Involvement of Akt and the antiapoptotic protein Bcl-xL in the inhibition of apoptosis of dendritic cells by Leishmania mexicana.

The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.

Effect of Prophylactic Vaccination with the Membrane-Bound Acid Phosphatase Gene of Leishmania mexicana in the Murine Model of Localized Cutaneous Leishmaniasis.

Leishmaniasis is a disease caused by an intracellular protozoan parasite of the genus Leishmania. Current treatments for leishmaniasis are long, toxic, and expensive and are not available in some endemic regions. Attempts to develop an effective vaccine are feasible, but no vaccine is in active clinical use. In this study, the LmxMBA gene of Leishmania mexicana was selected as a possible vaccine candidate using the reverse vaccinology approach, and the prophylactic effect generated by DNA vaccination with this gene in a murine model of cutaneous leishmaniasis was evaluated. The results showed that prophylactic vaccination with pVAX1::LmxMBA significantly reduced the size of the lesion and the parasitic load on the footpad, compared to the control groups. At a histological level, a smaller number of parasites were evident in the dermis, as well as the absence of connective tissue damage. Mice immunized with plasmid pVAX1::LmxMBA induced immunity characterized by an increase in the IgG2a/IgG1 > 1 ratio and a higher rate of lymphocyte proliferation. In this study, immunization with the plasmid promoted an improvement in the macroscopic and microscopic clinical manifestations of the experimental infection by L. mexicana, with a T helper 1 response characterized by an IgG2a/IgG1 > 1 ratio and high lymphoproliferative response. These findings support immunization with the plasmid pVAX1::LmxMBA as a preventive strategy against cutaneous infection of L. mexicana.

Chemical inhibition of ß-glucocerebrosidase does not affect phagocytosis and early containment of Leishmania by murine macrophages.

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.

Key role of the macrophage microtubule network in the intracellular lifestyle of Leishmania amazonensis.

We conducted a study to decipher the mechanism of the formation of the large communal Leishmania amazonensis-containing parasitophorous vacuole (PV) and found that the macrophage microtubule (MT) network dynamically orchestrates the intracellular lifestyle of this intracellular parasite. Physical disassembly of the MT network of macrophage-like RAW 264.7 cells or silencing of the dynein gene, encoding the MT-associated molecular motor that powers MT-dependent vacuolar movement, by siRNA resulted in most of the infected cells hosting only tight parasite-containing phagosome-like vacuoles randomly distributed throughout the cytoplasm, each insulating a single parasite. Only a minority of the infected cells hosted both isolated parasite-containing phagosome-like vacuoles and a small communal PV, insulating a maximum of two to three parasites. The tight parasite-containing phagosome-like vacuoles never matured, whereas the small PVs only matured to a small degree, shown by the absence or faint acquisition of host-cell endolysosomal characteristics. As a consequence, the parasites were unable to successfully complete promastigote-to-amastigote differentiation and died, regardless of the type of insulation.

Differential Regulation of l-Arginine Metabolism through Arginase 1 during Infection with Leishmania mexicana Isolates Obtained from Patients with Localized and Diffuse Cutaneous Leishmaniasis.

l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of leishmaniasis. Infection with Leishmania mexicana can cause two distinct clinical manifestations: localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In this work, we analyzed in an in vivo model the capacity of two L. mexicana isolates, one obtained from a patient with LCL and the other from a patient with DCL, to regulate the metabolism of l-arginine through Arg-1 and NOS2. Susceptible BALB/c mice were infected with L. mexicana isolates from both clinical manifestations, and the evolution of the infection as well as protein presence and activity of Arg-1 and NOS2 were evaluated. The lesions of mice infected with the DCL isolate were bigger, had higher parasite loads, and showed greater protein presence and enzymatic activity of Arg-1 than the lesions of mice infected with the LCL isolate. In contrast, NOS2 protein synthesis was poorly or not induced in the lesions of mice infected with the LCL or DCL isolate. The immunochemistry analysis of the lesions allowed the identification of highly parasitized macrophages positive for Arg-1, while no staining for NOS2 was found. In addition, we observed in lesions of patients with DCL macrophages with higher parasite loads and stronger Arg-1 staining than those in lesions of patients with LCL. Our results suggest that L. mexicana isolates obtained from patients with LCL or DCL exhibit different virulence or pathogenicity degrees and differentially regulate l-arginine metabolism through Arg-1.

