Crystal structure of Q4D6Q6, a conserved kinetoplastid-specific protein from Trypanosoma cruzi.

Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ßßαßßαßß topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction.

Comparative study of different forms of Jellein antimicrobial peptide on Leishmania parasite.

Typically, antimicrobial peptides (AMPs) are short positive charged peptides serving a key role in innate immunity as well as antimicrobial activity. Discovering novel therapeutic agents is considered as an undeniable demand due to increasing microbial species with antibiotic resistance. In this direction, the unique ability of AMPs to modulate immune responses highlighted them as novel drug candidates in the field of microbiology. Patients affected by leishmaniasis; a neglected tropical disease, confront serious problems for their treatment including resistance to common drugs as well as toxicity and high cost of therapy. So, there is a need for development of new drug candidates to control the diseases. Jellein, a peptide derived from royal jelly of honeybee has been shown to have promising effect against several bacterial and fungal species. In current study, anti-leishmanial effect of Jellein and its lauric acid conjugated form was investigated against two forms of Leishmania major (L. major) parasite. Moreover, cytotoxic effect of these peptides was studied in THP1 cell line and human Red Blood Cells (RBCs). Furthermore, the mechanism of action of peptides on L. major promastigotes was assessed through different methods. The results demonstrated that, conjugation of lauric acid to Jellein not only had no effect on the elevation of antimicrobial activity but also halted it completely. Moreover, Jellein caused a limitation in the number of L. major promastigotes by pore formation as well as changing the membrane potential rather than induction of apoptosis or activation of caspases.

Effectiveness of amiodarone in treatment of cutaneous leishmaniasis caused by Leishmania major.

Development of new chemotherapeutic agents is an essential issue in the treatment and control of a disease. This study aimed to evaluate the anti-leishmanial activity of amiodarone, an antiarrhythmic class III drug, against Leishmania major, the most prevalent etiological agent of cutaneous leishmaniasis in the old world. The proliferation of promastigotes and intracellular amastigotes in the absence or presence of amiodarone was estimated, in an in vitro study. For in vivo study, five weeks after infection of BALB/c mice with L. major, when the lesions appeared at the injection site, the mice were divided into four groups (n = 6 each); treatment was conducted for 28 consecutive days with vehicle, amiodarone at 40 mg/kg orally and glucantime at 60 mg/kg intraperitoneally. Therapy with amiodarone reduced the size of lesions compared to the untreated group after 12 days. Amiodarone decreased the parasite load and inflammatory responses, particularly the macrophages containing amastigotes, and enhanced granulation tissue formation in the dermis and subcutaneous area. The Tumor necrosis factor-α and Interleukin-6 levels were significantly lower in the cell culture supernatants of the inguinal lymph node in the amiodarone treated group compared to the vehicle and untreated groups. Amiodarone significantly increased the activity of glutathione peroxidase in comparison to the vehicle and untreated groups but did not affect the plasma levels of superoxide dismutase, malondialdehyde, adiponectin, and ferric reducing ability of plasma. Therefore, the anti- L. major activity and immunomodulatory effects of amiodarone reduced the parasitic load and enhanced wound healing in cutaneous leishmaniasis in BALB/c mice. Amiodarone reduced the lesion surface area, but it did not cure it completely.

In vitro evaluation of the anti-leishmanial activity and toxicity of PK11195.

BACKGROUND: Leishmaniasis, one of the most neglected diseases, is a serious public health problem in many countries, including Brazil. Currently available treatments require long-term use and have serious side effects, necessitating the development of new therapeutic interventions. Because translocator protein (TSPO) levels are reduced in Leishmania amazonensis-infected cells and because this protein participates in apoptosis and immunomodulation, TSPO represents a potential target for Leishmania chemotherapy. The present study evaluated PK11195, a ligand of this protein, as an anti-leishmanial agent. OBJECTIVE: To evaluate the leishmanicidal activity of PK11195 against L. amazonensis in infected CBA mouse macrophages in vitro. METHODS: The viability of axenic L. amazonensis, Leishmania major, and Leishmania braziliensis promastigotes was assessed after 48 h treatment with PK11195 (0.2-400 µM). Additionally, intracellular parasite viability was evaluated to determine IC50 values and the number of viable parasites in infected macrophages treated with PK11195 (50-100 µM). Infected macrophages were then treated with PK11195 (25-100 µM) to determine the percentage of L. amazonensis-infected cells and the number of parasites per infected cell. Electron microscopy was used to investigate morphological changes caused by PK11195. The production of free oxygen radicals, nitric oxide, and pro-inflammatory cytokines was also evaluated in infected macrophages treated with PK11195 and primed or not primed with IFN-γ. FINDINGS: Median IC50 values for PK11195 were 14.2 µM for L. amazonensis, 8.2 µM for L. major, and 3.5 µM for L. braziliensis. The selective index value for L. amazonensis was 13.7, indicating the safety of PK11195 for future testing in mammals. Time- and dose-dependent reductions in the percentage of infected macrophages, the number of parasites per infected macrophage, and the number of viable intracellular parasites were observed. Electron microscopy revealed some morphological alterations suggestive of autophagy. Interestingly, MCP-1 and superoxide levels were reduced in L. amazonensis-infected macrophages treated with PK11195. MAIN CONCLUSIONS: PK11195 causes the killing of amastigotes in vitro by mechanisms independent of inflammatory mediators and causes morphological alterations within Leishmania parasites, suggestive of autophagy, at doses that are non-toxic to macrophages. Thus, this molecule has demonstrated potential as an anti-leishmanial agent.

