Protease activated receptor signaling is required for African trypanosome traversal of human brain microvascular endothelial cells.
BACKGROUND
Using 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.


RESULTS
Using 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 Galpha(q) 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
Together, the data support a role, in part, for GPCRs as molecular targets for parasite proteases that lead to the activation of Galpha(q)-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.
Grab
Dennis J
DJ
Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America. dgrab1@jhmi.edu
Garcia-Garcia
Jose C
JC
Nikolskaia
Olga V
OV
Kim
Yuri V
YV
Brown
Amanda
A
Pardo
Carlos A
CA
Zhang
Yongqing
Y
Becker
Kevin G
KG
Wilson
Brenda A
BA
de A Lima
Ana Paula C
AP
Scharfstein
Julio
J
Dumler
J Stephen
JS
D000818:Animals; D001426:Bacterial Proteins / Q000633:toxicity; D001427:Bacterial Toxins / Q000633:toxicity; D002460:Cell Line; D042783:Endothelial Cells / Q000469:parasitology*; D020868:Gene Silencing; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction; D016833:Trypanosoma brucei rhodesiense / Q000472:pathogenicity*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C062258:Pasteurella multocida toxin; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2