Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease

J Clin Invest. 2006 Oct;116(10):2739-47. doi: 10.1172/JCI27798. Epub 2006 Sep 21.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood-Brain Barrier / cytology
  • Blood-Brain Barrier / metabolism
  • Blood-Brain Barrier / parasitology
  • Calcium / metabolism
  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology*
  • Cathepsins / antagonists & inhibitors
  • Cathepsins / metabolism
  • Cell Communication / drug effects
  • Cell Communication / physiology
  • Cell Movement / drug effects
  • Cell Movement / physiology*
  • Cells, Cultured
  • Culture Media, Conditioned / pharmacology
  • Cysteine Endopeptidases / metabolism*
  • Cysteine Proteinase Inhibitors / pharmacology
  • Dipeptides / pharmacology
  • Egtazic Acid / analogs & derivatives
  • Egtazic Acid / pharmacology
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism
  • Endothelial Cells / parasitology
  • Estrenes / pharmacokinetics
  • Humans
  • Leucine / analogs & derivatives
  • Leucine / pharmacology
  • Naphthalenes / pharmacology
  • Protozoan Proteins / metabolism
  • Pyrrolidinones / pharmacokinetics
  • Trypanosoma / enzymology*
  • Trypanosoma / metabolism
  • Trypanosoma brucei brucei / enzymology
  • Trypanosoma brucei brucei / metabolism
  • Trypanosoma brucei gambiense / enzymology
  • Trypanosoma brucei gambiense / metabolism
  • Trypanosoma brucei rhodesiense / enzymology
  • Trypanosoma brucei rhodesiense / metabolism
  • Vinyl Compounds / pharmacology

Substances

  • Culture Media, Conditioned
  • Cysteine Proteinase Inhibitors
  • Dipeptides
  • Estrenes
  • N-pip-phenylalanine-homophenylalanine-vinyl sulfone phenyl
  • Naphthalenes
  • Protozoan Proteins
  • Pyrrolidinones
  • Vinyl Compounds
  • 1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
  • 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester
  • Egtazic Acid
  • Cathepsins
  • Cysteine Endopeptidases
  • trypanopain Tb
  • Leucine
  • calphostin C
  • E 64
  • Calcium