Intact Parasites Research Articles

Trypanosoma rangeli: Characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53 ± 0.12 nmol P i/h × 10 7 cells). The ATP hydrolysis was stimulated by MgCl 2 and the Mg-dependent ecto-ATPase activity was 5.24 ± 0.64 nmol P i/h × 10 7 cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl 2 was replaced by MnCl 2, but not by CaCl 2, SrCl 2, and ZnCl 2. The apparent K m for Mg-ATP2- was 0.53 ± 0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4′-diisothiocyanostylbene 2′-2′-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg 2+-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.

Analysis of T-cell responses in malaria-exposed and non-exposed donors using Plasmodium falciparum asexual blood stages enriched by a simple centrifugation method

Several studies have reported on similar in vitro cellular responses to different malaria-antigen preparations in both malaria-primed and un-primed donors. Whether intact live parasites can exert a distinct type of response in either of the two groups is not well known. In this study, we developed a simple three-step centrifugation method for simultaneous enrichment of early and late blood stages from Plasmodium falciparum cultures. Such enriched P. falciparum fractions and other antigen preparations were used to stimulate lymphocytes from malaria-exposed and non-exposed individuals to examine the proliferative activity and expansion of CD3 +, γδ +, CD4 +, and CD8 + T cells. While lymphocytes from malaria non-exposed donors proliferated relatively higher than those from malaria-exposed donors in response to most antigens tested, the enriched fractions of live parasites exerted higher proliferative responses on cells from the latter donors. This suggests the existence of memory cells in the malaria-exposed donors, but not in the non-exposed ones. Flow cytometric analysis revealed a higher percentage expansion of CD4 + T cells in the responding cells of the exposed donors than the non-exposed ones. Taken together, this study reports on a simple method that simultaneously enriches for intact live early and late blood stages of P. falciparum parasites. Moreover, the study revealed higher expansion CD4 + T cells in the exposed individuals than the non-exposed in response to live malaria parasites and not to other parasite–antigen preparations.

Deoxyuridine Triphosphate Nucleotidohydrolase as a Potential Antiparasitic Drug Target

This paper describes a structure-activity study to identify novel, small-molecule inhibitors of the enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) from parasitic protozoa. The successful synthesis of a variety of analogues of dUMP is described in which the substituents are introduced at the 3'- and 5'-positions, together with variation in the heteroatom at the 5'-position. The compounds were assayed against recombinant Plasmodium falciparum and Leishmania major enzymes and the human enzyme to give a measure of selectivity. The compounds were also tested in vitro against the intact parasites P. falciparum and L. donovani. A number of potent and selective inhibitors of the P. falciparum dUTPase that show drug-like properties and represent good leads for future development were identified. The best inhibitors included the compounds 5'-tritylamino-2',5'-dideoxyuridine (2j) (Ki = 0.2 microM) and 5'-O-triphenylsilyl-2',3'-didehydro-2',3'-dideoxyuridine (5h) (Ki = 1.3 microM), with selectivity greater than 200-fold compared to the human enzyme. Structural features important for antiplasmodial activity were determined. The correlation observed between the inhibition of the enzyme and the inhibition of the parasite growth in vitro demonstrates that the P. falciparum dUTPase constitutes a valid and attractive novel target for the development of much-needed new antimalarial drugs.

989. Toward Multivalent Adeno-Based Vaccine for Malaria: Effects of Promoter Strength on Immunogenicity and Vaccine Efficacy

Malaria is the most devastating parasitic disease affecting humans. Each year 300-500 million people become infected with the parasite and more than 1.5 million people, primarily children, die of the disease. The feasibility of a malaria vaccine is supported by the demonstration of protective immunity following exposure to the intact Plasmodium parasite.

Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

The proteins that mediate polyamine translocation into eukaryotic cells have not been identified at the molecular level. To define the polyamine transport pathways in eukaryotic cells we have cloned a gene, LmPOT1, that encodes a polyamine transporter from the protozoan pathogen, Leishmania major. Sequence analysis of LmPOT1 predicted an unusual 803-residue polytopic protein with 9-12 transmembrane domains. Expression of LmPOT1 cRNA in Xenopus laevis oocytes revealed LmPOT1 to be a high affinity transporter for both putrescine and spermidine, whereas expression of LmPOT1 in Trypanosoma brucei stimulated putrescine uptake that was sensitive to inhibition by pentamidine and proton ionophores. Immunoblot analysis established that LmPOT1 was expressed predominantly in the insect vector form of L. major, and immunofluorescence demonstrated that LmPOT1 was localized predominantly to the parasite plasma membrane. To our knowledge this is the first molecular identification and characterization of a cell surface polyamine transporter in eukaryotic cells.

Manipulación genética y el estudio del parásito protozoario Leishmania.

During the last 15 years, many aspects of the functional genomics of Leishmania have been revealed due to advances in DNA transfection, gene disruption and complementation through homologous recombination, and efficient strategies for the selection of transfected cells. These strategies have provided information about gene expression and protein function in the context of the intact parasite. The genome of Leishmania shows a marked deficiency of known transcription initiation factors, and gene expression is regulated almost entirely at the post-transcriptional level through trans-splicing of mRNAs and novel control mechanisms involving differential processing of 3'-untranslated regions (3'-UTRs) of mRNAs. Therefore, gene transfection represents a useful tool for the identification and functional analysis of genes of interest as well as the mechanisms that direct their regulation. The development of genetic manipulation systems has provided opportunities for the study of genes involved in virulence, intracellular survival and drug resistance of Leishmania, as well as for the functional validation of specific parasite proteins as new chemo- and immunotherapeutic targets. The current review presents recent advances in genetic manipulations that permit structural, functional and phenotypic analyses and by means of gene deletion and complementation using the methods of gene transfection.

The Schistosoma mansoni soluble proteome: a comparison across four life-cycle stages

Differential analysis of immune responses to schistosomes has routinely been performed using complex mixtures of soluble proteins from various life-cycle stages, on the assumption that these differed significantly in composition. Proteomic techniques now allow us to characterise and compare such mixtures. The soluble proteins from cercariae, lung-schistosomula, adult worms and eggs of Schistosoma mansoni were separated by high-resolution two-dimensional electrophoresis and the resulting images analysed using appropriate software. A high degree of quantitative and qualitative similarity in spot pattern was revealed across the life-cycle, greatest between adjacent stages. To initiate mapping of these soluble proteomes, the 40 most abundant spots in each preparation, accounting for 21-46% of the total protein, were subjected to peptide fingerprinting by mass spectrometry. On average 55% of the spots were identified, but overall, these comprised only 32 different protein species. With one exception all proteins originated in the cytosol and 24 of the 32 had previously been pinpointed by virtue of their immunoreactivity, including four of the WHO priority vaccine candidates. The similarity in composition between the four preparations means that they are unlikely to discriminate adequately between immune responses to different life-cycle stages and argues strongly for the need to identify true stage-specific marker proteins. Equally, it is difficult to reconcile the abundance and immunogenicity of such cytosolic proteins with their status as vaccine candidates, as it is unlikely they will be accessible to the immune system in an intact parasite.

The Leishmania chagasi proteasome: role in promastigotes growth and amastigotes survival within murine macrophages

Proteasomes are multisubunit proteases that exist universally among eukaryotes. They have multiple proteolytic activities and are believed to have important roles in regulating cell cycle, selective intracellular proteolysis, and antigen presentation. Here we have partially purified Leishmania chagasi proteasome. The L. chagasi proteasome rich fraction displayed the typical features of eukaryotic 20S proteasome complexes, being active towards peptidyl substrates with hydrophobic and acidic residues, and sensitive to the proteasome-specific inhibitor lactacystin. We have shown that lactacystin, or its active form clasto-lactacystin β-lactone, but not E-64, blocks the in vitro growth of L. chagasi promastigotes, demonstrating that the interference with parasite growth is due to the lack of proteasome activity. Furthermore, pre-treatment of L. chagasi promastigotes with lactacystin did not prevent parasite entry in host cells, but markedly restricted its intracellular survival. These results demonstrate that intact parasite proteasome function is required for replication of L. chagasi and for amastigotes survival inside the vertebrate host cell.

A Mg-dependent ecto-ATPase is increased in the infective stages of Trypanosoma cruzi.

