Cloning,characterization and preliminary crystallographic analysis of Leishmania hypoxanthine–guanine phosphoribosyltransferase

Hypoxanthine–guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a Leishmania tarentolae genomic library and the sequence determined. The L. tarentolae hgprt gene contains a 633-nucleotide open reading frame that encodes a 23.4-kDa protein. A pairwise alignment of the different HGPRT's sequences revealed a 26%–53% sequence identity with the Leishmania sequences and 87% identity to the HGPRT of Leishmania donovani. A recombinant protein was expressed in Escherichia coli, purified to homogeneity and found to retain enzymatic activity. The steady-state kinetic parameters were determined for the recombinant enzyme and the enzyme is active as a homodimer in solution. Single crystals were obtained for the L. tarentolae HGPRT representing the first Leishmania HGPRT crystallized and initial crystallographic data were collected. The crystals obtained belong to the orthorhombic space group (P2₁2₁2₁) with unit cell parameters a=58.104 Å, b=85.443 Å and c=87.598 Å and diffract to a resolution of 2.3 Å. The availability of the HGPRT enzyme from Leishmania and its crystallization suitable for X-ray diffraction data collection should provide the basis for a functional and structural analysis of this enzyme, which has been proposed as a potential target for rational drug design, in a Leishmania model system.     

Expression,Purification, and Evaluation of Anti-IL-2Rα Antibody Secreted by Leishmania tarentolae

International Journal of Peptide Research and Therapeutics - Producing a functional anti-IL-2Rα antibody in Leishmania tarentolae, a trypanosomatid protozoan non-pathogenic to human, is a...     

Inhibition of Leishmania (Leishmania) amazonensis and Rat Arginases by Green Tea EGCG, (+)-Catechin and (?)-Epicatechin: A Comparative Structural Analysis of Enzyme-Inhibitor Interactions

Epigallocatechin-3-gallate (EGCG), a dietary polyphenol (flavanol) from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+)-catechin and (−)-epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L) and rat liver arginase (ARG-1). The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+)-catechin and (−)-epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+)-catechin (IC₅₀ = 0.8 µM) followed by (−)-epicatechin (IC₅₀ = 1.8 µM), gallic acid (IC₅₀ = 2.2 µM) and EGCG (IC₅₀ = 3.8 µM). Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC₅₀ values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.     

Redox-active dinitrodiphenylthioethers against Leishmania: Synthesis,structure–activity relationships and mechanism of action studies

BTB 06237 (2-[(2,4-dichloro-5-methylphenyl)sulfanyl]-1,3-dinitro-5-(trifluoromethyl) benzene), a compound previously identified through QSAR pharmacophore development and a virtual screen of the Maybridge database, possesses potent and selective activity against Leishmania parasites. In the present study, several analogs of BTB 06237 were synthesized and analyzed for activity against Leishmania axenic amastigotes, their ability to reduce the level of parasitemia in peritoneal macrophages, and their ability to generate reactive oxygen species (ROS) in L. donovani promastigotes. It was found that an aromatic ring must be present in the position occupied by the 2,4-dichloro-5-methylphenyl group in the lead compound, but changing the functional groups generally has little effect on the antileishmanial potency. Alterations to the 1,3-dinitro-5-(trifluoromethyl)benzene ring have more influence on antiparasitic activity with two aromatic nitro groups and a third electron-withdrawing group being required. This structural requirement corresponds with redox potential, the ability to generate ROS in the parasites, and dissipation of the mitochondrial membrane potential. Finally, we used this collection of data to design a new antileishmanial compound with strong activity in vitro and improved properties as an antileishmanial candidate.     

Identification, purification, and characterization of a secretory serine protease in an Indian strain of Leishmania donovani

An aprotinin sensitive serine protease was identified in the culture supernatant of the Indian strain of Leishmania donovani (MHOM/IN/1983/AG83). The protease was subsequently purified and characterized. The apparent molecular mass of the enzyme was 115 kDa in SDS-PAGE under non-reducing condition, while on reduction it showed a 56 kDa protein band indicating that the protease is a dimeric protein. The purified enzyme was optimally active at the pH and temperature of 7.5 and 28 degrees C, respectively. Assays of thermal stability indicated that the enzyme preserved 59% of activity even after pretreatment at 42 degrees C for 1 h. The purified protease was not glycosylated and its isoelectric pI was 5.0. N-alpha-p-tosyl-L-arginine methylester (TAME) appeared to be relatively better substrate among the commonly used synthetic substrates. The enzyme was inhibited by Ca(2+) and Mn(2+), but activated by Zn(2+). The protease could play important role(s) in the pathogenesis of visceral leishmaniasis or kala-azar.     

Neutrophil granulocytes--Trojan horses for Leishmania major and other intracellular microbes?

