Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers

Abstract

The apicomplexan parasite Toxoplasma gondii invades tissues and traverses nonpermissive biological barriers in infected humans and other vertebrates. Following ingestion, the parasite penetrates the intestinal wall and disseminates to immuneprivileged sites such as the brain parenchyma, after crossing the blood-brain barrier. In the present study, we have established a protocol for high-purification of primary mouse brain endothelial cells to generate stably polarised monolayers that allowed assessment of cellular barrier traversal by T. gondii. We report that T. gondii tachyzoites translocate across polarised monolayers of mouse brain endothelial cells and human intestinal Caco2 cells without significantly perturbing barrier impermeability and with minimal change in transcellular electrical resistance.

Integrin-dependent Migratory Switches Regulate the Translocation of Toxoplasma-infected Dendritic Cells Across Brain Endothelial Monolayers

“…1b) and high transendothelial electrical resistance (TEER) (Fig. 1c), consistent with characteristics of cerebral endothelium [32]. Importantly, T. gondii-challenged DCs transmigrated across polarised MBEC and bEnd.3 monolayers, at significantly higher frequencies compared with unchallenged DCs (Fig.…”

“…DCs migrate into the brain parenchyma in an integrin-dependent fashion during toxoplasmosis [51]. However, it remains unclear if DCs can transport T. gondii into the CNS, as extracellular parasites can also invade and transmigrate across endothelium [52,32]. Here, we provide in vitro proof-of-concept that transmigration of DCs across highly-polarised primary endothelium is potentiated by infection.…”

“…The brain tissue was freed from the cerebellum, hypothalamus, olfactory bulb and kept in ice-cold PBS. Preparation of cells was performed as described [61], with modifications [32]. The tissue was digested in collagenase IV (1 mg/ml, Gibco) for 1 h at 37° C, homogenised and washed with wash buffer (PBS, 0.5% FBS and 2mM EDTA).…”

Toxoplasma gondii excreted/secreted proteases disrupt intercellular junction proteins in epithelial cell monolayers to facilitate tachyzoites paracellular migration

“…There are three possible routes for Toxoplasma transmigration such as (a) transcellular pathway, (b) within macrophages in a Trojan horse‐like mechanism and (c) paracellular migration. To date, only a few reports have considered the paracellular migration of the parasite as a pathway to reach the diverse organs of the infected host, although morphological changes in cell monolayers during the infection by the parasite have been reported (Briceno et al, 2016; Franklin‐Murray et al, 2020; Nogueira et al, 2016; Ross, Olivera, & Barragan, 2019). However, the entire process of migration, regulation, and molecules involved still uncharacterized.…”

“…Also, transmigration of T. gondii has been reported in Caco‐2 cells and murine brain endothelial cells (MBEC) without affecting the barrier integrity. However, when cell monolayers were treated with parasite lysates, distribution of ZO‐1 was modified with significative increase of permeability (measured using dextran) and high transmigration frequency of Toxoplasma , apparently due to a transient alteration of the TJ stability through FAK dysregulation (Ross et al, 2019); in addition, infection of HUVEC cells by Toxoplasma has been related with barrier dysregulation a modification of the cell morphology (Franklin‐Murray et al, 2020). A previous study characterized the migration of tachyzoites apparently through a paracellular route although without changes in cell barrier function and mediated by the interaction between ICAM‐1 from the host cell and parasite adhesin MIC2 (Barragan, Brossier, & Sibley, 2005).…”