Cytokine regulation of female-macrophage resistance to Leishmania mexicana parasites. Role of IL-12p70.

In previous studies, carried out in humans, we showed that females are resistant to Leishmania mexicana infection. We also showed that 17ß-estradiol (E2) induces killing of parasites inside of murine macrophages. In this work, we compared, for the first time, L mexicana survival inside of male (male BMDM) and female (female BMDM) bone marrow-derived macrophages (BMDM) treated in vitro with E2 or dihydrotestosterone (DHT). We also compared their levels of nitric oxide (NO), interleukin (IL)-6, IL-10, IL-12p70 and tumour necrosis factor (TNF-α). We found that female BMDM are a lot less susceptible to infection as compared with male BMDM. 17ß-estradiol induced killing of most parasites inside of female BMDM. Dihydrotestosterone, on the other hand, induced some parasite killing inside of some infected male BMDM. Interleukin-6 levels were higher in female BMDM treated with either hormone. Neither TNF-α nor IL-10 levels showed significant differences compared with sham controls. Interestingly IL-12p70 was more abundantly produced by sham female BMDM as compared with sham male BMDM. Only female BMDM treated with E2 trigger a robust IL-12p70 production, but it was significantly reduced in male BMDM. This suggests IL-12p70 is an important factor in female-macrophage resistance to L mexicana parasites.

Metabolomic Profile of BALB/c Macrophages Infected with Leishmania amazonensis: Deciphering L-Arginine Metabolism.

BACKGROUND: Leishmaniases are neglected tropical diseases that are caused by Leishmania, being endemic worldwide. L-arginine is an essential amino acid that is required for polyamines production on mammal cells. During Leishmania infection of macrophages, L-arginine is used by host and parasite arginase to produce polyamines, leading to parasite survival; or, by nitric oxide synthase 2 to produce nitric oxide leading to parasite killing. Here, we determined the metabolomic profile of BALB/c macrophages that were infected with L. amazonensis wild type or with L. amazonensis arginase knockout, correlating the regulation of L-arginine metabolism from both host and parasite. METHODS: The metabolites of infected macrophages were analyzed by capillary electrophoresis coupled with mass spectrometry (CE-MS). The metabolic fingerprints analysis provided the dual profile from the host and parasite. RESULTS: We observed increased levels of proline, glutamic acid, glutamine, L-arginine, ornithine, and putrescine in infected-L. amazonensis wild type macrophages, which indicated that this infection induces the polyamine production. Despite this, we observed reduced levels of ornithine, proline, and trypanothione in infected-L. amazonensis arginase knockout macrophages, indicating that this infection reduces the polyamine production. CONCLUSIONS: The metabolome fingerprint indicated that Leishmania infection alters the L-arginine/polyamines/trypanothione metabolism inside the host cell and the parasite arginase impacts on L-arginine metabolism and polyamine production, defining the infection fate.

Macrophage priming is dispensable for NLRP3 inflammasome activation and restriction of Leishmania amazonensis replication.

The NLRP3 inflammasome is activated in response to multiple stimuli and triggers activation of caspase-1 (CASP1), IL-1ß production, and inflammation. NLRP3 activation requires two signals. The first leads to transcriptional regulation of specific genes related to inflammation, and the second is triggered when pathogens, toxins, or specific compounds damage cellular membranes and/or trigger the production of reactive oxygen species (ROS). Here, we assess the requirement of the first signal (priming) for the activation of the NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) infected with Leishmania amazonensis. We found that BMDMs express the inflammasome components NLRP3, ASC, and CASP1 at sufficient levels to enable the assembly and activation of NLRP3 inflammasome in response to infection. Therefore, priming was not required for the formation of ASC specks, CASP1 activation (measured by fluorescent dye FAM-YVAD), and restriction of L. amazonensis replication via the NLRP3 inflammasome. By contrast, BMDM priming was required for CASP1 cleavage (p20) and IL-1ß secretion, because priming triggers robust up-regulation of pro-IL-1ß and CASP11 that are important for efficient processing of CASP1 and IL-1ß. Taken together, our data shed light into the cellular and molecular processes involved in activation of the NLRP3 in macrophages by Leishmania, a process that is important for the outcome of Leishmaniasis.

