Leishmania amazonensis: characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.     

Leishmania amazonensis: effects of heat shock on ecto-ATPase activity

In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.     

Leishmania amazonensis: Biological and biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities

The presence of Leishmania amazonensis ecto-nucleoside triphosphate triphosphohydrolase activities was demonstrated using antibodies against different NTPDase members by Western blotting, flow cytometry, and immunoelectron microscopy analysis. Living promastigote cells sequentially hydrolyzed the ATP molecule generating ADP, AMP, and adenosine, indicating that this surface enzyme may play a role in the salvage of purines from the extracellular medium. The L. amazonensis ecto-NTPDase activities were insensitive to Triton X-100, but they were enhanced by divalent cations, such as Mg(2+). In addition, the ecto-NTPDase activities decreased with time for 96 h when promastigotes were grown in vitro. On the other hand, these activities increased considerably when measured in living amastigote forms. Furthermore, the treatment with adenosine, a mediator of several relevant biological phenomena, induced a decrease in the reactivity with anti-CD39 antibody, raised against mammalian E-NTPDase, probably because of down regulation in the L. amazonensis ecto-NTPDase expression. Also, adenosine and anti-NTPDase antibodies induced a significant diminishing in the interaction between promastigotes of L. amazonensis and mouse peritoneal macrophages.     

Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro

Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.     

Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.     

Leishmania amazonensis: participation of regulatory T and B cells in the in vitro priming (PIV) of CBA/J spleen cells susceptible response

CBA/J mice are resistant to Leishmania major and susceptible to Leishmania amazonensis. Early events determine infection outcome. Until now, PIV (in vitro priming) immune response to L. amazonensis has not been assessed. Herein, we have shown that compared to L. major, L. amazonensis induced higher parasite burden associated to similar IL-4, IFN-gamma, and TNF-alpha mRNA expressions and IFN-gamma and IL-10 levels. Although similar amounts of IL-10 were detected, the frequency of intracellular IL-10 positive B cells was enhanced in spleen cells stimulated with anti-CD3/anti-CD28, or anti-CD3/anti-CD28 and L. amazonensis, compared to L. major-stimulation. Interestingly, IL-10- producing B cells were reduced in response to anti-CD3/anti-CD28 stimulation combined with L. major compared to the other groups. L. amazonensis may favor T regulatory cell development, since 40% of all the CD4+CD25+ were CD25(high) cells. These data suggest that in PIV, susceptibility to L. amazonensis is not related to Th cell polarization, but to the presence and activity of regulatory T and B cells.     

Photodynamic therapy for American cutaneous leishmaniasis: the efficacy of methylene blue in hamsters experimentally infected with Leishmania (Leishmania) amazonensis

The aim of this study was to investigate the effectiveness of Photodynamic Therapy (PDT) using Methylene Blue (MB) as the photosensitizing compound and a Light-Emitting Diode (LED) in American cutaneous leishmaniasis (ACL). Hamsters were experimentally infected with Leishmania (Leishmania) amazonensis. After the development of the lesions in the footpad, the animals were treated with MB three times a week for 3 months. Ten minutes after each application of MB, the lesions were irradiated with LED for 1 h. The lesions were evaluated weekly by the measurement of the hamster footpad thickness. At the end of the treatment the parasitic load was quantified in the regional lymph node of the hamsters. The treatment promoted a decrease in the thickness of infected footpad (P = 0.0001) and reduction in the parasitic load in the regional lymph node (P = 0.0007) of the animals from group treated with MB + LED. PDT using MB + LED in ACL caused by L. amazonensis shows a strong photodynamic effect. This therapy is very promising, once it is an inexpensive system and the own patient can apply it in their wound and in their house without the need of technical assistance.     

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 μM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 μM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.     

