Molecular Preadaptation to Antimony Resistance in Leishmania donovani on the Indian Subcontinent.

Antimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because of Leishmania donovani resistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations of L. donovani: the core group (CG) in the Gangetic plains and ISC1 in the Nepalese highlands. Sb resistance was only encountered within the CG, and a series of potential markers were identified. Here, we analyzed the development of resistance to trivalent antimonials (SbIII) upon experimental selection in ISC1 and CG strains. We observed that (i) baseline SbIII susceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIII resistance was higher for ISC1 parasites than for CG strains, and (iii) untargeted genomic and metabolomic analyses revealed molecular changes along the selection process: these were more numerous in ISC1 than in the CG. Altogether these observations led to the hypothesis that CG parasites are preadapted to SbIII resistance. This hypothesis was experimentally confirmed by showing that only wild-type CG strains could survive a direct exposure to the maximal concentration of SbIII The main driver of this preadaptation was shown to be MRPA, a gene involved in SbIII sequestration and amplified in an intrachromosomal amplicon in all CG strains characterized so far. This amplicon emerged around 1850 in the CG, well before the implementation of antimonials for VL chemotherapy, and we discuss here several hypotheses of selective pressure that could have accompanied its emergence.IMPORTANCE The "antibiotic resistance crisis" is a major challenge for scientists and medical professionals. This steady rise in drug-resistant pathogens also extends to parasitic diseases, with antimony being the first anti-Leishmania drug that fell in the Indian subcontinent (ISC). Leishmaniasis is a major but neglected infectious disease with limited therapeutic options. Therefore, understanding how parasites became resistant to antimonials is of commanding importance. In this study, we experimentally characterized the dynamics of this resistance acquisition and show for the first time that some Leishmania populations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

No stress--Hsp90 and signal transduction in Leishmania.

SUMMARY Hsp90 (a.k.a. Hsp83) plays a significant role in the life cycle control of the protozoan parasite Leishmania donovani. Rather than protecting Leishmania spp. against adverse and stressful environs, Hsp90 is required for the maintenance of the motile, highly proliferative insect stage, the promastigote. However, Hsp90 is also essential for survival and proliferation of the intracellular mammalian stage, the amastigote. Moreover, recent evidence shows Hsp90 and other components of large multi-chaperone complexes as substrates of stage-specific protein phosphorylation pathways, and thus as likely effectors of the signal transduction pathways in Leishmania spp. Future efforts should be directed towards the identification of the protein kinases and the critical phosphorylation sites as targets for novel therapeutic approaches.

Development of rebiana, a natural, non-caloric sweetener.

Rebiana is the common name for high-purity rebaudioside A, a natural non-calorie sweetener 200-300 times more potent than sucrose. It provides zero calories and has a clean, sweet taste with no significant undesirable taste characteristics. It is functional in a wide array of beverages and foods and can be blended with other non-calorie or carbohydrate sweeteners. It is stable under dry conditions, and has much better stability than aspartame or neotame in aqueous food systems. Studies undertaken for the development of a purification process and for the full characterization of the properties of rebiana are reported here.

Molecular modeling approaches for determining gene function: application to a putative poly-A binding protein from Leishmania amazonensis (LaPABP).

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 amino acids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy

Heat shock protein 90 homeostasis controls stage differentiation in Leishmania donovani.

The differentiation of Leishmania parasites from the insect stage, the promastigote, toward the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect-to-mammal transmission. We show here that inactivation of heat shock protein (Hsp) 90, with the use of the drugs geldanamycin or radicicol, mimics transmission and induces the differentiation from the promastigote to the amastigote stage. Geldanamycin also induces a growth arrest of cultured promastigotes that can be forestalled by overexpression of the cytoplasmic Hsp90. Moreover, we demonstrate that Hsp90 serves as a feedback inhibitor of the cellular heat shock response in Leishmania. Our results are consistent with Hsp90 homeostasis serving as cellular thermometer for these primitive eukaryotes, controlling both the heat shock response and morphological differentiation.

