Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Human African trypanosomiasis, or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is composed of brain microvascular endothelial cells (BMECs) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open up to parasite crossing. Because human BMECs express protease-activated receptors (PARs) that trigger calcium signals in BMECs, we hypothesized a functional link between parasite brucipain and BMEC PARs. Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open up and cross human BMECs. Using gene-profiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for brucipain and BMEC PARs in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease

Localisation of a multicatalytic protease complex in Trypanosoma brucei brucei

Trypanosoma brucei brucei are tsetse-transmitted, extracellular protozoan parasite that causes Nagana, a cattle disease similar to sleeping sickness caused in humans by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Proteases of these parasites have come under increasing scrutiny because of their importance in such aspects as disease pathogenesis and as potential drug targets. Recently, a multicatalytic protease complex, the proteasome, has also become of interest because of its potential involvement in cell-cycle development. The proteasome is the central protease of the non lysosomal ubiquitin dependent pathway of protein degradation. Here we report the subcellular localization of 20S proteasome of Trypanosoma brucei brucei, using subcellular fractionation and enzymatic assays. Homogenate prepared from bloodstream form of T. brucei was fractionated by differential centrifugation into nuclear [NU] fraction, a large-granule [LG] fraction, small-granule [SG] fraction, microsomal [MI] and soluble cytosolic [CY] fractions. Each of these subcellular fractions was enriched in a particular organelle. The identity of these organelles was evaluated by assaying for the activity of marker enzymes. The proteasome activity was measured in the fractions using Z-Gly-Gly-Leu-AMC as substrate, peptide aldehyde ZLeu-Leu-Leu-H as specific inhibitor. The enzymatic activity was detected mainly in the cytosolic fraction (6.7-fold enrichment relative to the whole homogenate), but also in the nuclear fraction (2.8-fold). Our findings indicate that the internal location of the trypanosomal proteasome may not be restricted to the cytosol and nuclei as has previously been demonstrated in the eukaryotic cells.Key words: Trypanosoma brucei, 20S proteasome. subcellular localizatio

Characterisation of the antitrypanosomal activity of peptidyl alpha-aminoalkyl phosphonate diphenyl esters.

Two groups of irreversible serine peptidase inhibitors, peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters, were examined for antitrypanosomal activity against the bloodstream form of Trypanosoma brucei brucei. Both peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters inhibited trypsin-like peptidases of the parasites and exhibited antitrypanosomal activity at micromolar concentrations. In live T. b. brucei, labelled analogues of both of these groups of inhibitors primarily targeted an 80-kDa peptidase, possibly a serine oligopeptidase known as oligopeptidase B. In an in vivo mouse model of infection, one of these inhibitors, carbobenzyloxyglycyl-4-amidinophenylglycine phosphonate diphenyl ester, was curative at 5 mg kg(-1) day(-1) but appeared toxic at higher doses. There was no significant correlation between the inhibitory potency (as evaluated against purified T. b. brucei oligopeptidase B) and the in vitro antitrypanosomal efficacy of either group of inhibitors, suggesting that these inhibitors were acting on multiple targets within the parasites, or had different cell permeability properties. These findings suggest that serine peptidases may represent novel chemotherapeutic targets in African trypanosomes