Effect of Two Different Isolates of Leishmania mexicana in the Production of Cytokines and Phagocytosis by Murine Dendritic Cells.

Species of the genus Leishmania are the causal agents of leishmaniasis, a disease with diametrically different clinical manifestations that have been attributed to the species and host immune response. Some Leishmania species, including Leishmania mexicana, are capable of causing both localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). Therefore, it is possible that intraspecific differences may exist that contribute to the development of distinct clinical forms. Dendritic cells (DC) are important host cells of Leishmania spp. parasites, and cytokine production and phagocytosis upon infection with the parasite are significant for the outcome of the disease. In the present study we analyzed the production of IL-12, TNF-α, and IL-10 by DC infected with L. mexicana amastigotes isolated from a patient with LCL (amastigote = Lac) and from a patient with DCL (amastigote = Diact) by murine DC. Furthermore, we compared the frequency of phagocytosis of L. mexicana amastigotes of each isolate by fluorescence and optical microscopy and by flow cytometry. We show that the infection of DC with Diact amastigotes elicited the secretion of IL-10, TNF-α, and IL-12 by DC to a major extent as compared to the infection with Lac amastigotes. On the other hand, Lac and Diact amastigotes were similarly phagocytosed by DC, but interestingly there were more vacuoles in DC infected with Diact amastigotes. Our results suggest that isolates from a same species of Leishmania, such as L. mexicana, with different degrees of virulence according to the clinical manifestation they cause, differ in their capacity to elicit cytokine production and form vacuoles in DC.

Development of Leishmania mexicana in Lutzomyia longipalpis in the absence of sugar feeding

The leishmaniases are caused by Leishmania parasites and transmitted through the bites of phlebotomine sand flies. During parasite development inside the vector's midgut, promastigotes move towards the stomodeal valve, a mechanism that is crucial for transmission. It has been reported that the sugar meal acquired by sand flies during feeding between bloodmeals is essential for the development and migration of parasites. We demonstrated that the distribution of Leishmania mexicana parasites was affected by the sugar meals obtained by the sand flies. Promastigote migration towards the cardia region seems to be only partially based on the stimuli provided by sugar molecules. In the absence of sugars, significant amounts of parasites developed in the hindgut. In addition, sugar meals were important for the survival of sand flies, especially during blood digestion, presumably supporting their energy requirements.

Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays.

The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are toxic, expensive, difficult to administer, and resistance to them is emerging. New therapeutics are urgently needed, however, screening the infective amastigote form of the parasite is challenging. Only certain species can be differentiated into axenic amastigotes, and compound activity against these does not always correlate with efficacy against the parasite in its intracellular niche. Methods used to assess compound efficacy on intracellular amastigotes often rely on microscopy-based assays. These are laborious, require specialist equipment and can only determine parasite burden, not parasite viability. We have addressed this clear need in the anti-leishmanial drug discovery process by producing a transgenic L. mexicana cell line that expresses the luciferase NanoLuc-PEST. We tested the sensitivity and versatility of this transgenic strain, in comparison with strains expressing NanoLuc and the red-shifted firefly luciferase. We then compared the NanoLuc-PEST luciferase to the current methods in both axenic and intramacrophage amastigotes following treatment with a supralethal dose of Amphotericin B. NanoLuc-PEST was a more dynamic indicator of cell viability due to its high turnover rate and high signal:background ratio. This, coupled with its sensitivity in the intramacrophage assay, led us to validate the NanoLuc-PEST expressing cell line using the MMV Pathogen Box in a two-step process: i) identify hits against axenic amastigotes, ii) screen these hits using our bioluminescence-based intramacrophage assay. The data obtained from this highlights the potential of compounds active against M. tuberculosis to be re-purposed for use against Leishmania. Our transgenic L. mexicana cell line is therefore a highly sensitive and dynamic system suitable for Leishmania drug discovery in axenic and intramacrophage amastigote models.

Apoptotic induction induces Leishmania aethiopica and L. mexicana spreading in terminally differentiated THP-1 cells.

