Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease.
In this study we investigated why bloodstream forms of Trypanosoma brucei gambiense cross human brain microvascular endothelial cells (BMECs), a human blood-brain barrier (BBB) model system, at much greater efficiency than do T. b. brucei. After noting that T. b. gambiense displayed higher levels of cathepsin L-like cysteine proteases, we investigated whether these enzymes contribute to parasite crossing. First, we found that T. b. gambiense crossing of human BMECs was abrogated by N-methylpiperazine-urea-Phe-homopheylalanine-vinylsulfone-benzene (K11777), an irreversible inhibitor of cathepsin L-like cysteine proteases. Affinity labeling and immunochemical studies characterized brucipain as the K11777-sensitive cysteine protease expressed at higher levels by T. b. gambiense. K11777-treated T. b. gambiense failed to elicit calcium fluxes in BMECs, suggesting that generation of activation signals for the BBB is critically dependant on brucipain activity. Strikingly, crossing of T. b. brucei across the BBB was enhanced upon incubation with brucipain-rich supernatants derived from T. b. gambiense. The effects of the conditioned medium, which correlated with ability to evoke calcium fluxes, were canceled by K11777, but not by the cathepsin B inhibitor CA074. Collectively, these in vitro studies implicate brucipain as a critical driver of T. b. gambiense transendothelial migration of the human BBB.
Nikolskaia
Olga V
OV
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
de A Lima
Ana Paula C
AP
Kim
Yuri V
YV
Lonsdale-Eccles
John D
JD
Fukuma
Toshihide
T
Scharfstein
Julio
J
Grab
Dennis J
DJ
D000818:Animals; D001812:Blood-Brain Barrier / Q000166:cytology / Q000378:metabolism / Q000469:parasitology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000469:parasitology; D004963:Estrenes / Q000493:pharmacokinetics; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives; D010879:Piperazines; D015800:Protozoan Proteins / Q000378:metabolism; D011760:Pyrrolidinones / Q000493:pharmacokinetics; D014105:Tosyl Compounds; D014345:Trypanosoma / Q000201:enzymology* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000378:metabolism; D014347:Trypanosoma brucei gambiense / Q000201:enzymology / Q000378:metabolism; D016833:Trypanosoma brucei rhodesiense / Q000201:enzymology / Q000378:metabolism; D014753:Vinyl Compounds / Q000494:pharmacology
D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D004963:Estrenes; D009281:Naphthalenes; D010879:Piperazines; D015800:Protozoan Proteins; D011760:Pyrrolidinones; D014105:Tosyl Compounds; D014753:Vinyl Compounds; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C487484:N-pip-phenylalanine-homophenylalanine-vinyl sulfone phenyl; D010649:Phenylalanine; D004533:Egtazic Acid; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C100684:trypanopain Tb; D007930:Leucine; C058819:calphostin C; C024974:E 64; D002118:Calcium