Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

Trypanosoma brucei gambiense is responsible for more than 90% of reported cases of human African trypanosomosis (HAT). Infection can last for months or even years without major signs or symptoms of infection, but if left untreated, sleeping sickness is always fatal. In the present study, different T. b. gambiense field isolates from the cerebrospinal fluid of patients with HAT were adapted to growth in vitro. These isolates belong to the homogeneous Group 1 of T. b. gambiense, which is known to induce a chronic infection in humans. In spite of this, these isolates induced infections ranging from chronic to silent in mice, with variations in parasitaemia, mouse lifespan, their ability to invade the CNS and to elicit specific immune responses. In addition, during infection, an unexpected early tropism for the brain as well as the spleen and lungs was observed using bioluminescence analysis. The murine models presented in this work provide new insights into our understanding of HAT and allow further studies of parasite tropism during infection, which will be very useful for the treatment and the diagnosis of the disease

Two Related Subpellicular Cytoskeleton-associated Proteins in Trypanosoma brucei Stabilize Microtubules

The subpellicular microtubules of the trypanosome cytoskeleton are cross-linked to each other and the plasma membrane, creating a cage-like structure. We have isolated, from Trypanosoma brucei, two related low-molecular-weight cytoskeleton-associated proteins (15- and 17-kDa), called CAP15 and CAP17, which are differentially expressed during the life cycle. Immunolabeling shows a corset-like colocalization of both CAPs and tubulin. Western blot and electron microscope analyses show CAP15 and CAP17 labeling on detergent-extracted cytoskeletons. However, the localization of both proteins is restricted to the anterior, microtubule minus, and less dynamic half of the corset. CAP15 and CAP17 share properties of microtubule-associated proteins when expressed in heterologous cells (Chinese hamster ovary and HeLa), colocalization with their microtubules, induction of microtubule bundle formation, cold resistance, and insensitivity to nocodazole. When overexpressed in T. brucei, both CAP15 and CAP17 cover the whole subpellicular corset and induce morphological disorders, cell cycle-based abnormalities, and subsequent asymmetric cytokinesis

Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique to Trypanosoma congolense▿ †

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis

Reactivity patterns of immunoreactive invariant trypanosome proteins during BALB/c mice infections.

<p>Four mice were infected with either a low (10³) or a high (10⁶) load of <i>Tbg</i>945b, <i>Tbg</i>1122b, <i>Tbg</i>1166b, <i>Tbg</i>1135b or <i>Tbg</i>1135c isolates and their sera collected at different time points were tested by Western blotting (1/100 dilution) against a strip loaded with recombinant protein: 0.5 µg PFR and ISG75, 1 µg ISG65, ISG64 and TgsGP and 2 µg calflagin. The data are representative of one immunoblot out of 4 mice tested. NI represents the control sera before infection.</p

Biological properties of type 1 <i>T. b. gambiense</i> field isolates.

<p>Biological properties of type 1 <i>T. b. gambiense</i> field isolates.</p

Analysis of <i>T. b. gambiense</i> organs and central nervous system invasion in BALB/c-infected mice.

<p>A. Immunohistochemical detection of trypanosomes in the brains of mice (n = 2, only results from one mouse are shown) infected for 4 months with 10⁶ parasites of the <i>Tbg</i>945b isolate (1, 2, 3) and of paralyzed mice (n = 2) treated with cyclophosphamide before infection with either 5×10⁶ parasites of subchronic <i>Tbg</i>1122c or <i>Tbg</i>1166c isolates. Paralysis occurred 10 and 6 months PI with <i>Tbg</i>1122c and <i>Tbg</i>1166c isolates respectively. Only results from <i>Tbg</i>1122c are shown (4, 5, 6). A1 and A4 correspond to an olfactory bulb coronal section, A2 to a forebrain section, A3 and A5 to brain stem sections and A6 to a cerebellum section. Whole brain invasion was observed with the chronic isolate at an advanced stage of the disease (4 months PI, death within 6–8 months). Invasion was restricted to the olfactory bulb and the brain stem (including the cerebellum for <i>Tbg</i>1122c) in paralyzed mice infected with the sub-chronic isolates after treatment with cyclophosphamide. No invasion was observed in mice (n = 2) infected for 9 months with 5×10⁶ parasites of subchronic <i>Tbg</i>1166c isolate (data not shown). B. Spatial distribution of R-Luc activity in animals developing a sub-chronic or a silent infection and treated with or without cyclophosphamide (+/−cyclo). BALB/c mice were either directly infected with 10⁶ LucR-<i>Tbg</i>1135b (n = 6) or LucR-<i>Tbg</i>1135c (n = 2) or treated 24 h before infection with cyclophosphamide (n = 2). At different time PI, mice were anaesthetized and injected intravenously (i.v., retro-orbital) or intraperitoneally (i.p.) with coelenterazine and BLI signals were recorded in real time with a Biospace Imaging System. The panels show dorsal and ventral images of 2 representative mice infected for 8–11 weeks: LucR-<i>Tbg</i>1135b (11 weeks), LucR-<i>Tbg</i>1135c (10 weeks), LucR-<i>Tbg</i>1135b+cyclo (9 weeks), LucR-<i>Tbg</i>1135+cyclo (8 weeks). C. Spatial distribution of R-Luc activity in organs removed from LucR-Tbg1135 infected BALB/c mice. The different organs shown in this figure were isolated from mice: (1) non infected (control) (2) infected for 18 weeks with 10⁶ LucR-<i>Tbg</i>1135b, (3) infected for 18 weeks with 10⁶ LucR-<i>Tbg</i>1135c, (4) pre-treated with cyclophosphamide and infected for 16 weeks with 10⁶ LucR-<i>Tbg</i>1135c. Quantification data of light emission signals for ROI delimitating each organ are given in photons/second/cm²/steradian (p/sec/cm²/sr).</p

PCR detection of Trypanosomes in the blood of BALB/c mice infected with 10³ parasites of the silent <i>Tbg</i>1135c isolate.

<p>Detection is based on the amplification of 3 <i>Trypanosoma brucei</i> specific gene targets. The + sign represents a positive result (2–3 out of 3 targets amplification). ± represents a doubtful result (1 out of 3 targets amplification).</p