Therapeutic efficacy of stable analogues of vasoactive intestinal peptide against pathogens.

Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide recently identified as a potential antimicrobial peptide. To overcome the metabolic limitations of VIP, we modified the native peptide sequence and generated two stable synthetic analogues (VIP51 and VIP51(6-30)) with better antimicrobial profiles. Herein we investigate the effects of both VIP analogues on cell viability, membrane integrity, and ultrastructure of various bacterial strains and Leishmania species. We found that the two VIP derivatives kill various non-pathogenic and pathogenic Gram-positive and Gram-negative bacteria as well as the parasite Leishmania major through a mechanism that depends on the interaction with certain components of the microbial surface, the formation of pores, and the disruption of the surface membrane. The cytotoxicity of the VIP derivatives is specific for pathogens, because they do not affect the viability of mammalian cells. Docking simulations indicate that the chemical changes made in the analogues are critical to increase their antimicrobial activities. Consequently, we found that the native VIP is less potent as an antibacterial and fails as a leishmanicidal. Noteworthy from a therapeutic point of view is that treatment with both derivatives increases the survival and reduces bacterial load and inflammation in mice with polymicrobial sepsis. Moreover, treatment with VIP51(6-30) is very effective at reducing lesion size and parasite burden in a model of cutaneous leishmaniasis. These results indicate that the VIP analogues emerge as attractive alternatives for treating drug-resistant infectious diseases and provide key insights into a rational design of novel agents against these pathogens.

Distinct roles in autophagy and importance in infectivity of the two ATG4 cysteine peptidases of Leishmania major.

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.

Aziridine-2,3-dicarboxylate-based cysteine cathepsin inhibitors induce cell death in Leishmania major associated with accumulation of debris in autophagy-related lysosome-like vacuoles.

The papain-like cysteine cathepsins expressed by Leishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)(2)]-based inhibitors, Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)(2) (compound 13b) and Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)(2) (compound 13e), reduced the growth and viability of Leishmania major and the infection rate of macrophages while not showing cytotoxicity against host cells. In the present study, we characterized the mode of action of inhibitors 13b and 13e in L. major. Both compounds targeted leishmanial cathepsin B-like cysteine cathepsin cysteine proteinase C, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors. Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential, and increased DNA fragmentation. Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes. The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death in Leishmania by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles.

Leishmania major: disruption of signal peptidase type I and its consequences on survival, growth and infectivity.

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival. The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.

Delta24(25)-sterol methenyltransferase: intracellular localization and azasterol sensitivity in Leishmania major promastigotes overexpressing the enzyme.

Trypanosomatids contain predominantly ergostane-based sterols, which differ from cholesterol, the main sterol in mammalian cells, in the presence of a methyl group in the 24 position. The methylation is initiated by S-adenosyl-L-methionine:Delta(24 (25))-sterol methenyltransferase, an enzyme present in protozoa, but absent in mammals. The importance of this enzyme is underscored by its potential as a drug target in the treatment of the leishmaniases. Here, we report studies concerning the intracellular distribution of sterol methenyltransferase in Leishmania major promastigotes and overexpressing cells using a specific antibody raised against highly purified recombinant protein. It was found by immunofluorescence and electron microscopy studies that in L. major wild-type cells sterol methenyltransferase was primarily associated to the endoplasmic reticulum. In addition to this location, the protein was incorporated into translucent vesicles presumably of the endocytic pathway. We also found in this study that cells overproducing the enzyme do not have increased resistance to the sterol methenyltransferase inhibitor 22, 26 azasterol.

Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes.

This study was undertaken to analyze the effect of an engineered, killer decapeptide (KP) on Leishmania major and Leishmania infantum promastigotes. The KP was synthesized on the basis of the sequence of a recombinant, single-chain anti-idiotypic antibody acting as a functional internal image of a yeast killer toxin. The evaluation of in vitro inhibitory activity of KP on L. major and L. infantum, release of intracellular green fluorescent protein (GFP) molecules by L. major, DNA fragmentation, and ultrastructural analysis (TEM) of L. infantum upon KP treatment were performed. KP presented antiproliferative and leishmanicidal activity with LC(50)/1 day of 58 and 72 microM for L. major and L. infantum, respectively. A dose-dependent decrease in proliferation and increase of killing of promastigotes was seen after KP treatment. No DNA fragmentation in L. infantum promastigotes or release of intracellular GFP molecules on peptide treatment of a GFP expressing L. major clone was demonstrated. Moreover the plasma-membrane was not disrupted, but, by TEM analysis, intracellular damage was observed.

The ultrastructure of the parasitophorous vacuole formed by Leishmania major.

Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes.

Cutting edge: neutrophil granulocyte serves as a vector for Leishmania entry into macrophages.

Macrophages (MF) are the final host cells for multiplication of the intracellular parasite Leishmania major (L. major). However, polymorphonuclear neutrophil granulocytes (PMN), not MF, are the first leukocytes that migrate to the site of infection and encounter the parasites. Our previous studies indicated that PMN phagocytose but do not kill L. major. Upon infection with Leishmania, apoptosis of human PMN is delayed and takes 2 days to occur. Infected PMN were found to secrete high levels of the chemokine MIP-1beta, which attracts MF. In this study, we investigated whether MF can ingest parasite-infected PMN. We observed that MF readily phagocytosed infected apoptotic PMN. Leishmania internalized by this indirect way survived and multiplied in MF. Moreover, ingestion of apoptotic infected PMN resulted in release of the anti-inflammatory cytokine TGF-beta by MF. These data indicate that Leishmania can misuse granulocytes as a "Trojan horse" to enter their final host cells "silently" and unrecognized.

Leishmania major proteophosphoglycans exist as membrane-bound and soluble forms and localise to the cell membrane, the flagellar pocket and the lysosome.

The Leishmania proteophosphoglycan belongs to a family of heterogeneous polypeptides of unusual composition and structure. Here we demonstrate the presence in the parasite of a membrane-bound hydrophobic form of proteophosphoglycan, in addition to the previously described water-soluble form secreted into the culture medium. Phosphatidylinositol-specific phospholipase C treatment of the hydrophobic form of proteophosphoglycan converted it into a water-soluble form, confirming that it has a functional glycosylphosphatidylinositol-anchor, compatible with it being the product of the proteophosphoglycan1 gene. Immunofluorescence, immunoelectron microscopy and surface labelling showed that proteophosphoglycan expression was variable in individual cells but that it was present on the surface of both amastigotes and promastigotes, in the flagellar pocket, in endosomes and in the multi-vesicular tubule which is the newly described lysosome.

Inhibition of intracellular proliferation of Leishmania parasites in vitro and suppression of skin lesion development in BALB/c mice by a novel lipid A analog (ONO-4007).

A synthetic lipid A analog (ONO-4007) exhibits antileishmanial activity by activating Leishmania-infected macrophages in experimental leishmaniasis. In the present in vitro study, ONO-4007 at concentrations between 0.01 and 1.00 mg/mL markedly inhibited the proliferation of Leishmania major and L. amazonensis promastigotes. Ultrastructurally, L. major-infected macrophages showed degenerated intracellular amastigotes after exposure to ONO-4007. Leishmania-infected macrophages treated with ONO-4007 showed poorly developed parasitophorous vacuoles. High levels of tumor necrosis factor-alpha were induced by ONO-4007 in Leishmania-infected macrophages. In this in vivo study, L. amazonensis-infected BALB/c mice were treated with a dose of 30 mg/kg of ONO-4007 by perilesional and peritoneal injections. The skin lesion size was assessed before treatment with ONO-4007 and at eight weeks after injection. The lesion size was significantly suppressed in mice perilesionally injected with ONO-4007 (P < 0.01) compared with the controls. The data from our present in vitro and in vivo studies indicate that ONO-4007 has an antileishmanial effect.

Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites.

