BackgroundIn order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells.Methodology/Principal findingsBy screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time.ConclusionA kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

We previously reported that macrophage arginase inhibits NO-dependent trypanosome killing in vitro and in vivo. BALB/c and C57BL/6 mice are known to be susceptible and resistant to trypanosome infection, respectively. Hence, we assessed the expression and the role of inducible NO synthase (iNOS) and arginase in these two mouse strains infected with Trypanosoma brucei brucei. Arginase I and arginase II mRNA expression was higher in macrophages from infected BALB/c compared with those from C57BL/6 mice, whereas iNOS mRNA was up-regulated at the same level in both phenotypes. Similarly, arginase activity was more important in macrophages from infected BALB/c vs infected C57BL/6 mice. Moreover, increase of arginase I and arginase II mRNA levels and of macrophage arginase activity was directly induced by trypanosomes, with a higher level in BALB/c compared with C57BL/6 mice. Neither iNOS expression nor NO production was stimulated by trypanosomes in vitro. The high level of arginase activity in T. brucei brucei-infected BALB/c macrophages strongly inhibited macrophage NO production, which in turn resulted in less trypanosome killing compared with C57BL/6 macrophages. NO generation and parasite killing were restored to the same level in BALB/c and C57BL/6 macrophages when arginase was specifically inhibited with Nω-hydroxy-nor-l-arginine. In conclusion, host arginase represents a marker of resistance/susceptibility to trypanosome infections.

Reactive nitrogen intermediates were synthesized spontaneously in cultures of macrophages from Trypanosoma brucei brucei-infected mice by an inducible nitric oxide (NO) synthase. This was inhibited by the addition of nitro-L-arginine. In this paper, we report the kinetics of the fixation of macrophage-derived NO on bovine serum albumin by using an enzyme-linked immunosorbent assay. S nitrosylation was confirmed by the Saville reaction, using mercuric chloride. It is known that reactive oxygen intermediates (ROI) are also synthesized by stimulated macrophages. The fact that NO is able to bind cysteine only under aerobic conditions led us to investigate the role of macrophage-derived ROI in the formation of S-nitrosylated proteins by activated macrophages. The immunoenzymatic signal decreased by 66 and 30% when superoxide dismutase and catalase, respectively, were added to the culture medium of macrophages from infected mice. In addition, the decrease in S-nitrosylated albumin formation correlated with the protection of extracellular trypanosomes from the cytostatic and cytotoxic activity of NO. Melatonin, a hydroxyl radical scavenger resulting from the decomposition of peroxynitrous acid, had no effect. All these data support the concept that an interaction between NO and ROI promoted the production of S-nitroso-albumin by activated macrophages from infected mice.

Arginase activity induction in macrophages is an escape mechanism developed by parasites to cope with the host's immune defense and benefit from increased host-derived growth factor production. We report that arginase expression and activity were induced in macrophages during mouse infection by , a natural parasite of this host. This induction was reproduced in vitro by excreted/secreted factors of the parasite. A mAb directed toKHC1, an orphan kinesin H chain from , inhibited excreted/secreted factor-mediated arginase induction. Anti-KHC1 Ab also inhibited growth, both in vitro and in vivo. Induction of arginase activity and parasite growth involved C-type lectin receptors, because mannose injection decreased arginase activity induction and parasite load in vitro and in vivo. Accordingly, the parasite load was reduced in mice lacking mannose receptor C-type 1. The KHC1 homolog showed high similarity with KHC1. Bioinformatics analysis revealed the presence of homologs of this gene in other trypanosomes, including pathogens for humans and animals. Host metabolism dysregulation represents an effective parasite mechanism to hamper the host immune response and modify host molecule production to favor parasite invasion and growth. Thus, this orphan kinesin plays an important role in promoting trypanosome infection, and its neutralization or the lock of its partner host molecules offers promising approaches to increasing resistance to infection and new developments in vaccination against trypanosomiasis.

African trypanosomiases, including the human disease referred to as 'sleeping sickness' and the animal diseases such as nagana, surra and dourine, are neglected vector-borne diseases that after years of research still need improved diagnosis and chemotherapy. Advances in proteomics offer new tools to define biomarkers, whose expression may reflect host-parasite interactions occurring during the infection. In this review, the authors first describe the current diagnostic tools used to detect a trypanosome infection during field surveys, and then discuss their interests, limits and further evolutions. The authors also report on the contribution of molecular diagnostics, and the recent advances and developments that make it suitable for fieldwork. The authors then explore the recent uses of proteomics technology to define host and parasite biomarkers that allow detection of the infection, the power and constraints of the technology. The authors conclude by discussing the urgent need to use the biomarkers discovered in order to develop tools to improve trypanosomiasis control in the near future.

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Trypanosomes from animals are potential pathogens for humans. Several human cases infected by Trypanosoma lewisi, a parasite of rats, have been reported. The number of these infections is possibly underestimated. Some infections were self-cured, others required treatment with drugs used in human African trypanosomosis. An in vitro evaluation of these drugs and fexinidazole, a new oral drug candidate, has been performed against T. lewisi in comparison with T. brucei gambiense. All have comparable activities against the two parasites. Suramin was not effective. In vivo, drugs were tested in rats immunosuppressed by cyclophosphamide. The best efficacy was obtained for fexinidazole, and pentamidine (15 mg/kg): rats were cured in 7 and 10 days respectively. Rats receiving nifurtimox-eflornithine combination therapy (NECT) or pentamidine (4 mg/kg) were cured after 28 days, while melarsoprol was weakly active. The identification of efficient drugs with reduced toxicity will help in the management of new cases of atypical trypanosomosis.

SummaryAntibodies directed against nitrosylated epitopes have been found in sera from patients suffering from human African trypanosomiasis (HAT) but not in sera from control subjects living in the same endemic area or African control subjects living in France. We conjugated amino acids to albumin by glutaraldehyde (conjugates) and then nitrosylated the conjugates. Both conjugates and nitrosylated conjugates were analysed by enzyme-linked immunosorbent assay (ELISA). We detected antibodies directed against nitrosylated l-cysteine and l-tyrosine conjugates; antibody levels were higher in stage II patients than in stage I. Patients with severe clinical signs had higher antibody levels, and antibody levels were highest in patients with major neurological signs. Antibody response was only associated with the IgM isotype. We evaluated antibody specificity and avidity by competition experiments using conjugates and nitrosylated conjugates. Avidity was around 2 ·10 )6 m for the S-nitroso-cysteine epitope and 2 · 10 )8 m for the S-nitroso-tyrosine epitope. Detection of circulating antibodies to S-nitroso-cysteine and S-nitroso-tyrosine epitopes provides indirect evidence for nitric oxide (NO) involvement in HAT and their levels are correlated with disease severity.keywords African trypanosomiasis, nitric oxide, anti-nitric oxide cysteine, anti-nitric oxide tyrosine