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.

Figure 1. Ca²⁺ oscillations of human BMECs in response to T. b. gambiense are mediated by papain-like cysteine proteases.

Fura-2/AM–loaded BMECs were mounted on a recording chamber, and 25–40 regions of interest representing individual cells were selected. Cells were exposed to bloodstream forms of T. b. gambiense (10⁶ parasites/ml) in HEPES-buffered HBSS, and real-time fluorescent images were captured by alternating excitation at 340 and 380 nm. (A) Time-lapse images of [Ca²⁺]_i changes presented between the time points marked with arrows 2 and 3 in B. Increased [Ca²⁺]_i indicated by color change from blue to red. (B) Kinetics of [Ca²⁺]_i changes. T. b. gambiense (Tb; 10⁶ parasites/ml) was added where indicated. To show functional integrity of human BMECs after T. b. gambiense treatment, 10 μM ATP was added at the end of the experiments at 1770 seconds as indicated. (C) To show the presence of parasites, during Ca²⁺ measurements a differential interference contrast image was taken at the time point marked by arrow 1 in B. Arrows show some of the motile T. b. gambiense. [Ca²⁺]_i changes were expressed as the 340:380 ratio. (D) K11777 (20 μM) was added to cells in 0.5% DMSO immediately before addition of parasites, and the variations of fluorescence were measured. DMSO alone had no effect on parasite-induced calcium changes. ATP (1 μM) was added at the end of the recording as a positive control of BMEC integrity. Unlabeled arrows indicate when a change to normal medium was made. Results are representative of 2 independent experiments.

Figure 2. The traversal of the human BBB model by T. b. gambiense requires calcium and the activity of papain-like cysteine proteases.

(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.

Figure 3. The main target of K11777 in T. b. gambiense is brucipain.

(A) Bloodstream forms of T. b. gambiense Triton X-100 lysates were prepared, and equal aliquots of the soluble fractions (equivalent to 3 × 10⁵ parasites) were incubated with 5 mM DTT (lane 1) or with 5 mM DTT and 20 μM E-64 (lane 2) for 15 minutes at room temperature, followed by incubation with 20 μM APC336, a biotinylated cysteine protease inhibitor related to K11777, for 30 minutes. The samples were diluted in SDS-PAGE sample buffer under reducing conditions and boiled for 5 minutes prior to separation by electrophoresis and transfer to nitrocellulose. The membranes were incubated with anti-biotin antibodies, and the reactive bands were visualized by chemiluminescence. (B) Immunoprecipitation of inhibitor-bound brucipain. Lysate (165 μg) of bloodstream forms of T. b. gambiense was incubated with 20 μM APC336 for 30 minutes and mixed with protein G agarose beads coated with nonimmune mouse serum (lane 1) or anti-brucipain antiserum (lane 2) for 2 hours. The beads were washed, then boiled for 5 minutes in SDS-PAGE sample buffer under reducing conditions prior to separation by electrophoresis and transfer to nitrocellulose. The membranes were incubated with avidin-alkaline phosphatase. The prestained molecular weight markers are shown at the right.

Figure 4. Cysteine protease activity in T. brucei subspecies.

(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.

Figure 5. Secretion products of T. b. gambiense enhance transendothelial migration of bloodstream forms of T. b. brucei.

(A) Bloodstream forms of T. b. brucei parasites (10⁶) were incubated for 3 hours at 37°C with human BMECs on Transwell inserts in medium or T. b. gambiense conditioned medium with 10% FBS. The percentage of parasites ± SEM that crossed in control medium or T. b. gambiense–conditioned medium relative to the 10⁶ parasites added is shown. *P < 0.05. (B) T. b. gambiense–conditioned medium was also incubated with DMSO (0.25 %) and 10 μM CA074 or K11777 for 15 minutes at 37°C prior to addition to BMEC monolayers. Bloodstream forms of T. b. brucei parasites (2 × 10⁶) were incubated for 3 hours at 37°C with BMECs on Transwell inserts in T. b. gambiense–conditioned medium with 10% FBS. The relative percentage of parasites ± SEM that crossed in the presence of inhibitor relative to DMSO controls is shown. *P < 0.05.

Figure 6. Cysteine proteases secreted by T. b. gambiense induce Ca²⁺ oscillations in human BMECs.

(A) Fura-2/AM–loaded BMECs were mounted on a microscope, and 25–40 regions of interest representing individual cells were selected. T. b. gambiense–conditioned medium (200 μl) was added to cells, and real-time fluorescent images were captured by alternating excitation at 340 and 380 nm. [Ca²⁺]_i changes were expressed as the 340:380 ratio. (B) K11777 (20 μM) was added to the Fura-2/AM–loaded endothelial cells prior to addition of conditioned medium (Tbg supernatant). ATP (1 μM) was added at the end of the recording as a positive control for human BMEC integrity. Unlabeled arrows indicate when a change to normal medium was made. Results are representative of 2 independent experiments.