Leishmaniasis develops after parasites establish themselves as amastigotes inside mammalian cells and start replicating. As relatively few parasites survive the innate immune defence, intracellular amastigotes spreading towards uninfected cells is instrumental to disease progression. Nevertheless the mechanism of Leishmania dissemination remains unclear, mostly due to the lack of a reliable model of infection spreading. Here, an in vitro model representing the dissemination of Leishmania amastigotes between human macrophages has been developed. Differentiated THP-1 macrophages were infected with GFP expressing Leishmania aethiopica and Leishmania mexicana. The percentage of infected cells was enriched via camptothecin treatment to achieve 64·1 ± 3% (L. aethiopica) and 92 ± 1·2% (L. mexicana) at 72 h, compared to 35 ± 4·2% (L. aethiopica) and 36·2 ± 2·4% (L. mexicana) in untreated population. Infected cells were co-cultured with a newly differentiated population of THP-1 macrophages. Spreading was detected after 12 h of co-culture. Live cell imaging showed inter-cellular extrusion of L. aethiopica and L. mexicana to recipient cells took place independently of host cell lysis. Establishment of secondary infection from Leishmania infected cells provided an insight into the cellular phenomena of parasite movement between human macrophages. Moreover, it supports further investigation into the molecular mechanisms of parasites spreading, which forms the basis of disease development.

Effect of 1,2,3-triazole salts, non-classical bioisosteres of miltefosine, on Leishmania amazonensis.

Here, we report the effect of new non-classical bioisosteres of miltefosine on Leishmania amazonensis. Fifteen compounds were synthesized and the compound dhmtAc, containing an acetate anion, a side chain of 10 carbon atoms linked to N-1 and a methyl group linked to N-3, showed high and selective biological activity against L. amazonensis. On the intracellular amastigotes, stages of the parasite related to human disease, the IC50 values were near or similar to the 1.0µM (0.9, 0.8 and 1.0µM on L. amazonensis-WT, and two transgenic L. amazonensis expressing GFP and RFP, respectively), being more active than miltefosine. Furthermore, dhmtAc did not show toxic effects on human erythrocytes and macrophages (CC50=115.9µM) being more destructive to the intracellular parasites (selectivity index>115). Promastigotes and intramacrophage amastigotes treated with dhmtAc showed low capacity for reversion of the effect of the compound. A study of the mechanism of action of this compound showed some features of metazoan apoptosis, including cell volume decreases, loss of mitochondrial membrane potential, ROS production, an increase in the intracellular lipid bodies, in situ labeling of DNA fragments by TUNEL labeling and phosphatidylserine exposure to the outerleaflet of the plasma membrane. In addition, the plasma membrane disruption, revealed by PI labeling, suggests cell death by necrosis. No increase in autophagic vacuoles formation in treated promastigotes was observed. Taken together, the data indicate that the bioisostere of miltefosine, dhmtAc, has promising antileishmanial activity that is mediated via apoptosis and necrosis.

Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα and NFκB activation.

Cutaneous leishmaniasis (CL) is the most common form of the leishmaniasis in humans. Ulcerative painless skin lesions are predominant clinical features of CL. Wider data indicate pain accompanies human leishmaniasis, out with areas of painless ulcerative lesions per se. In rodents, Leishmania (L.) major infection induces nociceptive behaviors that correlate with peripheral cytokine levels. However, the role of the spinal cord in pain processing after Leishmania infection has not been investigated. Balb/c mice received intraplantar (i.pl.) injection of Leishmania (L). amazonensis and hyperalgesia, edema, parasitism, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression, and NFκB activation were evaluated. The effects of intrathecal (i.t.) injection of morphine, TNFα, TNFα inhibitors (etanercept and adalimumab) and NFκB inhibitor (PDTC) were investigated. The present study demonstrates that Leishmania (L.) amazonensis infection in balb/c mice induces chronic mechanical and thermal hyperalgesia in an opioid-sensitive manner. Spinal cord TNFα mRNA expression increased in a time-dependent manner, peaking between 30 and 40 days after infection. At the peak of TNFα mRNA expression (day 30), there was a concomitant increase in TNFR1 and TNFR2 mRNA expression. TNFα i.t. injection enhanced L. (L.) amazonensis-induced hyperalgesia. Corroborating a role for TNFα in L. (L.) amazonensis-induced hyperalgesia, i.t. treatment with the TNFα inhibitors, etanercept and adalimumab inhibited the hyperalgesia. L. (L.) amazonensis also induced spinal cord activation of NFκB, and PDTC (given i.t.), also inhibited L. (L.) amazonensis-induced hyperalgesia, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression. Moreover, L. (L.) amazonensis-induced spinal cord activation of NFκB was also inhibited by etanercept and adalimumab as well as PDTC i.t. TREATMENT: These results demonstrate that endogenous spinal cord TNFα and NFκB activation contribute to L. (L.) amazonensis-induced hyperalgesia in mice. Thus, spinal cord TNFα and NFκB are potential therapeutic targets for Leishmania infection-induced pain.