Polymorphonuclear neutrophil granulocytes (PMNs) possess numerous effector mechanisms to kill ingested pathogens as the first line of defence. However, several microorganisms evade intracellular killing in neutrophils, survive and retain infectivity. There is increasing evidence that several pathogens even multiply within neutrophils. Taking Leishmania major as a prototypic intracellular pathogen, we suggest an evasion strategy that includes the manipulation of PMNs in such a way that the pathogens are able to use the granulocytes as host cells. The ability to survive and maintain infectivity in PMNs subsequently enables these organisms to establish productive infection. These organisms can use granulocytes as Trojan horses before they enter their definitive host cells, the macrophages.     

Phlebotomine sand flies and Leishmania parasites: friends or foes?

Leishmania parasites need phlebotomine sand flies to complete their life cycle and to propagate. This review looks at Leishmania-sand fly interactions as the parasites develop from amastigotes to infectious metacyclics, highlighting recent findings concerning the evolutionary adaptations that ensure survival of the parasites. Such adaptations include secretion of phosphoglycans, which protect the parasite from digestive enzymes; production of chitinases that degrade the stomodeal valve of the sand fly; secretion of a neuropeptide that arrests midgut and hindgut peristalsis; and attaching to the midgut to avoid expulsion.     

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

Leishmania infection in humans, dogs and sandflies was examined in the endemic visceral leishmaniasis (VL) municipality of Raposa, state of Maranh?o, Brazil. In this study, we examined Leishmania chagasi infection in the blood serum of both humans and Canis familiaris and the natural Leishmania sp. infection rate in the sandfly vector, Lutzomyia longipalpis. Enzyme-linked immunosorbent assay, indirect immunofluorescence reaction and polymerase chain reaction were performed to detect Leishmania infections in humans, dogs and sandflies, respectively. Overall, 186 out of 986 studied human beings were infected with L. chagasi parasites, representing an infection prevalence of 18.9%. An even higher infection rate was detected in dogs, where 66 (47.8%) out of 138 were infected. Among all Lu. longipalpis captured (n = 1,881), only 26.7% were females. The Leishmania infection frequency for the vector Lu. longipalpis was 1.56%. Remarkably, all infected sandflies were found in the peridomiciliary area. Furthermore, a high incidence of asymptomatic forms of VL in the human and canine populations was observed. The results of this study suggest autochthonous transmission of L. chagasi in this endemic area for visceral leishmaniasis because infection by Leishmania sp. was identified in all important elements of the transmission chain.     

Iron overload potentiates diet-induced hypercholesterolemia and reduces liver PPAR-α expression in hamsters

Iron stores and lipids are related to the development of cardiovascular disease. Given that peroxisome proliferator-activated receptor alpha (PPAR-α) regulates important physiological processes that impact lipid and glucose homeostasis, we decided to investigate the effects of iron overload on serum lipids and the liver expression of PPAR-α, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol 7α-hydroxylase. Hamsters were divided into four groups. The standard group (S) was fed the AIN-93M diet, the SI group was fed the diet and iron injections, the hypercholesterolemic group (H) was fed a standard diet containing cholesterol, and the HI group was fed a high-cholesterol diet and iron injections. Serum cholesterol in the HI group was higher than in the H group. Gene expression analysis of PPAR-α showed that the HI group had a lower PPAR-α expression than H. These data show that iron, when associated with a high-fat diet, can cause increased serum cholesterol levels, possibly due to a reduction in PPAR-α expression.     

Nucleoside hydrolase from Leishmania major. Cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-? crystal structure.

Protozoan parasites lack the pathway of the de novo synthesis of purines and depend on host-derived nucleosides and nucleotides to salvage purines for DNA and RNA synthesis. Nucleoside hydrolase is a central enzyme in the purine salvage pathway and represents a prime target for the development of anti-parasitic drugs. The full-length cDNA for nucleoside hydrolase from Leishmania major was cloned and sequence analysis revealed that the L. major nucleoside hydrolase shares 78% sequence identity with the nonspecific nucleoside hydrolase from Crithidia fasciculata. The L. major enzyme was overexpressed in Escherichia coli and purified to over 95% homogeneity. The L. major nucleoside hydrolase was identified as a nonspecific nucleoside hydrolase since it demonstrates the characteristics: 1) efficient utilization of p-nitrophenyl beta-D-ribofuranoside as a substrate; 2) recognition of both inosine and uridine nucleosides as favored substrates; and 3) significant activity with all of the naturally occurring purine and pyrimidine nucleosides. The crystal structure of the L. major nucleoside hydrolase revealed a bound Ca(2+) ion in the active site with five oxygen ligands from Asp-10, Asp-15 (bidentate), Thr-126 (carbonyl), and Asp-241. The structure is similar to the C. fasciculata IU-nucleoside hydrolase apoenzyme. Despite the similarities, the catalytic specificities differ substantially. Relative values of k(cat) for the L. major enzyme with inosine, adenosine, guanosine, uridine, and cytidine as substrates are 100, 0.5, 0.5, 27 and 0.3; while those for the enzyme from C. fasciculata are 100, 15, 14, 510, and 36 for the same substrates. Iminoribitol analogues of the transition state are nanomolar inhibitors. The results provide new information for purine and pyrimidine salvage pathways in Leishmania.     

