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.

Trypanosoma brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

“…12 Trypanosoma brucei expresses two Clan CA, Family C1 cysteine proteases; TbCatL and TbCatB. 15,16 Given these data, cardiomyocytes were incubated with/without TbCatL-specific inhibitor K11777 12 (10 µM) and spontaneous contractile events assessed within 60 s. Supernatant increased the contractile events to 137% of media levels (100.0 vs. 137.5 ± 5.4% cardiomyocytes with contractile events relative to media; media vs. supernatant; P < 0.05; Figure 5A ). K11777 inhibited this effect (96.8 ± 5.4 vs. 95.8 ± 2.5% cardiomyocytes with contractile events relative to media; media + K11777 vs. supernatant + K11777; P > 0.05; Figure 5A ).…”

“…A similar effect has been observed in BMECs, where [Ca 2+ ] i spontaneous releases occurred despite the removal of the parasite from the media. 12 It is clear therefore that in both cardiomyocytes and BMECs, the [Ca 2+ ] i alteration is mediated by a trypanosome-derived (secreted/excreted) factor.…”

“…In contrast, a significant body of recent work has focused on the neuro-pathogenesis of the disease showing that both an inflammatory response and trypanosome interaction with BBB cells are important. 11,12 Therefore, one inference is that the cardiac-related clinical signs in African trypanosomiasis result from the inflammatory response. Whilst this may indeed play a role, an alternative hypothesis of parasites interacting with cardiomyocytes and altering heart function is untested.…”

“…12 Given that trypanosomes affect host cell [Ca 2+ ] i dynamics, and the pivotal role of Ca 2+ in cardiomyocyte and heart function, the aim of this study was to utilize isolated cardiomyocytes and whole hearts to investigate the hypothesis that African trypanosomes alter intra-cardiomyocyte Ca 2+ handling and whole heart function.…”

Parasite Prolyl Oligopeptidases and the Challenge of Designing Chemotherapeuticals for Chagas Disease, Leishmaniasis and African Trypanosomiasis

“…The ability of T. brucei to cross the BBB seems to be driven by parasite proteases [76] . T. brucei cysteine proteases have been implicated in calcium-dependent signaling that promotes parasite transmigration [77, 78]. Host MMPs released during the inflammatory response against pathogen infection enhance collagen degradation in the BBB, a mechanism proposed in cerebral malaria [79], HAT [80], canine cerebral leishmaniasis [81] and Lyme disease [82].…”