Anti-Leishmania amazonensis activity of Serjania lethalis A. St.-Hil.

Extracts of Serjania lethalis A. St.-Hil leaves and stems were tested in order to identify potential agents against Leishmania amazonensis. The hexane fraction (HF) and dichloromethane subfractions (DDF and MDF) showed leishmanicidal effect. The anti-promastigote IC50 values were 10.29 (HF), 11.41 (DDF) and 28.33µg/mL (MDF); whereas those against amastigote were 7.2 (HF), 8.1 (DDF) and 6.5µg/mL (MDF). Among the fractions and subfractions assayed, only HF altered the cell cycle of the parasite, increasing 3-fold the number of cells in the sub-G0/G1 phase. HF also changed the parasite mitochondrial membrane potential (ΔΨm) and the percentage of annexin-V-propidium iodide positive promastigotes. Our evaluations of the IC50 values showed that HF, DDF and MDF decreased NO production in infected macrophages stimulated with IFN-γ and LPS. Moreover, HF increased the production of TNF-α in Leishmania infected macrophages. This paper reports for the first time the leishmanicidal activity of extracts and fractions of Serjania lethalis leaves and also characterizes its leishmanicidal and immunomodulatory properties.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics.

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Leishmania mexicana promastigotes down regulate JNK and p-38 MAPK activation: Role in the inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells.

Dendritic cells (DC) are one of the principal host cells of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. We have previously shown that the infection of monocyte-derived dendritic cells (moDC) with Leishmania mexicana inhibits campthotecin-induced apoptosis. Nevertheless, the mechanisms involved in the inhibition of apoptosis of dendritic cells by Leishmania have not been established. Mitogen-activated protein kinases (MAPK) are key participants in the process of apoptosis and different species of Leishmania have been shown to regulate these kinases. In the present study, we analyzed the effect of L. mexicana promastigotes in the activation of JNK and p38 MAP kinase and their participation in the inhibition of apoptosis. The infection of moDC with L. mexicana promastigotes diminished significantly the phosphorylation of the MAP kinases JNK and p38. The inhibition of both kinases diminished DNA fragmentation, but in a major extent was the reduction of DNA fragmentation when JNK was inhibited. The capacity of L. mexicana promastigotes to diminish MAP kinases activation is probably one of the strategies employed to delay apoptosis induction in the infected moDC and may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

Basal body multipotency and axonemal remodelling are two pathways to a 9+0 flagellum.

Eukaryotic cilia/flagella exhibit two characteristic ultrastructures reflecting two main functions; a 9+2 axoneme for motility and a 9+0 axoneme for sensation and signalling. Whether, and if so how, they interconvert is unclear. Here we analyse flagellum length, structure and molecular composition changes in the unicellular eukaryotic parasite Leishmania during the transformation of a life cycle stage with a 9+2 axoneme (the promastigote) to one with a 9+0 axoneme (the amastigote). We show 9+0 axonemes can be generated by two pathways: by de novo formation and by restructuring of existing 9+2 axonemes associated with decreased intraflagellar transport. Furthermore, pro-basal bodies formed under conditions conducive for 9+2 axoneme formation can form a 9+0 axoneme de novo. We conclude that pro-centrioles/pro-basal bodies are multipotent and not committed to form either a 9+2 or 9+0 axoneme. In an alternative pathway structures can also be removed from existing 9+2 axonemes to convert them to 9+0.

Effect of aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing amino acids on Leishmania spp. chemotaxis.

In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10-12 m), L-tryptophan (10-11 m), L-glutamine and L-glutamic acid (10-6 m), induced positive chemotactic responses, while L-alanine (10-7 m), L-methionine (10-11 and 10-7 m), L-tryptophan (10-11 m), L-glutamine (10-12 m) and L-glutamic acid (10-9 m) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10-11 m, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.