In this work, we describe the ability of living epimastigotes of Trypanosoma cruzi to hydrolyze extracellular ATP. In these intact parasites, there was a low level of ATP hydrolysis in the absence of any divalent metal (2.42 +/- 0.31 nmol Pi/h x 10(8) cells). ATP hydrolysis was stimulated by MgCl2, and the Mg-dependent ecto-ATPase activity was 27.15 +/- 2.91 nmol Pi/h x 10(8) cells. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The apparent Km for Mg-ATP2- was 0.61 mM, and free Mg2+ did not increase the ecto-ATPase activity. This ecto-ATPase activity was insensitive to the inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'.diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. A comparison among the Mg2+-ecto-ATPase activities of the three forms of T. cruzi showed that the noninfective epimastigotes were less efficient at hydrolyzing ATP than the infective trypomastigote and amastigote stages.

Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi

An ecto-NTP diphosphohydrolase (NTPDase) activity, insensitive to inhibitors of ATPases and phosphatases, was characterized on the surface of live Trypanosoma cruzi intact parasites. The enzyme exhibits broad substrate specificity, typical of NTPDases, and a high hydrolysis rate for GTP. A 2282 bp message encoding a full-length NTPDase was cloned by RT-PCR using epimastigote mRNA. A single protein was immunoprecipitated from [ 35S]methionine-labeled parasites using antibodies against Toxoplasma gondii NTPase I. This antibody localized an NTPDase on the external surface of all forms of T. cruzi, as seen by confocal immuno-fluorescence microscopy. The NTPDase could be part of the parasite’s purine salvage pathway. Additionally, trypomastigotes (infective form) presented a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes (non-infective form) presented a 1:1 ratio, suggesting a possible role for the NTPDase in the parasite’s virulence mechanisms.

Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound anti-parasitic activity

In this study, the efficacies of chemotherapy employing nitazoxanide (NTZ), albendazole (ABZ), and a NTZ/ABZ-combination against alveolar echinococcosis (AE) were investigated in an experimental murine model. Following secondary infection, meaning i.p. injection of 20 Echinococcus multilocularis metacestodes, the drugs were administered by intragastric inoculation on a daily bases for a period of 5 weeks. Treatment was started either immediately on the day of infection, or at 2 months p.i., respectively. Application of the NTZ/ABZ-combination starting at 2 months p.i. was proven to be most effective in terms of reducing parasite weight (from 4.42±1.03 to 1±0.05 g; P=0.001). Inspection of treated parasites by transmission electron microscopy showed that ABZ- and NTZ-treated metacestode tissues, respectively, were heterogeneous in that both largely intact parasites as well as severely altered metacestodes could be observed. NTZ/ABZ-combination treatment induced the most severe ultrastructural alterations, including massive reduction in length and number of microtriches, severely damaged tegumental architecture, and progressive loss of viability of the germinal layer, associated with encapsulation by host connective tissue. A comparative pharmacokinetic study in mice revealed that the application of ABZ and NTZ in combination resulted in a two- to four-fold increase of albendazole sulfoxide serum levels for the period of 4–8 h following drug uptake compared to application of ABZ alone. In a third experiment, mice were orally infected with E. multilocularis eggs, and treated with NTZ starting at 2 months p.i. This resulted in a significantly lower lesion number in treated versus untreated mice ( P=0.01). This investigation indicates the potential value for NTZ and/or a combined ABZ/NTZ chemotherapy against AE.

A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms

In this work, we describe how living cells of Trypanosoma brucei procyclic forms were able to hydrolyze extracellular p-nitrophenylphosphate ( pNPP). These intact parasites, which had their viability determined by motility and the Trypan blue method, presented a low level of pNPP hydrolysis in the absence of any divalent metal (0.72±0.07 nmol pNP/mg min). Interestingly, in the presence of 5 mM MgCl 2, ectophosphatase activity of 1.91±0.21 nmol pNP/mg min was observed. The ectophosphatase activity was also stimulated by MnCl 2, CoCl 2 and CuCl 2 but not by CaCl 2 and CdCl 2 and was inhibited by ZnCl 2. The addition of Mg 2+, Mn 2+, Co 2+ and Cu 2+ to extracellular medium increased the ectophosphatase activity in a dose-dependent manner. At 5 mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39±0.05 mM MgCl 2, 0.33±0.03 mM MnCl 2, 1.63±0.12 mM CoCl 2 and 2.04±0.33 mM CuCl 2. In the absence of any divalent metal (basal activity) the apparent K m for pNPP was 0.66±0.09 mM, while at saturating MgCl 2 concentrations the corresponding apparent K m for pNPP for Mg 2+-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.27±0.03 mM. The Mg 2+-stimulated pNPP hydrolysis was strongly inhibited by ZnCl 2 and vanadate, while the metal-independent basal phosphatase activity was less inhibited by these phosphotyrosyl phosphatase inhibitors.

