Raquel Álvarez-Velilla

Role of trypanosomatid's arginase in polyamine biosynthesis and pathogenesis.

L-Arginine is one of the precursor amino acids of polyamine biosynthesis in most living organisms including Leishmania parasites. L-Arginine is enzymatically hydrolyzed by arginase producing L-ornithine and urea. In Leishmania spp. and other trypanosomatids a single gene encoding arginase has been described. The product of this gene is compartmentalized in glycosomes and is the main source of L-ornithine for polyamine synthesis in these parasites. L-Ornithine is substrate of ornithine decarboxylase (ODC) - one of the key enzymes of polyamine biosynthesis and a validated target for therapeutic intervention - producing putrescine, which in turn is converted to spermidine by condensing with an aminopropyl group from decarboxylated S-adenosylmethionine. Unlike trypanosomatids, mammalian hosts have two arginases (arginase I and II), which have close structural and kinetic resemblances, but localize in different subcellular organelles, respond to different stimuli and have different immunological reactivity. Arginase I is a cytosolic enzyme, mostly expressed in the liver as a pivotal component of the urea cycle, providing in addition L-ornithine for polyamine synthesis. In contrast, arginase II localizes inside mitochondria and is metabolically involved in L-proline and L-glutamine biosynthesis. More striking is the role played by L-arginine as substrate for nitric oxide synthase (NOS2) in macrophages, the main route of clearance of many infectious agents including Leishmania and Trypanosoma cruzi. In infected macrophages L-arginine is catalysed by NOS2 or arginase, contributing to host defense or parasite killing, respectively. A balance between NOS2 and arginase activities is a crucial factor in the progression of the Leishmania infection inside macrophages. In response to T-helper type 2 (Th2) cytokines, resident macrophages induce arginase I inhibiting NO production from L-arginine, thereby promoting parasite proliferation. Conversely, the response to T-helper type 1 (Th1) cytokines is linked to NOS2 induction and parasite death. Moreover, induction of any of these enzymes is accompanied by suppression of the other. Specifically, arginase reduces NO synthesis by substrate depletion, and N(ω)-hydroxy-L-arginine, one of the intermediates of NOS2 catalysis, competitively inhibits arginase activity. In spite of abundant data concerning arginases in mammals as well their involvement in parasite killing, there are very few papers regarding the actual role of arginase in the parasite itself. This review is an update on the recent progress in research on leishmanial arginase including the role played by this enzyme in the establishment of infection in macrophages and the immune response of the host. A comparative study of arginases from other kinetoplatids is also discussed.

Infrared Fluorescent Imaging as a Potent Tool for In Vitro, Ex Vivo and In Vivo Models of Visceral Leishmaniasis

Background Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years. Methodology/Principal findings We generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks. Conclusions/Significance The near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

Indotecan (LMP400) and AM13-55: Two Novel Indenoisoquinolines Show Potential for Treating Visceral Leishmaniasis

ABSTRACT Visceral leishmaniasis is an emerging neglected tropical disease (NTD) caused by the protozoan Leishmania infantum in the countries bordering the Mediterranean Basin. Currently there is no effective vaccine against this disease, and the therapeutic approach is based on toxic derivatives of SbV. Therefore, the discovery of new therapeutic targets and the development of drugs designed to inhibit them comprise an extremely important approach to fighting this disease. DNA topoisomerases (Top) have been identified as promising targets for therapy against leishmaniasis. These enzymes are involved in solving topological problems generated during replication, transcription, and recombination of DNA. Being unlike that of the mammalian host, type IB DNA topoisomerase (TopIB) from Leishmania spp. is a unique bisubunit protein, which makes it very interesting as a selective drug target. In the present investigation, we studied the effect of two TopIB poisons with indenoisoquinoline structure, indotecan and AM13-55, on a murine BALB/c model of infected splenocytes with L. infantum, comparing their effectiveness with that of the clinically tested leishmanicidal drug paromomycin. Both compounds have high selectivity indexes compared with uninfected splenocytes. SDS-KCl-precipitable DNA-protein complexes in Leishmania promastigotes and in vitro cleaving assays confirmed that these drugs are Top poisons. The inhibitory potency of both indenoisoquinolines on L. infantum recombinant TopIB was assessed in vitro, with results showing that indotecan was the most active compound, preventing the relaxation of supercoiled DNA. Experimental infections in susceptible BALB/c mice treated with 2.5 mg/kg body weight/day once every other day for a total of 15 days showed that indotecan cleared more than 80% of the parasite burden of the spleen and liver, indicating promising activity against visceral leishmaniasis.

