Abstract
BackgroundUsing human brain microvascular endothelial cells (HBMECs) as an in vitro model for how African trypanosomes cross the human blood-brain barrier (BBB) we recently reported that the parasites cross the BBB by generating calcium activation signals in HBMECs through the activity of parasite cysteine proteases, particularly cathepsin L (brucipain). In the current study, we examined the possible role of a class of protease stimulated HBMEC G protein coupled receptors (GPCRs) known as protease activated receptors (PARs) that might be implicated in calcium signaling by African trypanosomes.Methodology/Principal FindingsUsing RNA interference (RNAi) we found that in vitro PAR-2 gene (F2RL1) expression in HBMEC monolayers could be reduced by over 95%. We also found that the ability of Trypanosoma brucei rhodesiense to cross F2RL1-silenced HBMEC monolayers was reduced (39%–49%) and that HBMECs silenced for F2RL1 maintained control levels of barrier function in the presence of the parasite. Consistent with the role of PAR-2, we found that HBMEC barrier function was also maintained after blockade of Gαq with Pasteurella multocida toxin (PMT). PAR-2 signaling has been shown in other systems to have neuroinflammatory and neuroprotective roles and our data implicate a role for proteases (i.e. brucipain) and PAR-2 in African trypanosome/HBMEC interactions. Using gene-profiling methods to interrogate candidate HBMEC pathways specifically triggered by brucipain, several pathways that potentially link some pathophysiologic processes associated with CNS HAT were identified.Conclusions/SignificanceTogether, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Gαq-mediated calcium signaling. The consequence of these events is predicted to be increased permeability of the BBB to parasite transmigration and the initiation of neuroinflammation, events precursory to CNS disease.
Highlights
Human African trypanosomiasis (HAT), commonly called sleeping sickness, is a vector-borne disease for which death is inevitable if the patient is untreated [1,2,3]
Using an in vitro model of the blood-brain barrier (BBB) consisting of human brain microvascular endothelial cells (HBMEC), we showed that human infective T. b. rhodesiense have a high potential for transendothelial migration, while animal infective T. b. brucei cross inefficiently [5]
After silencing of F2RL1 by RNA interference (RNAi), based on qRT-PCR targeting F2RL1 transcripts normalized to ACTB (b-actin transcripts), we found that protease activated receptors (PARs)-2 expression was reduced by over 95% (Fig 1A) in laser capture microdissection (LCM)-isolated HBMECs
Summary
Human African trypanosomiasis (HAT), commonly called sleeping sickness, is a vector-borne disease for which death is inevitable if the patient is untreated [1,2,3]. HAT is caused by two subspecies of African trypanosomes, Trypanosoma brucei rhodesiense and T. b. Gambiense causing East African and West African sleeping sickness, respectively. In classical late stage HAT (stage 2), the parasites invade the central nervous system (CNS) and the infected individuals suffer from progressive neurologic deterioration with concomitant psychiatric disorders, sleep disturbances, stupor, and coma. The role of the parasites in the pathogenesis of CNS lesions is not completely understood [4]. Rhodesiense have a high potential for transendothelial migration, while animal infective T. b. Brucei cross inefficiently [5].
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