Anti-Leishmania amazonensis activity of the terpenoid fraction from Eugenia pruniformis leaves.

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania and includes cutaneous, mucocutaneous and visceral clinical forms. Drugs currently available for leishmaniasis treatment present high toxicity, and development of parasite resistance. Plants constitute an important source of compounds with leishmanicidal potential. This study aimed to evaluate the anti-Leishmania amazonensis activity of the terpenoid fraction of Eugenia pruniformis leaves (TF-EpL). TF-EpL was active against the promastigote and intracellular amastigote forms of L. amazonensis with IC50(24h) value of 43.60µg/mL and 44.77µg/mL, respectively. TF-EpL altered the cell cycle of the parasite, increasing 2.32-fold the cells in the Sub-G0/G1 phase. TF-EpL also changed the ΔΨm and increased ROS and the number of annexin-V-PI positive promastigotes, which suggests incidental death. ß-sitosterol, ursolic acid, corosolic acid and asiatic acid were isolated from TF-EpL. The results showed the antileishmanial activity of E. pruniformis terpenoids and its potential for further studies as a source of new drugs for leishmaniasis.

Endocytosis and Exocytosis in Leishmania amazonensis Are Modulated by Bromoenol Lactone.

In the protozoan pathogen Leishmania, endocytosis, and exocytosis occur mainly in the small area of the flagellar pocket membrane, which makes this parasite an interesting model of strikingly polarized internalization and secretion. Moreover, little is known about vesicle recognition and fusion mechanisms, which are essential for both endo/exocytosis in this parasite. In other cell types, vesicle fusion events require the activity of phospholipase A2 (PLA2), including Ca2+-independent iPLA2 and soluble, Ca2+-dependent sPLA2. Here, we studied the role of bromoenol lactone (BEL) inhibition of endo/exocytosis in promastigotes of Leishmania amazonensis. PLA2 activities were assayed in intact parasites, in whole conditioned media, and in soluble and extracellular vesicles (EVs) conditioned media fractions. BEL did not affect the viability of promastigotes, but reduced the differentiation into metacyclic forms. Intact parasites and EVs had BEL-sensitive iPLA2 activity. BEL treatment reduced total EVs secretion, as evidenced by reduced total protein concentration, as well as its size distribution and vesicles in the flagellar pocket of treated parasites as observed by TEM. Membrane proteins, such as acid phosphatases and GP63, became concentrated in the cytoplasm, mainly in multivesicular tubules of the endocytic pathway. BEL also prevented the endocytosis of BSA, transferrin and ConA, with the accumulation of these markers in the flagellar pocket. These results suggested that the activity inhibited by BEL, which is one of the irreversible inhibitors of iPLA2, is required for both endocytosis and exocytosis in promastigotes of L. amazonensis.

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

Trans- ß -Caryophyllene: An Effective Antileishmanial Compound Found in Commercial Copaiba Oil (Copaifera spp.).

This study investigated the leishmanicidal activity against Leishmania amazonensis of four commercial oils from Copaifera spp. named as C1, C2, C3, and C4, the sesquiterpene and diterpene pools obtained from distilling C4, and isolated ß -caryophyllene (CAR). Copaiba oils chemical compositions were analyzed by gas chromatography and correlated with biological activities. Diterpenes-rich oils C2 and C3 showed antipromastigote activity. Sesquiterpenes-rich C1 and C4, and isolated CAR presented a dose-dependent activity against intracellular amastigotes, with IC50s of 2.9 µ g/mL, 2.3 µ g/mL, and 1.3 µ g/mL (6.4 µ M), respectively. Based on the highest antiamastigote activity and the low toxicity to the host cells, C4 was steamdistillated to separate pools of sesquiterpenes and diterpenes. Both pools were less active against L. amazonensis and more toxic for the macrophages than the whole C4 oil. The leishmanicidal activity of C3 and C4 oils, as well as C4 fractions and CAR, appears to be independent of nitric oxide production by macrophages. This study pointed out ß -caryophyllene as an effective antileishmanial compound and also to its role as potential chemical marker in copaiba oils or fractions derived thereof, aiming further development of this rainforest raw material for leishmaniasis therapy.

Different secreted phosphatase activities in Leishmania amazonensis.