Appraisal of a Leishmania major Strain Stably Expressing mCherry Fluorescent Protein for Both In Vitro and In Vivo Studies of Potential Drugs and Vaccine against Cutaneous Leishmaniasis

Background Leishmania major cutaneous leishmaniasis is an infectious zoonotic disease. It is produced by a digenetic parasite, which resides in the phagolysosomal compartment of different mammalian macrophage populations. There is an urgent need to develop new therapies (drugs) against this neglected disease that hits developing countries. The main goal of this work is to establish an easier and cheaper tool of choice for real-time monitoring of the establishment and progression of this pathology either in BALB/c mice or in vitro assays. To validate this new technique we vaccinated mice with an attenuated Δhsp70-II strain of Leishmania to assess protection against this disease. Methodology We engineered a transgenic L. major strain expressing the mCherry red-fluorescent protein for real-time monitoring of the parasitic load. This is achieved via measurement of fluorescence emission, allowing a weekly record of the footpads over eight weeks after the inoculation of BALB/c mice. Results In vitro results show a linear correlation between the number of parasites and fluorescence emission over a range of four logs. The minimum number of parasites (amastigote isolated from lesion) detected by their fluorescent phenotype was 10,000. The effect of antileishmanial drugs against mCherry+L. major infecting peritoneal macrophages were evaluated by direct assay of fluorescence emission, with IC50 values of 0.12, 0.56 and 9.20 µM for amphotericin B, miltefosine and paromomycin, respectively. An experimental vaccination trial based on the protection conferred by an attenuated Δhsp70-II mutant of Leishmania was used to validate the suitability of this technique in vivo. Conclusions A Leishmania major strain expressing mCherry red-fluorescent protein enables the monitoring of parasitic load via measurement of fluorescence emission. This approach allows a simpler, faster, non-invasive and cost-effective technique to assess the clinical progression of the infection after drug or vaccine therapy.

Gimatecan and other camptothecin derivatives poison Leishmania DNA-topoisomerase IB leading to a strong leishmanicidal effect

The aim of this work is the in vitro and ex vivo assessment of the leishmanicidal activity of camptothecin and three analogues used in cancer therapy: topotecan (Hycantim®), gimatecan (ST1481) and the pro-drug irinotecan (Camptosar®) as well as its active metabolite SN-38 against Leishmania infantum. The activity of camptothecin and its derivatives was studied on extracellular L. infantum infrared-emitting promastigotes and on an ex vivo murine model of infected splenocytes with L. infantum fluorescent amastigotes. In situ formation of SDS/KCl precipitable DNA-protein complexes in Leishmania promastigotes indicated that these drugs are DNA topoisomerase IB poisons. The inhibitory potency of camptothecin derivatives on recombinant L. infantum topoisomerase IB was assessed in vitro showing that gimatecan is the most active compound preventing the relaxation of supercoiled DNA at submicromolar concentrations. Cleavage equilibrium assays in Leishmania topoisomerase IB show that gimatecan changes the equilibrium towards cleavage at much lower concentrations than the other camptothecin derivatives and that this effect persists over time. Gimatecan and camptothecin were the most powerful compounds preventing cell growth of free-living L. infantum promastigotes within the same concentration range. All these compounds killed L. infantum splenocyte-infecting amastigotes within the nanomolar range. The amastigote form showed higher sensitivity to topoisomerase IB poisons (with high therapeutic selectivity indexes) than free-living promastigotes. All the compounds assayed poisoned L. infantum DNA topoisomerase IB leading to a strong leishmanicidal effect. Camptothecin derivatives are suitable for reducing the parasitic burden of ex vivo infected splenocytes. The selectivity index of gimatecan makes it a promising drug against this neglected disease.

