Marcelo Genestra

Nitric oxide biosynthesis by Leishmania amazonensis promastigotes containing a high percentage of metacyclic forms

Due to the diversity of its physiological and pathophysiological functions and general ubiquity, the study of nitric oxide (NO) has become of great interest. In this work, it was demonstrated that Leishmania amazonensis promastigotes produces NO, a free radical synthesized from l-arginine by nitric oxide synthase (NOS). A soluble NOS was purified from L. amazonensis promastigotes by affinity chromatography (2′, 5′-ADP-agarose) and on SDS-PAGE the enzyme migrates as a single protein band of 116.2 (±6) kDa. Furthermore, the presence of a constitutive NOS was detected through indirect immunofluorescence using anti-cNOS and in NADPH consumption assays. The present work show that NO production, detected as nitrite in culture supernatant, is prominent in promastigotes preparations with high number of metacyclic forms, suggesting an association with the differentiation and the infectivity of the parasite.

Comparative analysis of the nitric oxide production by Leishmania sp.

The present report explores a comparative analysis of nitric oxide (NO·) production by three different species of Leishmania (L. amazonensis, L. braziliensis and L. chagasi). Among these species, L. braziliensis produced the highest amount of NO·, measured in the supernatants of promastigotes cultures as nitrite, a stable by-product derived from NO·. We have previously described the expression of a constitutive nitric oxide synthase (cNOS) in L. amazonensis promastigotes and axenic amastigotes. Comparing those results with the present work, using immunofluorescence assay, it was shown that both L. braziliensis and L. chagasi also express a cNOS. Immunostaining experiments showed that promastigotes from early passages of these species in culture had a strong immunoreactivity against anti-cNOS and anti-endothelial cell NOS, in comparison with the same parasite cultured for long time, suggesting a correlation between the NO· production and the presence of metacyclic forms prominent in those newly isolated parasites. These data corroborate findings of a higher NO· production by those parasites, following the growth curve. The relationship between the two NO·-generating systems in the parasite and in their host cell warrants further investigation. The presence of cNOS raises the possibility of a similar type of cross-talk or down-regulation between the NO· signaling systems in host cells and the lower eukaryotic-like Leishmania sp.

Recentes avanços da quimioterapia das leishmanioses: moléculas intracelulares como alvo de fármacos

Leishmanioses sao doencas causadas por protozoarios do genero Leishmania sp., que se apresentam na forma promastigota ou amastigota; os promastigotas infectam o inseto vetor e os amastigotas sao as formas infectivas antes presentes em macrofagos humanos. O tratamento utilizado pela clinica tem se mostrado ineficaz; os farmacos utilizados se apresentam na forma injetavel, o que dificulta o tratamento, ja que o paciente, para ser tratado, deve ser internado para as aplicacoes - que sao dolorosas - e assim muitos pacientes desistem do tratamento. Outra razao para a internacao e a monitoracao dos efeitos colaterais causados pelos farmacos. Por estas razoes a quimioterapia para leishmaniose tem sido objeto de estudo de muitos laboratorios de pesquisa, os quais tem testado outras substâncias quimicas e extratos de plantas com a finalidade de se encontrar novos agentes leishmanicidas com menores efeitos colaterais e otima biodisponibilidade. Alem disso, pesquisam-se, tambem, outras formas farmaceuticas que viabilizem a aplicacao desses farmacos, o que tornaria desnecessaria a internacao do paciente para se efetuar o tratamento. Este artigo visa discutir os recentes avancos da quimioterapia utilizada para as leishmanioses e, tambem, apresentar os aspectos farmacologicos e bioquimicos da participacao de moleculas intracelulares do parasita como alvos de farmacos.

Kinetoplastid membrane protein-11 exacerbates infection with Leishmania amazonensis in murine macrophages

In Leishmania amazonensis, kinetoplastid membrane protein-11 (KMP-11) expression increases during meta-cyclogenesis and is higher in amastigotes than in promastigotes, suggesting a role for this protein in the infection of the mammalian host. We show that the addition of KMP-11 exacerbates L. amazonensis infection in peritoneal macrophages from BALB/c mice by increasing interleukin (IL)-10 secretion and arginase activity while reducing nitric oxide (NO) production. The doses of KMP-11, the IL-10 levels and the intracellular amastigote loads were strongly, positively and significantly correlated. The increase in parasite load induced by KMP-11 was inhibited by anti-KMP-11 or anti-IL-10 neutralising antibodies, but not by isotype controls. The neutralising antibodies, but not the isotype controls, were also able to significantly decrease the parasite load in macrophages cultured without the addition of KMP-11, demonstrating that KMP-11-induced exacerbation of the infection is not dependent on the addition of exogenous KMP-11 and that the protein naturally expressed by the parasite is able to promote it. In this study, the exacerbating effect of KMP-11 on macrophage infection with Leishmania is for the first time demonstrated, implicating it as a virulence factor in L. amazonensis. The stimulation of IL-10 production and arginase activity and the inhibition of NO synthesis are likely involved in this effect.