The heat shock protein 90 of Leishmania donovani.

The 90-kDa heat shock protein (Hsp90) of Leishmania donovani is a highly abundant cytoplasmic protein and is involved in a variety of cellular processes. Pharmacological deactivation of Hsp90 leads to growth arrest and induces the synthesis of heat shock proteins. Moreover, treatment of promastigote parasites with Hsp90 inhibitors induces the synthesis of amastigote-specific marker proteins and a morphological alteration similar to axenic amastigote differentiation. We propose a role for Hsp90 in the feedback control of the cellular stress response and in the control of the parasite's life cycle.

Use of genetic complementation to identify gene(s) which specify species-specific organ tropism of Leishmania.

We have employed a genetic complementation screening to identify genetic markers of heat stress tolerance and visceralisation of Leishmania infection. Leishmania major, which has a low thermotolerance and which causes cutaneous lesions, was transfected with a cosmid library of L. donovani DNA. The recombinant parasites were then screened either for thermotolerance or selected by repeated passage in BALB/c mice. Cosmids which conferred selective advantage were isolated. Several strategies were tested to identify the gene(s) within the cosmids responsible for the observed selective advantages. Of the approaches tested, the complete sequence analysis of the cosmids and subsequent screening of defined candidate ORFs proved to be the method of choice. Other approaches, such as creation of sub-libraries or transposon insertion strategies proved to be unsuccessful.

Heat shock protein 100 and the amastigote stage-specific A2 proteins of Leishmania donovani.

HSP100 protein in Leishmania spp. plays an important role for the survival and integrity of intracellular amastigotes. The A2 proteins of L. donovani are functionally linked to HSP100. There is evidence for an interdependence between these two proteins, which are both expressed predominantly in the amastigote stage of Leishmania donovani. Mutant strains lacking either of these proteins display very similar phenotypes, i.e. loss of virulence both in vivo and in vitro. Also, both proteins colocalise specifically to small foci within the cytoplasm of amastigotes.

[Thrombolytic delay in myocardial infarction and primary care]. / Retraso trombolítico del infarto de miocardio y atención primaria.

OBJECTIVES: To analyse the effect of a primary care consultation at a health centre or at home and to determine the effect of the use of the pre-hospital electrocardiogram on thrombolytic delay. DESIGN: Analytical cross-sectional study. SETTING: La Safor county (136,000 inhabitants), Valencia, Spain. PATIENTS: Sample of 137 patients from the area admitted for acute myocardial infarction. INTERVENTION: None. MEASUREMENTS AND RESULTS: Multivariate analysis through Cox regression models of the thrombolytic delay, comparing the patients who attended a primary care centre (40, 29.2%) and those who called out a doctor to their home (26, 19.0%) with those who attended hospital (71, 51.8%). The thrombolysis proportions in the groups were analysed with logistic regression. Patients referred from primary care arrived at hospital later than those who attended directly, although a greater thrombolytic delay was only seen in those visited at home (RR 0.25, 95% CI 0.09-0.71). A primary care electrocardiogram (14 patients, 10.2%) reduced the thrombolytic delay (RR 8.81, 95% CI 1.20-64.91) by reducing intra-hospital delay. There were no differences between the groups for the thrombolysis proportion (67 patients, 48.9%). CONCLUSIONS: Patients with infarction seen in primary care reach hospital later. Calling and waiting for the doctor at home increases the thrombolytic delay. An electrocardiogram on the infarction patients who attend a health centre reduces thrombolytic delay by reducing intra-hospital delay.

