Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to ‘freeze' gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific ‘sensory' genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiatio

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability

MS in South Asians in England: early disease onset and novel pattern of myelin autoimmunity.

BACKGROUND: Epidemiological studies describe a latitude gradient for increased MS prevalence and a preponderance of disease in Caucasian individuals. However, individuals from other ethnic backgrounds and low-risk regions can acquire a raised risk through migration. Nearly a fifth of the London population is of Asian/Asian-British origin and a significant proportion of referrals are from this group. METHODS: We investigated whether there were differences in timing, presentation, severity, and immunology of disease (with respect to CD4 myelin epitope recognition) between individuals in London with MS who were either of S. Asian or Caucasian origin. Individuals of S. Asian origin with MS were compared with healthy S. Asian controls, individuals with MS and of Caucasian origin and Caucasian controls. RESULTS: Age at MS onset is significantly lower in the S. Asian group, attributable to earlier onset specifically in UK-born individuals, though clinical presentation is similar. Analysis of CD4 autoimmunity to myelin antigens shows disease in S. Asian individuals to encompass recognition of novel epitopes; immunity to MBP116-130 in S. Asian individuals was highly disease-specific. CONCLUSIONS: These findings emphasize the need to define disease profiles across ethnicities and identify environmental triggers conferring acquired risk. Such findings must inform choices for immunotherapeutic interventions suitable for all, across ethnicities

Comparison of gene expression patterns among Leishmania braziliensis clinical isolates showing a different in vitro susceptibility to pentavalent antimony

Introduction. Evaluation of Leishmania drug susceptibility depends on in vitro SbV susceptibility assays, which are labour-intensive and may give a biased view of the true parasite resistance. Molecular markers are urgently needed to improve and simplify the monitoring of SbV-resistance. We analysed here the gene expression profile of 21 L. braziliensis clinical isolates in vitro defined as SbV-resistant and -sensitive, in order to identify potential resistance markers. Methods. The differential expression of 13 genes involved in SbV metabolism, oxidative stress or housekeeping functions was analysed during in vitro promastigote growth. Results. Expression profiles were up-regulated for 5 genes only, each time affecting a different set of isolates (mosaic picture of gene expression). Two genes, ODC (ornithine decarboxylase) and TRYR (trypanothione reductase), showed a significantly higher expression rate in the group of SbV-resistant compared to the group of SbV-sensitive parasites (P<0·01). However, analysis of individual isolates showed both markers to explain only partially the drug resistance. Discussion. Our results might be explained by (i) the occurrence of a pleiotropic molecular mechanism leading to the in vitro SbV resistance and/or (ii) the existence of different epi-phenotypes not revealed by the in vitro SbV susceptibility assays, but interfering with the gene expression pattern

Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

BACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular

Comparative Gene Expression Analysis throughout the Life Cycle of Leishmania braziliensis: Diversity of Expression Profiles among Clinical Isolates

Leishmania is a group of parasites (Protozoa, Trypanosomatidae) responsible for a wide spectrum of clinical forms. Among the factors explaining this phenotypic polymorphism, parasite features are important contributors. One approach to identify them consists in characterizing the gene expression profiles throughout the life cycle. In a recent study, the transcriptome of 3 Leishmania species was compared and this revealed species-specific differences, albeit in a low number. A key issue, however, is to ensure that the observed differences are indeed species-specific and not specific of the strains selected for representing the species. In order to illustrate the relevance of this concern, we analyzed here the gene expression profiles of 5 clinical isolates of L. braziliensis at seven time points of the life cycle. Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics: one Leishmania strain is not necessarily representative of a given species

Antimonial Resistance in Leishmania donovani Is Associated with Increased In Vivo Parasite Burden

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis (VL). Antimonials (SSG) have long been the first-line treatment against VL, but have now been replaced by miltefosine (MIL) in the Indian subcontinent due to the emergence of SSG-resistance. Our previous study hypothesised that SSG-resistant L. donovani might have increased in vivo survival skills which could affect the efficacy of other treatments such as MIL. The present study attempts to validate these hypotheses. Fourteen strains derived from Nepalese clinical isolates with documented SSG-susceptibility were infected in BALB/c mice to study their survival capacity in drug free conditions (non-treated mice) and in MIL-treated mice. SSG-resistant parasites caused a significant higher in vivo parasite load compared to SSG-sensitive parasites. However, this did not seem to affect the strains' response to MIL-treatment since parasites from both phenotypes responded equally well to in vivo MIL exposure. We conclude that there is a positive association between SSG-resistance and in vivo survival skills in our sample of L. donovani strains which could suggest a higher virulence of SSG-R strains compared to SSG-S strains. These greater in vivo survival skills of SSG-R parasites do not seem to directly affect their susceptibility to MIL. However, it cannot be excluded that repeated MIL exposure will elicit different adaptations in these SSG-R parasites with superior survival skills compared to the SSG-S parasites. Our results therefore highlight the need to closely monitor drug efficacy in the field, especially in the context of the Kala-azar elimination programme ongoing in the Indian subcontinent

ATP13A2 deficiency disrupts lysosomal polyamine export

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome—a parkinsonism with dementia1—and early-onset Parkinson’s disease2. ATP13A2 offers protection against genetic and environmental risk factors of Parkinson’s disease, whereas loss of ATP13A2 compromises lysosomes3. However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system

Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.

Membrane protein insertion is an essential cellular process. The broad biophysical and topological range of membrane proteins necessitates multiple insertion pathways, which remain incompletely defined. Here, we have discovered a new membrane protein insertion pathway, identified the class of substrates it handles, explained why other known pathways do not work for these substrates and reconstituted the pathway using purified components