Protozoan parasites Leishmania alternate between a flagellated promastigote form and an amastigote form. In their mammalian hosts, Leishmania survive and multiply in macrophages. Both forms can be internalized by these host cells at different stages of the infectious process and eventually establish themselves within parasitophorous vacuoles exhibiting phagolysosomal properties. To determine whether the biogenesis of these organelles differs according to the parasitic stage used to initiate infection, we compared their formation kinetics after phagocytosis of either metacyclic promastigotes or amastigotes of L. amazonensis or of L. major by mouse bone-marrow-derived macrophages pre-exposed or not to IFN-gamma. After 10 minutes of contact, an accumulation of F-actin was observed around the promastigotes and amatigotes undergoing phagocytosis or those that had already been internalized. This accumulation was transient and rapidly disappeared at later times. At 30 minutes, most of the promastigotes were located in long, narrow organelles that were exactly the same shape as the parasites. The latter were elongated with their cell bodies near to the macrophage nucleus and their flagella towards the periphery. This suggests that promastigote phagocytosis mainly occurs in a polarized manner, with the cell body entering the macrophages first. Most, if not all, of the phagocytosed promastigotes were located in organelles that rapidly acquired phagolysosomal properties. At 30 minutes, lamp-1, macrosialin, cathepsins B and D were detected in 70-98% of these compartments and about 70% of them were surrounded by rab7p. These late endosome/lysosome 'markers' were recruited through fusion with late endocytic compartments. Indeed, when late endosomes/lysosomes were loaded with fluorescein dextran, 81-98% of the promastigote-harbouring compartments contained the endocytic tracer 30 minutes after infection. Electron microscopy of infected macrophages previously loaded with peroxidase confirmed that the phagosomes rapidly fused with late endocytic compartments. When the amastigote stage of L. amazonensis was used to initiate infection, the kinetics of acquisition of the different late endosome/lysosome 'markers' by the phagosomes were similar to those measured after infection with metacyclics. However, more rab7p(+)-phagosomes were observed at early time points (e.g. 90% were rab7p(+) at 30 minutes). The early endosome 'markers', EEA1 and the transferrin receptor, were hardly detected in parasite-containing compartments regardless of the parasitic stage used to infect macrophages and the time after infection. In conclusion, both metacyclic- and amastigote-containing phagosomes fuse with late endosomes/lysosomes within 30 minutes. However, with L. amazonensis, the time required for the formation of the huge parasitophorous vacuoles, which are characteristic of this species, was much shorter after infection with amastigotes than after infection with metacyclic promastigotes. This indicates that the initial fusions with late endosomes/lysosomes are followed by a stage-specific sequence of events.

Rastreamento da localização do glicoconjugado LPG na cinética da interação L. Major/Macrófago

A leishmaniose é uma doença endêmica que está distribuída por todo o mundo e é causada pelo protozoário do gênero Leishmania. Este parasita existe em duas formas: promastigotas (flagelados) no intestino do inseto vetor, e amastigotas (intracelulares) que vivem e se multiplicam dentro de macrófagos no hospedeiro vertebrado. As formas promastigotas, quando são inoculadas no hospedeiro vertebrado pelo inseto vetor, infectam macrófagos. Estas células são infectadas pelo parasita após um processo de reconhecimento mútuo entre as superficies do protozoário e da célula hospedeira. Questões cruciais sobre como as moléculas da superficie do parasita participam desse processo têm sido exaustivamente estudadas e, aos poucos respondidas. Sabe-se que in vitro todos os promastigotas são capazes de se aderir e entrar na células hospedeiras. Porém, apenas os que passaram pelo processo de metaciclogênese, os metacíclicos, são infectantes. Estudos demonstraram que nesse processo de diferenciação ocorrem modificações na mais abundante molécula expressa na superfície do parasita, o lipofosfoglicano (LPG). Nesse estudo, usamos o anticorpo 79.3, marcador de LPG, a fim de observar a distribuição dessa molécula durante a fase inicial de adesão e posterior interiorização de formas promastigotas por macrófago peritonial de camundongo. Por imunofluorescência, foi possível observar a marcação das superficies dos parasitas aderidos aos macrófagos. Comparamos então a entrada e a destruição dos procíclicos (promastigotas não infectantes) com a sobrevivência e manutenção da infecção fato sugere que a molécula é secretada durante a interação parasitacélula.