In vitro sodium nitroprusside‐mediated toxicity towards Leishmania amazonensis promastigotes and axenic amastigotes

Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose‐dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L−1 SNP for 24 h showed about 75% growth inhibition, and 97–100% when the cultures were treated with >2 mmol L−1 SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25–8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP‐mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP‐mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal‐independent oxidation of biomolecules to free radical intermediates. Copyright

Trypanothione Reductase Activity is Prominent in Metacyclic Promastigotes and Axenic Amastigotes of Leishmania amazonesis. Evaluation of its Potential as a Therapeutic Target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N′-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.

Effect of L-arginine analogs and a calcium chelator on nitric oxide (NO) production by Leishmania sp.

Leishmania amazonensis, L. braziliensis and L. chagasi promastigotes were grown in the presence of l-arginine analogs such as Nω-nitro-l-arginine methyl ester (l-NAME), NG-nitro-l-arginine (l-NNA) and d-arginine (an inactive l-arginine isomer), besides an intracellular calcium chelator [ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetra acetic acid; EGTA] to verify the importance of l-arginine metabolism and the cofactors for these parasites. The parasites growth curve was followed up and the culture supernatants were used to assay nitric oxide (NO˙) production by the Griess reaction. The results showed a significant effect of l-arginine analogs on NO˙ production by all Leishmania species studied, especially l-NAME, an irreversible inhibitor of the constitutive nitric oxide synthase (cNOS). When L. amazonensis promastigotes were pre-incubated with l-NAME, the infection range of the murine macrophages was lowered to 61% in 24 h and 19% after 48 h. These data demonstrated that the parasite NO˙ pathway is important to the establishment of the infection.

Leishmania amazonensis trypanothione reductase: Evaluation of the effect of glutathione analogs on parasite growth, infectivity and enzyme activity

Trypanothione reductase (TR) is a major enzyme in trypanosomatids. Its substrate, trypanothione is a molecule containing a tripeptide (L-glutamic acid-cysteine-glycine) coupled to a polyamine, spermidine. This redox system (TR/Trypanothione) is vital for parasite survival within the host cell and has been described as a good target for chemotherapy anti-Leishmania. The use of tripeptides analogs of glutathione would result in a decrease in trypanothione synthesis and as a consequence in TR activity. In this work, besides the enzyme potential inhibition, it also evaluated the influence of those analogs on parasite growth and on its infective capacity. The results showed a significant effect on parasite growth and infectivity and in addition TR activity was highly inhibited. These results are very promising, suggesting a potential use of those analogs as therapeutic drugs against experimental diseases caused by trypanosomatids.

Cloning and expression of trypanothione reductase from a New World Leishmania species.

Trypanothione disulfide (T[S]2), an unusual form of glutathione found in parasitic protozoa, plays a crucial role in the regulation of the intracellular thiol redox balance and in the defense against oxidative stress. Trypanothione reductase (TR) is central to the thiol metabolism in all trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei and Leishmania. Here we report the cloning, sequencing and expression of the TR encoding gene from L. (L.) amazonensis. Multiple protein sequence alignment of all known trypanosomatid TRs highlights the high degree of conservation and illustrates the phylogenetic relationships. A 3D homology model for L. amazonensis TR was constructed based on the previously reported Crithidia fasciculata structure. The purified recombinant TR shows enzyme activity and in vivo expression of the native enzyme could be detected in infective promastigotes, both by Western blotting and by immunofluorescence.

Amidine Derivatives and Leishmania amazonensis: an Evaluation of the Effect of Nitric Oxide (NO) Production on the Parasite-macrophage Interaction

Previous work has demonstrated that N-N′-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes and Trypanosoma evansi trypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infections supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production by L. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present in Leishmania parasites for leishmanicidal drugs.