The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters.

Heme (iron protoporphyrin IX) is an important molecule involved in many biological reactions, including oxygen transport, respiration, photosynthesis and drug detoxification. Trypanosoma cruzi parasites, the etiological agent of Chagas' disease, take up heme from the environment to supply their nutritional needs because they do not synthesize this cofactor. However, the mechanisms involved in heme transport across biological membranes are poorly understood. Indeed, in T. cruzi, no heme transporter has yet been characterized. In the present work, we evaluate the heme uptake processes by T. cruzi epimastigotes using fluorescent heme-analogues. Heme uptake decreased significantly when cells were pretreated with different concentrations of SnPPIX, PdMPIX or ZnMPIX, this observed competition suggests that they are taken up by the same transport system. We studied the growth behavior of epimastigotes using the same heme-analogues and the treatments with SnPPIX or PdMPIX impaired cell growth but when heme was added to the culture medium the observed inhibition was partially reversed. In addition, we tested how the heme uptake processes are affected by the presence of different transporter inhibitors. When the cells were treated with inhibitors and then incubated with heme, heme uptake decreased significantly for all treatments. These results constitute a strong indication for the existence of a protein associated with porphyrin transport in T. cruzi, possibly ATP-binding cassette transporters (ABC-transporter).

Pulcherrimasaponin, from the leaves of Calliandra pulcherrima, as adjuvant for immunization in the murine model of visceral leishmaniasis.

A novel triterpenoidal saponin, called pulcherrimasaponin (CP05), isolated from the leaves of Calliandra pulcherrima Benth. shows remarkable similarities to the previously described potent adjuvant, QS21 saponin (Quillaja saponaria Molina). On the basis of chemical and physicochemical evidence, its structure was established as [3beta,16alpha,28[2E,6S[2E,6S(2E,6S)]]]-olean-12-en-28-oic acid 3-[[O-alpha-l-arabinopyranosyl-(1-->2)-O-alpha-l-arabinopyranosyl-(1-->6)-2-(acetylamino)-2-deoxy-beta-d-glucopyranosyl]oxy]-16-hydroxy-O-beta-d-glucopyranosyl-(1-->3)-O-[O-beta-d-xylopyranosyl-(1-->3)-beta-d-xylopyranosyl-(1-->4)-O-6-deoxy-alpha-l-mannopyranosyl-(1-->2)-6-O-[6-[[2-O-2,6-dimethyl-1-oxo-6-(beta-d-xylopyranosyloxy)-2,7-octadienyl]-[(6-deoxy-beta-d-glucopyranosyl)oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-beta-d-xylopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-beta-d-glucopyranosyl ester. In vivo toxicity assays disclosed similar and transitory local swelling and loss of hair but no lethality for mice. The haemolytic index was higher for QS21 (5 microg/ml) than for CP05 (13 microg/ml). Mouse vaccination with either CP05 or QS21 in combination with the fucose-mannose ligand (FML) antigen of Leishmania donovani showed anti-FML responses, significantly enhanced over the saponin and saline controls, in IgM, IgG, IgG1, IgG2a, IgG2b and IgG3. Antibody levels were similar for both vaccines in most subtypes. However, QS21-FML vaccine showed a 1.5 to 2.1 proportional increase over the CP05-FML vaccine in IgG, IgG2a and IgG3 responses. The delayed type of hypersensitivity against leishmanial antigen was impressively increased for CP05-FML and for QS21-FML-treated animals over controls (p<0.005). Enhancement was similar for both vaccines (p<0.05). The safety analysis and the effect on humoral and cellular immune responses demonstrated that the novel Calliandra pulcherrima Benth. CP05 saponin is a potential candidate for a vaccine adjuvant.