A molécula de LPG foi constante e uniformemente identificada em toda a superficie do parasita. Importante fato foi a presença do LPG na área de adesão com a célula hospedeira. A marcação permaneceu intensa durante toda a entrada do parasita. Após a completa interiorização, o parasita continua com a superfície marcada e secretando o LPG. A molécula parece então ser sequestrada de dentro do vacúolo parasitóforo em vesículas, que se apresentam distribuidas por todo o citoplasma do macrófago. Além disto, experimentos com Western bloting demonstraram o aparecimento de uma nova banda de 21kDa, depois de 24 horas de interação com a célula hospedeira. Estes resultados mostram que a molécula de LPG esta envolvida e parece ser de grande importância nos primeiros momentos da invasão dos promastigotas no macrófago. Além do mais, o comportamento do LPG dentro do macrófago infectado sugere sua participação em possíveis mecanismos que garantam a permanência do parasita dentro da célula hospedeira.

Rastreamento da localização do glicoconjugado LPG na cinética da interação L. Major/Macrófago / Tracking the location of glucoconjuged LPG on the kinetics of interaction L. Major/macrophages

A leishmaniose é uma doença endêmica que está distribuída por todo o mundo e é causada pelo protozoário do gênero Leishmania. Este parasita existe em duas formas: promastigotas (flagelados) no intestino do inseto vetor, e amastigotas (intracelulares) que vivem e se multiplicam dentro de macrófagos no hospedeiro vertebrado. As formas promastigotas, quando são inoculadas no hospedeiro vertebrado pelo inseto vetor, infectam macrófagos. Estas células são infectadas pelo parasita após um processo de reconhecimento mútuo entre as superficies do protozoário e da célula hospedeira. Questões cruciais sobre como as moléculas da superficie do parasita participam desse processo têm sido exaustivamente estudadas e, aos poucos respondidas. Sabe-se que in vitro todos os promastigotas são capazes de se aderir e entrar na células hospedeiras. Porém, apenas os que passaram pelo processo de metaciclogênese, os metacíclicos, são infectantes. Estudos demonstraram que nesse processo de diferenciação ocorrem modificações na mais abundante molécula expressa na superfície do parasita, o lipofosfoglicano (LPG). Nesse estudo, usamos o anticorpo 79.3, marcador de LPG, a fim de observar a distribuição dessa molécula durante a fase inicial de adesão e posterior interiorização de formas promastigotas por macrófago peritonial de camundongo. Por imunofluorescência, foi possível observar a marcação das superficies dos parasitas aderidos aos macrófagos. Comparamos então a entrada e a destruição dos procíclicos (promastigotas não infectantes) com a sobrevivência e manutenção da infecção fato sugere que a molécula é secretada durante a interação parasitacélula. A molécula de LPG foi constante e uniformemente identificada em toda a superficie do parasita. Importante fato foi a presença do LPG na área de adesão com a célula hospedeira. A marcação permaneceu intensa durante toda a entrada do parasita. Após a completa interiorização, o parasita continua com a superfície marcada e secretando o LPG. A molécula parece então ser sequestrada de dentro do vacúolo parasitóforo em vesículas, que se apresentam distribuidas por todo o citoplasma do macrófago. Além disto, experimentos com Western bloting demonstraram o aparecimento de uma nova banda de 21kDa, depois de 24 horas de interação com a célula hospedeira. Estes resultados mostram que a molécula de LPG esta envolvida e parece ser de grande importância nos primeiros momentos da invasão dos promastigotas no macrófago. Além do mais, o comportamento do LPG dentro do macrófago infectado sugere sua participação em possíveis mecanismos que garantam a permanência do parasita dentro da célula hospedeira.

Cysteine protease inhibitors as chemotherapy: lessons from a parasite target.

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

The antileishmanial activity of novel oxygenated chalcones and their mechanism of action.

Our previous studies have shown that licochalcone A, an oxygenated chalcone, has antileishmanial and antimalarial activities, and alters the ultrastructure and function of the mitochondria of Leishmania spp. parasites. The present study was designed to investigate the antileishmanial activity and the mechanism of action of a group of new oxygenated chalcones. The tested oxygenated chalcones inhibited the in-vitro growth of Leishmania major promastigotes and Leishmania donovani amastigotes. Treatment of hamsters infected with L. donovani with intraperitoneal administration of two oxygenated chalcones resulted in a significant reduction of parasite load in the liver and the spleen compared with untreated control animals. The oxygenated chalcones also inhibited the respiration of the parasite and the activity of mitochondrial dehydrogenases. Electron microscopic studies illustrated that they altered the ultrastructure of the mitochondria of L. major promastigote. The data clearly indicate that this group of oxygenated chalcones has a strong antileishmanial activity and might be developed into a new antileishmanial drug. The antileishmanial activity of oxygenated chalcones might be the result of interference with function of the parasite mitochondria.