LaRbp38: a Leishmania amazonensis protein that binds nuclear and kinetoplast DNAs

Leishmania amazonensis causes a wide spectrum of leishmaniasis. There are no vaccines or adequate treatment for leishmaniasis, therefore there is considerable interest in the identification of new targets for anti-leishmania drugs. The central role of telomere-binding proteins in cell maintenance makes these proteins potential targets for new drugs. In this work, we used a combination of purification chromatographies to screen L. amazonensis proteins for molecules capable of binding double-stranded telomeric DNA. This approach resulted in the purification of a 38kDa polypeptide that was identified by mass spectrometry as Rbp38, a trypanosomatid protein previously shown to stabilize mitochondrial RNA and to associate with nuclear and kinetoplast DNAs. Western blotting and supershift assays confirmed the identity of the protein as LaRbp38. Competition and chromatin immunoprecipitation assays confirmed that LaRbp38 interacted with kinetoplast and nuclear DNAs in vivo and suggested that LaRbp38 may have dual cellular localization and more than one function.     

Leishmania amazonensis: effects of oral treatment with copaiba oil in mice

Leishmaniasis is a severe public-health problem, with high rates of morbidity and mortality. Efforts to find new, effective and safe oral agents for the treatment of leishmaniasis have been ongoing for several decades, in order to avoid the problems with the currently used antimonials. In the present study, we found that a copaiba oil oral treatment (Group IV) caused a significant reduction in the average lesion size (1.1 ± 0.4 mm) against Leishmania amazonensis lesions compared with untreated mice (Group I) (4.4 ± 1.3 mm). To prove the safety of the oil, the toxicity and genotoxicity were also determined. Histopathological evaluation did not reveal changes in the copaiba oil-treated animals compared to the control animals. In the mutagenicity evaluation, (micronucleus test) the dose tested (2000 mg/kg) showed no genotoxic effects. Morphological and ultrastructural analyses demonstrated notable changes in parasite cells treated with this oleoresin. The main ultrastructural effect was mitochondrial swelling. We also demonstrated that in vitro copaiba oil treatment of L. amazonensis led to an increase in plasma membrane permeability, and depolarization in the mitochondrial membrane potential in parasite cells. Although the mechanism of action of the oleoresin is still unclear, these findings indicate that copaiba oil is a possible new drug, which would provide a safer, shorter, less-expensive, and more easily administered treatment for leishmaniasis.     

Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.     

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3′-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.     

Leishmania (Leishmania) amazonensis: experimental cutaneous leishmaniasis associated with systemic amyloidosis in mice

We infected Swiss and C57BL/6 female mice in the left hind footpad with 10⁴Leishmania (L.) amazonensis promastigotes in stationary phase. The macroscopic examination showed a nodular non-ulcerated lesion at the site of inoculation and hepatic and spleenic enlargement. Histopathologically, the primary lesion showed an extensive liquefactive necrosis and inflammatory infiltrate, mainly consisting of macrophages filled with amastigotes, and rare lymphocytes. The inflammatory reaction in liver, spleen and kidney showed amyloid deposits. Additionally, C57BL/6 had accentuated amyloidosis in both ovarian cortical and medullar region and inflammatory infiltrates in the pancreas and adrenal gland.     

Genomic organization of telomeric and subtelomeric sequences of Leishmania (Leishmania) amazonensis

Telomeres are DNA-protein complexes that protect linear chromosomes from degradation and fusions. Telomeric DNA is repetitive and G-rich, and protrudes towards the end of the chromosomes as 3'G-overhangs. In Leishmania spp., sequences adjacent to telomeres comprise the Leishmania conserved telomere associated sequences (LCTAS) that are around 100 bp long and contain two conserved sequence elements (CSB1 and CSB2), in addition to non-conserved sequences. The aim of this work was to study the genomic organization of Leishmania (Leishmania) amazonensis telomeric/subtelomeric sequences. Leishmania amazonensis chromosomes were separated in a single Pulsed Field Gel Electrophoresis (PFGE) gel as 25 ethidium bromide-stained bands. All of the bands hybridized with the telomeric probe (5'-TTAGGG-3')3 and with probes generated from the conserved subtelomeric elements (CSB1, CSB2). Terminal restriction fragments (TRF) of L. amazonensis chromosomes were analyzed by hybridizing restriction digested genomic DNA and chromosomal DNA separated in 2D-PFGE with the telomeric probe. The L. amazonensis TRF was estimated to be approximately 3.3 kb long and the telomeres were polymorphic and ranged in size from 0.2 to 1.0 kb. Afa I restriction sites within the conserved CSB1 elements released the telomeres from the rest of the chromosome. Bal 31-sensitive analysis confirmed the presence of terminal Afa I restriction sites and served to differentiate telomeric fragments from interstitial internal sequences. The size of the L. amazonensis 3' G-overhang was estimated by non-denaturing Southern blotting to be approximately 12 nt long. Using similar approaches, the subtelomeric domains CSB1 and CSB2 were found to be present in a low copy number compared to telomeres and were organized in blocks of 0.3-1.5 kb flanked by Hinf I and Hae III restriction sites. A model for the organization of L. amazonensis chromosomal ends is provided.     