Trypanosomatids topoisomerase re-visited. New structural findings and role in drug discovery

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Target-based vs. phenotypic screenings in Leishmania drug discovery: A marriage of convenience or a dialogue of the deaf?

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First evidence of intraclonal genetic exchange in trypanosomatids using two Leishmania infantum fluorescent transgenic clones.

Background The mode of reproduction in Leishmania spp has been argued to be essentially clonal. However, recent data (genetic analysis of populations and co-infections in sand flies) have proposed the existence of a non-obligate sexual cycle in the extracellular stage of the parasite within the sand fly vector. In this article we propose the existence of intraclonal genetic exchange in the natural vector of Leishmania infantum. Methodology/Principal findings We have developed transgenic L. infantum lines expressing drug resistance markers linked to green and red fluorescent reporters. We hypothesized whether those cells with identical genotype can recognize each other and mate. Both types of markers were successfully exchanged within the sand fly midgut of the natural vector Phlebotomus perniciosus when individuals from these species were fed with a mixture of parental clones. Using the yellow phenotype and drug resistance markers, we provide evidence for genetic exchange in L. infantum. The hybrid progeny appeared to be triploid based on DNA content analysis. The hybrid clone analyzed was stable throughout the complete parasite life cycle. The progress of infections by the hybrid clone in BALB/c mice caused a reduction in parasite loads in both spleen and liver, and provided weight values similar to those obtained with uninfected mice. Spleen arginase activity was also significantly reduced relative to parental strains. Conclusions/Significance A L. infantum hybrid lineage was obtained from intraclonal genetic exchange within the midgut of the natural vector, suggesting the ability of this parasite to recognize the same genotype and mate. The yellow hybrid progeny is stable throughout the whole parasite life cycle but with a slower virulence, which correlates well with the lower arginase activity detected both in vitro and in vivo infections.

Synthesis of Marine α-Methoxylated Fatty Acid Analogs that Effectively Inhibit the Topoisomerase IB from Leishmania donovani with a Mechanism Different from that of Camptothecin

Sponges biosynthesize α-methoxylated fatty acids with unusual biophysical and biological properties and in some cases they display enhanced anticancer activities. However, the antiprotozoal properties of the α-methoxylated fatty acids have been less studied. In this work, we describe the total synthesis of (5Z,9Z)-(±)-2-methoxy-5,9-eicosadienoic acid (1) and its acetylenic analog (±)-2-methoxy-5,9-eicosadiynoic acid (2), and report that they inhibit (EC50 values between 31 and 22 µM) the Leishmania donovani DNA topoisomerase IB enzyme (LdTopIB). The inhibition of LdTopIB (EC50 = 53 µM) by the acid (±)-2-methoxy-6-icosynoic acid (12) was studied as well. The potency of LdTopIB inhibition followed the trend 2 > 1 > 12, indicating that the effectiveness of inhibition depends on the degree of unsaturation. All of the studied α-methoxylated fatty acids failed to inhibit the human topoisomerase IB enzyme (hTopIB) at 100 µM. However, the α-methoxylated fatty acids were capable of inhibiting an active but truncated LdTopIB with which camptothecin (CPT) cannot interact suggesting that the methoxylated fatty acids inhibit LdTopIB with a mechanism different from that of CPT. The diunsaturated fatty acids displayed low cytotoxicity towards Leishmania infantum promastigotes (EC50 values between 260 and 240 µM), but 12 displayed a better cytotoxicity towards Leishmania donovani promastigotes (EC50 = 100 µM) and a better therapeutic index.

Trypanosomatids see the light: recent advances in bioimaging research

The use of genetically engineered pathogens that express fluorescent or luminescent proteins represents a huge stride forward in the understanding of trypanosomatid-borne tropical diseases. Nowadays, such modified microorganisms are being used to screen thousands of compounds under a target-free (phenotypic) approach. In addition, experimental infections with transgenic parasites drastically reduce the number of animals required for preclinical studies, because no animal needs to be put down to assess its parasite load. Finally, the use of fluorescent parasites is contributing to unraveling genetic exchange events between trypanosomatid strains. This phenomenon is important for understanding the mechanism by which traits such as virulence, tissue tropism, and drug resistance are transferred, as well as the emergence of novel strains.