Blood-brain barrier traversal by African trypanosomes requires calcium signaling induced by parasite cysteine protease
Olga V. Nikolskaia, Ana Paula C. de A. Lima, Yuri V. Kim, John D. Lonsdale-Eccles, Toshihide Fukuma, Julio Scharfstein, Dennis J. Grab
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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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 4

Cysteine protease activity in T. brucei subspecies.

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Cysteine protease activity in T. brucei subspecies. (A) ...
(A) Lysates of T. b. gambiense (t) or T. b. brucei (Tbb) bloodstream-form parasites were prepared, and the protease activity of equal aliquots (equivalent to 3.3 μg/ml) was determined using Z-Phe-Arg-AMC as a substrate. Substrate hydrolysis was followed by continuous recording of increase in fluorescence, and the initial velocities (V0) were calculated by linear regression of the hydrolysis curves. To discriminate the activity of papain-like cysteine proteases from other proteases (e.g., serine proteases), samples were incubated with 2 μM E-64 and the residual activity was measured. Cathepsin B– or cathepsin L–like activities were discriminated from each other by incubating the samples with 2 μM CA074 and 2 μM K11777 (to inhibit cathepsin B– and cathepsin L–like enzymes, respectively). (B) Conditioned medium of bloodstream-form trypanosomes. Both T. brucei subspecies described in A were washed in serum-free HMI-9 medium and incubated (2 × 107 parasites/ml) for 1 hour at 37°C. Parasite-conditioned medium was prepared, and the protease activities of 200-μl aliquots were determined as described in A. (C) Lysates of bloodstream forms of T. b. gambiense, T. b. rhodesiense (Tbr), and T. b. brucei (equivalent to 2 × 106 parasites) were incubated with 5 mM DTT and 20 μM APC336 for 30 minutes at room temperature, separated by SDS-PAGE, and transferred to nitrocellulose. The reactive bands were visualized after incubation with anti-biotin antibodies followed by addition of the substrates for chemiluminescence.