Leishmania has strong acid phosphatase activity on the external surface of the plasma membrane and secreted into the extracellular milieu. Secreted acid phosphatase (sAcP), which is the most abundant secreted protein of Leishmania, is also a virulence factor that plays a role in vertebrate infection and survival in sand flies. In this study, we characterized the secreted phosphatase activities in Leishmania amazonensis. Both acidic and alkaline secreted phosphatase activities were observed with ß-glycerophosphate and p-nitrophenyl phosphate (p-NPP) hydrolysis and were inhibited with sodium tartrate and sodium orthovanadate. Cytochemical labeling revealed a significant difference in the localization of the electron-dense precipitates depending on the substrate. ß-Glycerophosphate electron-dense precipitates were concentrated on both the cell surface and flagellar pocket, whereas p-NPP labeling occurred primarily within intracellular organelles. Orthovanadate-treated metacyclic promastigotes were less infective and were confined to a tight parasitophorous vacuole (PV), which is not characteristic of this Leishmania species. Based on the results, we characterized the presence of different secreted phosphatase activities in L. amazonensis, the influence of the substrate in cytochemical labeling, and the potential involvement of secreted phosphatase activity in both PV maturation and amastigote survival.

Leishmanicidal activity of Himatanthus sucuuba latex against Leishmania amazonensis.

Himatanthus sucuuba (HsL) latex exhibited a potent leishmanicidal activity against intracellular amastigotes of Leishmania amazonensis, a causative agent of cutaneous leishmaniasis. HsL inhibited intracellular amastigote growth in a dose-dependent manner (IC(50)=15.7microg/mL). Moreover, HsL increased nitric oxide (NO) and Tumor Nuclear Factor-alpha (TNF-alpha) and decreased Transforming Growth Factor-beta (TGF-beta) production in macrophages. As assessed by plasma membrane integrity and mitochondrial activity, HsL showed low toxicity for host macrophages. HsL in vivo was active by the oral route, reducing the parasite load in established footpad lesions after only five doses. In summary, these findings support HsL as an interesting candidate for further evaluations regarding its potential application as a therapeutical agent against Leishmania.

Novel role for the double-stranded RNA-activated protein kinase PKR: modulation of macrophage infection by the protozoan parasite Leishmania.

The evolution of Leishmania infection depends on the balance between microbicidal and suppressor macrophage functions. Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a classic antiviral protein, is able to regulate a number of signaling pathways and macrophage functions. We investigated the possible role of PKR in the modulation of Leishmania infection. Our data demonstrated that Leishmania amazonensis infection led to PKR activation and increased PKR levels. Consistently, in macrophages from PKR knockout 129Sv/Ev mice and RAW-264.7 cells stably expressing a dominant-negative (DN) construct of PKR (DN-PKR), L. amazonensis infection was strongly reduced. The treatment of infected macrophages with the synthetic double-stranded RNA poly(I:C), a potent PKR inductor, increased L. amazonensis intracellular proliferation. This effect was reversed by 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, as well as by the expression of DN-PKR. NO release induced by dsRNA treatment was inhibited by L. amazonensis through NF-kappaB modulation. PKR activation induced by dsRNA also resulted in IL-10 production, whose neutralization with specific antibody completely abrogated L. amazonensis proliferation. Our data demonstrated a new role of PKR in protozoan parasitic infection through IL-10 modulation.

NF-kappaB-mediated repression of iNOS expression in Leishmania amazonensis macrophage infection.

Host invasion by pathogens is frequently associated with the activation of nuclear factor kappaB (NF-kappaB), which modulates the expression of genes involved in the immunological response of the host. However, pathogens may also subvert these mechanisms to secure their survival. We describe the effect of Leishmania amazonensis infection on NF-kappaB transcriptional factor activation in macrophages and the subsequent reduction in inducible nitric oxide synthase (iNOS) expression. L. amazonensis promastigote infection activates the p50/p50 NF-kappaB complex, a classic transcriptional repressor. Interestingly, L. amazonensis promotes the change of the classical p65/p50 NF-kappaB dimer induced by LPS, leading to the p50/p50 NF-kappaB complex activation in macrophages stimulated with LPS. Moreover, this parasite promotes the reduction of p65 total levels in infected macrophages. All these effects contribute to the observation that this parasite is able to restrain the NF-kappaB-dependent transcriptional activity induced by LPS. Strikingly, L. amazonensis reduces the mRNA levels of the iNOS in addition to protein expression and the production of nitric oxide in LPS-stimulated macrophages. Accordingly, as revealed by reporter-gene assays, L. amazonensis-induced iNOS repression requires NF-kappaB sites in the iNOS promoter region. In summary, our results suggest that L. amazonensis has developed an adaptive strategy to escape from host defense by activating the NF-kappaB repressor complex p50/p50. The activation of this specific host transcriptional response negatively regulates the expression of iNOS, favoring the establishment and success of L. amazonensis infection.