Retraso trombolítico del infarto de miocardio y atención primaria / Thrombolytic delay in myocardial infarction and primary care

Objetivo. Analizar la influencia de la visita en atención primaria en un centro de salud o en el domicilio y determinar la utilidad del electrocardiograma prehospitalario en el retraso trombolítico del infarto de miocardio. Diseño. Estudio transversal. Emplazamiento. Comarca de la Safor (136.000 habitantes), Valencia (España). Pacientes. Muestra de 137 pacientes ingresados por infarto agudo de miocardio procedentes de la comunidad. Intervenciones. Ninguna. Mediciones y resultados. Análisis multivariante mediante modelos de regresión de Cox del retraso trombolítico comparando los pacientes que acudieron a un centro de atención primaria (40 [29,2 por ciento]) y los que llamaron al médico al domicilio (26 [19,0 por ciento]) con los que acudieron el hospital (71 [51,8 por ciento]). Análisis de la proporción de trombólisis en los grupos mediante regresión logística. Los pacientes remitidos desde atención primaria llegaron más tarde al hospital que los que acudieron directamente, aunque sólo se observó un mayor retraso trombolítico en los visitados en el domicilio (RR 0,25; IC del 95 por ciento, 0,09-0,71). La realización de un electrocardiograma en atención primaria (14 pacientes [10,2 por ciento]) redujo el retraso trombolítico (RR 8,81; IC del 95 por ciento, 1,20-64,91) al disminuir el retraso intrahospitalario. No hubo diferencias en la proporción de trombólisis (67 pacientes [48,9 por ciento]) entre los grupos. Conclusiones. Los pacientes con infarto visitados en atención primaria llegan más tarde al hospital. Llamar y esperar al médico en el domicilio incrementa el retraso trombolítico. La realización de un electrocardiograma a los pacientes con infarto que acuden a un centro de salud reduce el retraso trombolítico al disminuir el retraso intrahospitalario (AU)

Expression and subcellular localization of cpn60 protein family members in Leishmania donovani.

We have identified two diverged members of the cpn60 gene family in Leishmania donovani, causative agent of Indian Kala Azar. One of the genes, cpn60.1, although actively transcribed, is not expressed to detectable levels of protein in cultured L. donovani. The other gene, cpn60.2, which, compared with cpn60.1, shows a higher sequence conservation with the hsp60 genes from Trypanosoma brucei and Trypanosoma cruzi is expressed constitutively in cultured promastigotes. The abundance of the gene product, Cpn60.2, increases by 2.5-fold under heat stress and in axenic amastigotes of L. donovani. Cpn60.2 is also found enriched in mitochondrial cell fractions and localizes to the mitochondrial matrix. We conclude that Cpn60.2 is the major mitochondrial chaperonin in Leishmania.

A novel role for 100 kD heat shock proteins in the parasite Leishmania donovani.

Heat shock proteins of the 100 kD family have been known to confer general stress tolerance in yeast and plants. Several protozoan parasites possess genes for Hsp100 proteins. In Leishmania species the protein is expressed under heat stress and during the mammalian stage, the amastigote. We show here that replacement of the clpB gene which encodes Hsp100 does not affect thermotolerance or general viability in Leishmania donovani insect stages (promastigotes) nor in axenically cultured mammalian stages (amastigotes). However, its expression is required for normal development of the parasite inside mammalian host cells. Hsp100 appears to function as an antagonist of amastigote-to-promastigote differentiation and a promoter of full amastigote development.

Uniform distribution of transcription complexes over the entire Leishmania donovani clpB (hsp 100) gene locus.

We have analyzed the RNA polymerase density on the Leishmania donovani clpB gene locus. Our results show an even distribution of RNA polymerase over the clpB locus indicating an undiscriminative transcription. We conclude that, unlike the hsp70 genes, the clpB gene is not transcribed individually, but rather as part of a larger, polycistronic transcription unit.

Leishmania donovani heat shock protein 100. Characterization and function in amastigote stage differentiation.