2,4-Diaminopyrimidines as inhibitors of Leishmanial and Trypanosomal dihydrofolate reductase

This paper describes the synthesis of 4′-substituted and 3′,4′-disubstituted 5-benzyl-2,4-diaminopyrimidines as selective inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were then assayed against the recombinant parasite and human enzymes. Some of the compounds showed good activity. They were also tested against the intact parasites using in vitro assays. Good activity was found against Trypanosoma cruzi, moderate activity against Trypanosoma brucei and Leishmania donovani. Molecular modeling was undertaken to explain the results. The leishmanial enzyme was found to have a more extensive lipophilic binding region in the active site than the human enzyme. Compounds which bound within the pocket showed the highest selectivity.

Membrane orientation of laminin binding protein.

Earlier we presented several lines of evidence that a 67-kDa laminin binding protein (LBP) in Leishmania donovani, that is different from the putative mammalian 67-kDa laminin receptor, may play an important role in the onset of leishmaniasis, as these parasites invade macrophages in various organs after migrating through the extracellular matrix. Here we describe the membrane orientation of this Leishmania laminin receptor. Flow cytometric analysis using anti-LBP Ig revealed its surface localization, which was further confirmed by enzymatic radiolabeling of Leishmania surface proteins, autoradiography and Western blotting. Efficient incorporation of LBP into artificial lipid bilayer, as well as its presence in the detergent phase after Triton X-114 membrane extraction, suggests that it may be an integral membrane protein. Limited trypsinization of intact parasite and subsequent immunoblotting of trypsin released material using laminin as primary probe revealed that a major part of this protein harbouring the laminin binding site is oriented extracellularly. Carboxypeptidase Y treatment of the whole cell, as well as the membrane preparation, revealed that a small part of the C-terminal is located in the cytosol. A 34-kDa transmembrane part of LBP could be identified using the photoactive probe, 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID). Partial sequence comparison of the intact protein to that with the trypsin-released fragment indicated that N-terminal may be located extracellularly. Together, these results suggest that LBP may be an integral membrane protein, having significant portion of N-terminal end as well as the laminin binding site oriented extracellularly, a membrane spanning domain and a C-terminal cytosolic end.

Genetic characterization of glucose transporter function in Leishmania mexicana.

Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This deltalmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the deltalmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. deltalmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in deltalmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in deltalmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite.

Expression of a novel Leishmania gene encoding a histone H1-like protein in Leishmania major modulates parasite infectivity in vitro.

We describe identification and characterization of a novel two-copy gene of the parasitic protozoan Leishmania that encodes a nuclear protein designated LNP18. This protein is highly conserved in the genus Leishmania, and it is developmentally regulated. It is an alanine- and lysine-rich protein with potential bipartite nuclear targeting sequence sites. LNP18 shows sequence similarity to H1 histones of trypanosomatids and of higher eukaryotes and in particular with histone H1 of Leishmania major. The nuclear localization of LNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by using antibodies raised against the recombinant protein as probes. The antibodies recognized predominantly a 18-kDa band or a 18-kDa-16-kDa doublet. Photochemical cross-linking of intact parasites followed by Western blot analysis provided evidence that LNP18 is indeed a DNA-binding protein. Generation of transfectants overexpressing LNP18 allowed us to determine the role of this protein in Leishmania infection of macrophages in vitro. These studies revealed that transfectants overexpressing LNP18 are significantly less infective than transfectants with the vector alone and suggested that the level of LNP18 expression modulates Leishmania infectivity, as assessed in vitro.

Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major.

Macrophages are the major target cell population of the obligate intracellular parasites Leishmania. Although polymorphonuclear neutrophil granulocytes (PMN) are able to internalize Leishmania promastigotes, these cells have not been considered to date as host cells for the parasites, primarily due to their short life span. In vitro coincubation experiments were conducted to investigate whether Leishmania can modify the spontaneous apoptosis of human PMN. Coincubation of PMN with Leishmania major promastigotes resulted in a significant decrease in the ratio of apoptotic neutrophils as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low m.w. DNA fragments, and annexin V staining. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in PMN. The inhibition of PMN apoptosis depended on viable parasites because killed Leishmania or a lysate of the parasites did not have antiapoptotic effect. L. major did not block, but rather delayed the programmed cell death of neutrophils by approximately 24 h. The antiapoptotic effect of the parasites could not be transferred by the supernatants, despite secretion of IL-8 by PMN upon coculture with L. major. In vivo, intact parasites were found intracellularly in PMN collected from the skin of mice 3 days after s.c. infection. This finding strongly suggests that infection with Leishmania prolongs the survival time of neutrophils also in vivo. These data indicate that Leishmania induce an increased survival of neutrophil granulocytes both in vitro and in vivo.

Entamoeba histolytica-induced dephosphorylation in host cells.

Activation of host cell protein tyrosine phosphatases (PTPases) and protein dephosphorylation is an important mechanism used by various microorganisms to deactivate or kill host defense cells. To determine whether protein tyrosine dephosphorylation played a role in signaling pathways affecting Entamoeba histolytica-mediated host cell killing, we investigated the involvement of PTPases during the attachment of E. histolytica to target cells. We observed a rapid decrease in cellular protein tyrosine levels in Jurkat cells, as measured with an antiphosphotyrosine monoclonal antibody, following adherence to E. histolytica. Ameba-induced protein dephosphorylation was contact dependent and required intact parasite, since blocking amebic adherence with galactose inhibited tyrosine dephosphorylation and amebic lysates had no effect on phosphotyrosine levels. Moreover, disruption of amebic adherence with galactose promoted recovery of phosphorylation in Jurkat cells, indicating that dephosphorylation precedes target cell death. The evidence suggests that ameba-induced dephosphorylation is mediated by host cell phosphatases. Prior treatment of Jurkat cells with phenylarsine oxide, a PTPase inhibitor, inhibited ameba-induced dephosphorylation. We also found proteolytic cleavage of the PTPase 1B (PTP1B) in Jurkat cells after contact with amebae. The calcium-dependent protease calpain is responsible for PTP1B cleavage and enzymatic activation. Pretreatment of Jurkat cells with calpeptin, a calpain inhibitor, blocked PTP1B cleavage and inhibited ameba-induced dephosphorylation. In addition, inhibition of Jurkat cell PTPases with phenylarsine oxide blocked Jurkat cell apoptosis induced by E. histolytica. These results suggest that E. histolytica-mediated host cell death occurs by a mechanism that involves PTPase activation.

Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors.

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of Leishmania. However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of Leishmania. Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.

Inhibition of Parasite Protein Kinase C by New Antileishmanial Imidazolidin-2-one Compounds

The protein kinase C (PKC) family of isoenzymes mediate a wide range of signal transduction pathways in many different cells lines. Little is known regarding the presence and functional roles of PKC in Leishmania spp. Here we report the inhibition of parasite PKC by new imidazolidinone compounds. The most active derivative 7 showed an important activity (IC 50 =9.9 μM) against the clinical relevant stage of parasites in comparison with Glucantime ® (IC 50 =464.5 μM), without inducing toxicity on human fibroblast cells (IC 50 =102 μM). Pretreatment of intact parasites with 10 μM of compound 7 inhibited 80% of PKC activity. At the same concentration, this compound inhibited 70% of the parasite-host cell invasion process. An in vivo model showed that compound 7 reduced the liver parasite burden by 25% and spleen parasite burden by 44%. These results provide the first evidence that PKC plays a critical role in the invasion process. Thus Leishmania PKC activity could be a relevant therapeutic target and the imidazolidinones novel antileishmanial candidates.