaluate the damage produced by the amoebic proteases, we preincubated the trophozoites and their CM with E‐64 or Aprotinin and observed that the cysteine proteases inhibitor but not the serine proteases exerted a protective effect on the endothelial monolayer.…”