We report the cloning and molecular analysis of the Leishmania donovani clpB gene. The protein-coding region is highly conserved compared with its L. major homologue, while 5'- and 3'-flanking DNA sequences display considerable divergence. The encoded mRNA has an unusually long 5'-leader sequence typical for RNAs, which are translated preferentially under heat stress. The gene product, a 100-kDa heat shock protein, Hsp100, becomes abundant only during sustained heat stress, but not under common chemical stresses. Hsp100 associates into trimeric complexes and is found mostly in a cytoplasmic, possibly membrane-associated, localization as determined by immune electron microscopy. Hsp100 shows immediate early expression kinetics during axenic amastigote development. In its absence, expression of at least one amastigote stage-specific protein family is impaired.

Chemical Stress does not Induce Heat Shock Protein Synthesis in Leishmania donovani.

The cellular heat shock response in kinetoplastid protozoa is regulated exclusively at a post-transcriptional level. The heat-inducibility of heat shock protein synthesis is retained under actinomycin C(1) which indicates an inducible translation of heat shock mRNAs. We have also assessed the ability of various chemicals known to be effective triggers of the heat shock response in higher eukaryotes to induce heat shock protein synthesis in Leishmania donovani. None of the tested chemicals elicited a stress response. We propose that the lack of transcription regulation in the kinetoplastida precludes a stress response under chemical stress.

Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite.

In Leishmania major a 100-kDa heat shock protein, Hsp100, is abundant in the intracellular amastigote stage which persists in the mammalian host. A replacement of both clpB alleles which encode Hsp100 does not affect promastigote viability under standard culture conditions but impairs thermotolerance in vitro. In experimental infections of BALB/c inbred mice, the lack of Hsp100 in the gene replacement mutants results in a markedly delayed lesion development compared with that in infections with wild-type L. major. Overexpression of exogenous clpB gene copies can partly restore virulence to the gene replacement mutants. Genetic-selection experiments also reveal a strong pressure for Hsp100 expression in the mammalian stage. This requirement for Hsp100 was also observed in in vitro infection experiments with mouse peritoneal macrophages. These experiments indicated a role for Hsp100 during the development from the promastigote to the amastigote stage. Our results suggest an important role for this parasite heat shock protein during the initial stages of a mammalian infection.

Autocrine regulation of apoptosis and bcl-2 expression by nerve growth factor in early differentiating cerebellar granule neurons involves low affinity neurotrophin receptor.

Cerebellar granule neurons produce homogenous cultures that provide a unique opportunity for quantifying the apoptosis by propidium iodide- and deoxynucleotidyl transferase-flow cytometry combined analysis and for studying its regulation by neurotrophins. Nerve growth factor (NGF) was found to promote postmitotic survival by preventing apoptosis of newly formed and early differentiated granule neurons. This regulation could be through protein bcl-2, which was underexpressed in apoptotic granule neurons and up-regulated by NGF in a dose-dependent manner. Antibodies against low affinity NGF receptors (p75NTR) mimicked the effects of NGF, suggesting that this receptor, which is transiently expressed at high levels in postmitotic granule neurons, is involved in apoptosis signaling. Since these neurons constitutively produce NGF, this is the first demonstration of an autocrine regulation of apoptosis in the CNS. Preliminary results strongly suggest that neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF) are also involved in the regulation of cell death, by first promoting necrosis and then protecting the remaining cells from apoptosis. In contrast, NGF may protect against two forms of cell death and act preferentially at early stages of granule neuron development. The possibility that these neurotrophins may act in parallel and/or in sequence to regulate survival of developing granule neurons through different mechanisms is discussed in the light of findings on neurotrophin and p75NTR patterns, and p75NTR/high affinity Trk receptor coexpression.

Transcription of the Leishmania major Hsp70-I gene locus does not proceed through the noncoding region.

Primary transcripts in kinetoplastid protozoa are generally assumed to be multicistronic. We have analyzed the transcription in the gene locus which encodes the 70-kDa heat shock protein by using nuclear run-on analysis. We find that RNA synthesis in the Hsp70-I gene locus either is terminated or pauses within the intergenic region approximately 250 nt downstream of the polyadenylation site. We therefore propose a discontinuous mode of transcription in the Hsp70 genes of Leishmania major.