Structural,molecular motions,and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.     

Synthesis and anti-leishmanial activity of 1-aryl-β-carboline derivatives against Leishmania donovani

β-carbolines from various natural and synthetic sources have been known to show diverse biological activities. As a part of our current ongoing project to search for potent natural product-derived anti-leishmanial compounds, we have synthesized a series of substituted 1-aryl-β-carboline derivatives. A total of 22 compounds were synthesized and tested in vitro against Leishmania donovani, out of which 6 compounds (4, 5, 10, 11, 19 and 22) showed notably more activity than the standard miltefosine (IC(50) 12.07±0.82 μM), with compound 4 being the most potent (IC(50) 2.16±0.26 μM).     

Epidemiological and clinical features of Leishmania (Viannia) braziliensis American cutaneous and mucocutaneous leishmaniasis in the State of Espírito Santo, Brazil

Between 1985 and 2000, epidemiological surveys of the American tegumentary leishmaniasis (ATL) were carried out in several rural and urban communities in Espírito Santo, Brazil. A total of 100 stocks of Leishmania (comprising isolates from both human and canine hosts with ATL) were identified by two methods of molecular characterization, using specific monoclonal antibodies and multilocus enzyme electrophoresis. Parasite isolates from 19 municipalities were found to belong to the same zymodeme and serodeme type as of the Leishmania (Viannia) braziliensis reference strain. In contrast, our genotyping studies have shown intra-specific variation among these parasites (comparisons of the variability of the internal transcribed spacers between the small and large subunits of the rRNA genes of the 22 stocks studied revealed at least 11 genotypes). Two main clusters of L. (V.) braziliensis genotypes were observed, representing parasites collected from different endemic regions in the state, where transmission reflects distinct eco-epidemiological features. Infection with this pathogen was associated with the characteristic disease forms, but neither the clinical outcome nor the response to treatment could be related to the genetic polymorphism of the isolates, as defined by using the proposed methodology.     

Diurnal rhythms of common α-subunit mRNA expression in the pars tuberalis of hamsters and chickens

To clarify whether the common -subunit of glycoprotein hormones is involved in photic signal transduction, -subunit mRNA levels in the pars tuberalis (PT) of both hamsters and chickens were estimated at different time points of the day/night cycle by laser capture microdissection (LCM) and real-time quantitative polymerase chain reaction (PCR). Distinct diurnal rhythms were found for -subunit mRNA expression in both species. In the hamster PT, -subunit mRNA levels gradually increased during the dark phase; the diurnal peak was found at time (ZT) 21. The lowest value was obtained at ZT 5 during the day. In the chicken PT, -subunit mRNA levels were maintained at a low constant level at night between ZT 13 and 21. Thus, -subunit mRNA expression in the PT depends on the light–dark cycle and may be controlled by the pineal hormone melatonin. The effect of various photoperiods on the hamster PT was examined by real-time PCR, immunohistochemistry, and electron microscopy. In hamsters kept under short photoperiod (L/D=8 h:16 h) or complete darkness, a dramatic decrease of -subunit mRNA level was induced, and the PT-specific cells accumulated glycogen-like particles and enlarged secretory granules. Under long photoperiods (L/D=16 h:8 h), however, the -subunit mRNA level was elevated and the PT-specific cells exhibited highly active features, i.e., piles of lamellar cisternae of rough endoplasmic reticulum and well-developed Golgi complexes. The -subunit synthesized by the PT-specific cells may therefore participate in the circadian and seasonal regulation of endocrine activities.     

Seasonal changes of thermoregulatory efficiency in Djungarian hamsters

In their natural habitat, Djungarian hamsters are faced with dramatic seasonal changes. This requires various morphological and physiological adaptations allowing cope with harsh climate and food shortage, particularly in winter. These seasonal changes are controlled by the photoperiod and can be observed also in the laboratory at room temperature. The aim of the present study was to investigate if the efficiency of thermoregulation also depends on the photoperiod. For this reason, Djungarian hamsters were transferred to short-day conditions (SDC) with 8 h light and 16 h darkness. Two-thirds of the animals were classified as responders showing the typical seasonal changes – decrease of body mass, fur change, testes regression, vagina closing. The total activity per day did not change but, the nocturnal activity was spread over the longer dark time. The body temperature decreased, and the animals showed regular daily torpor. To investigate the thermoregulatory efficiency, body temperatures were correlated with motor activity. The obtained regression coefficients describe formally the effect of motor activity on body temperature, a measure for the efficiency of thermoregulation. In SDC, the coefficients were elevated, both during rest and activity, i.e. the same amount of activity did produce a larger increase in body temperature. Under field conditions, this might be an additional mechanism to compensate the bigger in winter heat loss. Also, the high coefficients may support the increase in body temperature at the end of a torpor phase by a bout of motor activity. The results show that, seasonal changes of thermoregulatory efficiency are an effective accessory way to cope with different temperatures in hamsters’ natural environment.