Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools

Trypanosoma congolense is a parasite responsible for severe disease of African livestock. Its life cycle is complex and divided into two phases, one in the tsetse fly vector and one in the bloodstream of the mammalian host. Molecular tools for gene function analyses in parasitic organisms are essential. Previous studies described the possibility of completing the entire T. congolense life cycle in vitro. However, the model showed major flaws including the absence of stable long-term culture of the infectious bloodstream forms, a laborious time-consuming period to perform the cycle and a lack of genetic tools. We therefore aimed to develop a standardized model convenient for genetic engineering. We succeeded in producing long-term cultures of all the developmental stages on long-term, to define all the differentiation steps and to finally complete the whole cycle in vitro. This improved model offers the opportunity to conduct phenotype analyses of genetically modified strains throughout the in vitro cycle and also during experimental infections

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

Un modèle prion (études génétique et fonctionnelle de la protéine HET-s chez le champignon filamenteux Podospora anserina)

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] → [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide

Erythrophagocytosis of desialylated red blood cells is responsible for anaemia during Trypanosoma vivax infection.

International audienceTrypanosomal infection-induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in-depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced-anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans-sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans-sialidases is vital to develop a control strategy against the disease or parasite

Identification of Trans-Sialidases as a Common Mediator of Endothelial Cell Activation by African Trypanosomes

<div><p>Understanding African Trypanosomiasis (AT) host-pathogen interaction is the key to an “anti-disease vaccine”, a novel strategy to control AT. Here we provide a better insight into this poorly described interaction by characterizing the activation of a panel of endothelial cells by bloodstream forms of four African trypanosome species, known to interact with host endothelium. <i>T. congolense</i>, <i>T. vivax</i>, and <i>T. b. gambiense</i> activated the endothelial NF-κB pathway, but interestingly, not <i>T. b. brucei</i>. The parasitic TS (trans-sialidases) mediated this NF-κB activation, remarkably <i>via</i> their lectin-like domain and induced production of pro-inflammatory molecules not only <i>in vitro</i> but also <i>in vivo</i>, suggesting a considerable impact on pathogenesis. For the first time, TS activity was identified in <i>T. b. gambiense</i> BSF which distinguishes it from the subspecies <i>T. b. brucei.</i> The corresponding TS were characterized and shown to activate endothelial cells, suggesting that TS represent a common mediator of endothelium activation among trypanosome species with divergent physiopathologies.</p></div

Kinetics of activation of murine and human endothelial cells by <i>T. congolense</i>, <i>T. b. gambiense</i> and <i>T. vivax</i> BSF.

<p>Murine (M) (A, B, and C) and human (H) (D, E, and F) endothelial cells from brain, lung, BM (bone marrow), spleen, PLN (peripheral lymph node), thymus, appendix, umbilical vein (HUVEC), intestine and skin were cultivated for 2, 6, 16 and 24 h with BSF <i>T. congolense</i> IL3000 (A and D), <i>T. vivax</i> Y486 (B and E) and <i>T. b. gambiense</i> 1135 (C and F). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#s2" target="_blank">Results</a> were similar with <i>T. congolense</i> STIB910 strain, <i>T. b. gambiense</i> LiTat strain, and <i>T. b. brucei</i> 427 strain. Percentage of activated endothelial cells at each time point is represented as mean value±SD of at least three independent experiments. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710.s001" target="_blank">Fig. S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710.s002" target="_blank">S2</a>, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003710#ppat.1003710.s007" target="_blank">Table S1</a>.</p

Involvement of TS lectin domain and α-2,3 linked sialic acids in BAE activation.

<p>(A) Effect of inactivation of catalytic site of TcoTS-A1 on BAE activation. 10 µg/ml of the active and inactive TcoTS-A1 were incubated for 16 h with BAE. (B) Ligand binding specificity of the lectins. (C) Activation of BAE by the lectins WGA, Mal, SNA, ConA and succinylated WGA after 16 h of incubation. Lectin concentration is indicated on the X-axis. (D) Competitive effect of TcoTS-A1 on MAL binding to BAE. Ratios of median fluorescence intensity (MFI) of BAE in the presence of TcoTS-A1, Myricetin or TcoTS-A1 pre-incubated with myricetin, over MFI of BAE with SNA-FITC or MAL-fluorescein alone. Data are expressed as mean values±SD of three independent experiments.</p