Leishmania amazonensis: multiple receptor-ligand interactions are involved in amastigote infection of human dendritic cells

In their mammalian hosts, Leishmania are obligate intracellular parasites that reside in macrophages and dendritic cells (DCs). In the present study, we have investigated in vitro the mechanisms of entry into human DCs of Leishmania amazonensis amastigotes isolated from lesions in nude mice (Am nude). The DC infection rate with Am nude was approximately 36%, while opsonization of Am nude with normal human serum and infected human serum increased the DC infection rates to 60% and 62%, respectively. Heat inactivation and depletion of antibodies in sera brought the DC infection rate down to 40%. The DC infection rate was inhibited after pre-treatment of Am nude with heparin. We were unable to implicate mannose-fucose receptors in the uptake of Am nude by DCs. Our data suggest that the ability of L. amazonensis amastigotes to infect human DCs involves the participation of at least three multiple receptor-ligand interactions, antibodies/FcR, complement components/CR and proteoglycans/heparin-binding protein.     

Liposomal-lupane system as alternative chemotherapy against cutaneous leishmaniasis: Macrophage as target cell

Leishmania amazonensis causes human diseases that range from self-healing to diffusion cutaneous lesions. The chemotherapy of leishmaniasis requires long-term treatment and has been based on the use of pentavalent antimonials. Liposomes have been used as antileishmanial drug carries and have adjuvant activity in vaccines against several microorganisms, representing an important option to the development of new therapeutics for the disease. In this study, we developed a liposomal formulation containing lupane [3β,6β,16β-trihydroxylup-20(29)-ene], isolated from fruits of Combretum leprosum with pharmacological properties as antinociceptive, anti-inflammatory, antiulcerogenic and antileishmanial activities. The aim of the present study was to evaluate the efficacy of liposomal-lupane in L. amazonensis-infection model. Liposomes were prepared by the extrusion method with DPPC, DPPS and cholesterol at 5:1:4 weight ratio. The lupane (2 mg/mL) was added to the lipid mixture, solubilized in chloroform and dried under nitrogen flow. The activity of liposomal-lupane was conducted in vitro with mouse peritoneal infected macrophages. Furthermore, mice were infected in the right hind footpad with 10⁵ stationary growth phase of L. amazonensis promastigotes. After 6 weeks, animals were treated with liposomal-lupane for 15 days by intraperitoneal injection. The evolution of disease was monitored weekly by measuring footpad thickness with a caliper. Three days after the treatment, peritoneal macrophages were collected, plated and production of the cytokines IL-10 and IL-12 was evaluated in supernatants of the cultures after 24 h. The results indicate that the liposomal system containing lupane achieved here is a promising tool to confer antileishmanial activity to infected macrophages.     

