Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease
Olga V. Nikolskaia, … , Julio Scharfstein, Dennis J. Grab
Olga V. Nikolskaia, … , Julio Scharfstein, Dennis J. Grab
Published October 2, 2006
Citation Information: J Clin Invest. 2006;116(10):2739-2747. https://doi.org/10.1172/JCI27798.
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Research Article Infectious disease

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

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.

Authors

Olga V. Nikolskaia, Ana Paula C. de A. Lima, Yuri V. Kim, John D. Lonsdale-Eccles, Toshihide Fukuma, Julio Scharfstein, Dennis J. Grab

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Figure 2

The traversal of the human BBB model by T. b. gambiense requires calcium and the activity of papain-like cysteine proteases.

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The traversal of the human BBB model by T. b. gambiense requires calcium...
(A) Bloodstream forms of T. b. gambiense parasites (106) were incubated with human BMECs on Transwell inserts for 3 hours. The number of parasites at the lower chamber was subsequently estimated by counting in a hemacytometer. The percentage of parasites ± SEM that crossed relative to the 106 parasites added is shown. Experiments were performed in triplicate. To show the role of [Ca2+]i, the BMECs were incubated with 15 μM BAPTA-AM for 30 minutes and washed twice prior to adding parasites. Controls were performed by adding medium containing 0.5% DMSO. (B) Bloodstream-form parasites (106) were added to the endothelial cells and incubated for 3 hours at 37°C, after which the percentage of parasites that crossed was estimated as in A. DMSO (0.5%), K11777 (20 μM), and E-64d (20 μM) were added to human BMEC monolayers immediately before the addition of parasites. (C) Bloodstream-form parasites (106) were added to inserts containing human BMECs or empty inserts (No BMECs) and incubated for 3 hours at 37°C, after which the percentage of parasites that crossed was estimated as in A. DMSO (0.5%) and K11777 (1–20 μM) were added to insert monolayers immediately before the addition of parasites. *P < 0.05. The groups showing statistical significance against all other groups in the same graph are marked. In B, K11777 and E-64d are significantly different from medium or DMSO; in C, K11777 is significantly different from DMSO. There is no significant difference between DMSO and K11777 in wells with no BMECs.