Leishmania tarentolae: Utility as an in vitro model for screening of antileishmanial agents

Primary screens for antileishmanial compounds use Leishmania species pathogenic to humans that must be handled under biosafety conditions that cannot be adopted or guaranteed everywhere. Leishmania tarentolae, a parasite isolated from the gecko Tarentolae annularis, has not been considered pathogenic to humans. Promastigotes of L. tarentolae have been previously used as a eukaryotic expression system for the production of recombinant proteins and in the amplification of genes involved in resistance to antileishmanial drugs. To validate the use of this Leishmania species in the screening of antileishmanial drugs, the sensitivity of axenic and intracellular amastigotes of L. tarentolae was compared to the sensitivity showed by Leishmania species causative of human leishmaniasis. The ability of L. tarentolae to grow as axenic amastigotes is first described while its ability to infect several mammalian cells has been confirmed. L. tarentolae amastigotes offer a suitable model for the in vitro screening of compounds for antileishmanial activity.     

Leishmania amazonensis: metabolic adaptations induced by resistance to an ABC transporter blocker

We compared growth rate, cell glucose turnover and expression of ATP-binding-cassette (ABC) transporters in Leishmania amazonensis (LTB0016; LTB) versus LTB(160) selected for resistance against the ABC transporter blocker glibenclamide. Additionally, we evaluated the influence of drug-resistance on Leishmania sensitivity against 2-mercaptoacetate and 2-deoxyglucose. Our data demonstrate that (1) LTB(160) and LTB constitutively express ABC transporters for neutral substrates, (2) glibenclamide resistance induces the expression of organic anion ABC transporters, members of the drug resistance associated transporters subfamily, (3) LTB(160) parasites use less glucose as energy substrate and exhibit a slower glucose uptake than LTB cells, and (4) LTB(160) parasites are less sensitive to 2-mercaptoacetate and 2-deoxyglucose than the glibenclamide-sensitive Leishmania LTB. Together these and previous results indicate that the metabolic adaptations expressed in drug-resistant LTB(160) differ from those described for mammalian drug resistant cells and constitute general mechanisms that underlie drug resistance in Leishmania and may be helpful for identifying alternative strategies to circumvent drug resistance in leishmaniasis.     

Leishmania donovani: assessment of leishmanicidal effects of herbal extracts obtained from plants in the visceral leishmaniasis endemic area of Bihar, India

One obstacle faced in the effective control of visceral leishmaniasis (VL) is the limited number of available treatment options. Furthermore, control efforts have been hindered further by the emergence of Leishmania resistance to many of the available drugs. In this study, we investigated the anti-leishmanial properties of 30 medicinally important plants from the VL endemic area of Bihar, India and compared them to two available anti-leishmanial drugs (sodium antimony gluconate and amphotericin B) and two plant lectins (phytohemagglutinin and concanavalin A) on Leishmania donovani promastigotes in vitro at 24 and 48 h after initiation of culture. We identified eight plant extracts in addition to phytohemagglutinin and amphotericin B that significantly inhibited the growth of promastigotes (p < 0.03). We further studied the minimum effective concentrations as well as the effect on axenic amastigotes viability and the cell cytotoxicity on human peripheral blood of four (Agave americana, Azadirachta indica, Eclipta alba and Piper longum) of the eight plant extracts that induced significant promastigotes killing (p = 0.00098). Effect-based dose finding analysis revealed that the threshold concentration of A. americana required to eliminate L. donovani after 24 h was 0.05 mg/ml. A. indica and P. longum plant extracts eliminated L. donovani promastigotes after 48 h at concentrations of 0.1 and 0.5 mg/ml, respectively. E. alba eliminated the promastigotes at a concentration of 0.5 mg/ml within 24 h. The axenic amastigote killing response was 1.90-, 2.52- and 1.3-fold higher than the promastigote killing response with A. indica, A. americana and E. alba plant extracts, respectively. A. americana and A. indica, respectively, led to approximate 2.5- and 1.3-fold declines in mitochondrial dehydrogenase activity compared with control. E. alba stimulation resulted in an up-regulation of dehydrogenase activity (p = 0.00329). The CSA from P. longum was found to be least cytotoxic; the observed difference in mitochondrial activity was insignificant (p = 0.16314). Further studies may reveal the pharmacological significance of many of the plants with anti-leishmanial properties identified in the present study.     

LaTBP1: a Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP.