Metabolomics to unveil and understand phenotypic diversity between pathogen populations

Visceral leishmaniasis is caused by a parasite called Leishmania donovani, which every year infects about half a million people and claims several thousand lives. Existing treatments are now becoming less effective due to the emergence of drug resistance. Improving our understanding of the mechanisms used by the parasite to adapt to drugs and achieve resistance is crucial for developing future treatment strategies. Unfortunately, the biological mechanism whereby Leishmania acquires drug resistance is poorly understood. Recent years have brought new technologies with the potential to increase greatly our understanding of drug resistance mechanisms. The latest mass spectrometry techniques allow the metabolome of parasites to be studied rapidly and in great detail. We have applied this approach to determine the metabolome of drug-sensitive and drug-resistant parasites isolated from patients with leishmaniasis. The data show that there are wholesale differences between the isolates and that the membrane composition has been drastically modified in drug-resistant parasites compared with drug-sensitive parasites. Our findings demonstrate that untargeted metabolomics has great potential to identify major metabolic differences between closely related parasite strains and thus should find many applications in distinguishing parasite phenotypes of clinical relevance

Serotonin and corticosterone rhythms in mice exposed to cigarette smoke and in patients with COPD:implication for COPD-associated neuropathogenesis

The circadian timing system controls daily rhythms of physiology and behavior, and disruption of clock function can trigger stressful life events. Daily exposure to cigarette smoke (CS) can lead to alteration in diverse biological and physiological processes. Smoking is associated with mood disorders, including depression and anxiety. Patients with chronic obstructive pulmonary disease (COPD) have abnormal circadian rhythms, reflected by daily changes in respiratory symptoms and lung function. Corticosterone (CORT) is an adrenal steroid that plays a considerable role in stress and anti-inflammatory responses. Serotonin (5-hydroxytryptamine; 5HT) is a neurohormone, which plays a role in sleep/wake regulation and affective disorders. Secretion of stress hormones (CORT and 5HT) is under the control of the circadian clock in the suprachiasmatic nucleus. Since smoking is a contributing factor in the development of COPD, we hypothesize that CS can affect circadian rhythms of CORT and 5HT secretion leading to sleep and mood disorders in smokers and patients with COPD. We measured the daily rhythms of plasma CORT and 5HT in mice following acute (3 d), sub-chronic (10 d) or chronic (6 mo) CS exposure and in plasma from non-smokers, smokers and patients with COPD. Acute and chronic CS exposure affected both the timing (peak phase) and amplitude of the daily rhythm of plasma CORT and 5HT in mice. Acute CS appeared to have subtle time-dependent effects on CORT levels but more pronounced effects on 5HT. As compared with CORT, plasma 5HT was slightly elevated in smokers but was reduced in patients with COPD. Thus, the effects of CS on plasma 5HT were consistent between mice and patients with COPD. Together, these data reveal a significant impact of CS exposure on rhythms of stress hormone secretion and subsequent detrimental effects on cognitive function, depression-like behavior, mood/anxiety and sleep quality in smokers and patients with COPD

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

Sterol 14α-demethylase mutation leads to amphotericin B resistance in Leishmania mexicana

Amphotericin B has emerged as the therapy of choice for use against the leishmaniases. Administration of the drug in its liposomal formulation as a single injection is being promoted in a campaign to bring the leishmaniases under control. Understanding the risks and mechanisms of resistance is therefore of great importance. Here we select amphotericin B-resistant Leishmania mexicana parasites with relative ease. Metabolomic analysis demonstrated that ergosterol, the sterol known to bind the drug, is prevalent in wild-type cells, but diminished in the resistant line, where alternative sterols become prevalent. This indicates that the resistance phenotype is related to loss of drug binding. Comparing sequences of the parasites’ genomes revealed a plethora of single nucleotide polymorphisms that distinguish wild-type and resistant cells, but only one of these was found to be homozygous and associated with a gene encoding an enzyme in the sterol biosynthetic pathway, sterol 14α-demethylase (CYP51). The mutation, N176I, is found outside of the enzyme’s active site, consistent with the fact that the resistant line continues to produce the enzyme’s product. Expression of wild-type sterol 14α-demethylase in the resistant cells caused reversion to drug sensitivity and a restoration of ergosterol synthesis, showing that the mutation is indeed responsible for resistance. The amphotericin B resistant parasites become hypersensitive to pentamidine and also agents that induce oxidative stress. This work reveals the power of combining polyomics approaches, to discover the mechanism underlying drug resistance as well as offering novel insights into the selection of resistance to amphotericin B itself

Evaluation of Leishmania donovani Protein Disulfide Isomerase as a Potential Immunogenic Protein/Vaccine Candidate against Visceral Leishmaniasis

In Leishmania species, Protein disulfide isomerase (PDI) - a redox chaperone, is reported to be involved in its virulence and survival. This protein has also been identified, through proteomics, as a Th1 stimulatory protein in the soluble lysate of a clinical isolate of Leishmania donovani (LdPDI). In the present study, the molecular characterization of LdPDI was carried out and the immunogenicity of recombinant LdPDI (rLdPDI) was assessed by lymphocyte proliferation assay (LTT), nitric oxide (NO) production, estimation of Th1 cytokines (IFN-γ and IL-12) as well as IL-10 in PBMCs of cured/endemic/infected Leishmania patients and cured L. donovani infected hamsters. A significantly higher proliferative response against rLdPDI as well as elevated levels of IFN-γ and IL-12 were observed. The level of IL-10 was found to be highly down regulated in response to rLdPDI. A significant increase in the level of NO production in stimulated hamster macrophages as well as IgG2 antibody and a low level of IgG1 in cured patient's serum was observed. Higher level of IgG2 antibody indicated its Th1 stimulatory potential. The efficacy of pcDNA-LdPDI construct was further evaluated for its prophylactic potential. Vaccination with this construct conferred remarkably good prophylactic efficacy (∼90%) and generated a robust cellular immune response with significant increases in the levels of iNOS transcript as well as TNF-α, IFN-γ and IL-12 cytokines. This was further supported by the high level of IgG2 antibody in vaccinated animals. The in vitro as well as in vivo results thus indicate that LdPDI may be exploited as a potential vaccine candidate against visceral Leishmaniasis (VL)

Evaluation of Nephroprotective and Immunomodulatory Activities of Antioxidants in Combination with Cisplatin against Murine Visceral Leishmaniasis

Leishmaniasis, a neglected tropical disease (NTD) caused by Leishmania, has been put on the World Health Organization agenda for eradication as a part of their Special Programme for Tropical Diseases Research. Visceral leishmaniasis (VL) is a life-threatening disease when no treatment is given. Most of the drugs still used to treat VL are often expensive, difficult to administer, have serious side effects, and several are becoming ineffective because of increasing parasite resistance. Cisplatin is a first-generation platinum-containing drug, used in the treatment of various solid tumors. We have for the first time characterized the in vivo effect of cisplatin in murine experimental visceral leishmaniasis, but at higher doses it is nephrotoxic. Considering the above findings, the present study was designed to evaluate the protective efficacy of the drug in combination with various antioxidants to reduce or prevent cisplatin-induced nephrotoxicity. Drug treatment induces a higher secretion of Th1 cytokines, diminution in parasite burden, and the supplementation of antioxidants which are antagonists of the toxicity helps in reducing the nephrotoxicity

WR279,396, a Third Generation Aminoglycoside Ointment for the Treatment of Leishmania major Cutaneous Leishmaniasis: A Phase 2, Randomized, Double Blind, Placebo Controlled Study

Cutaneous leishmaniasis is due to a small parasite (Leishmania) that creates disfiguring sores, and affects more than one million persons (mainly children) each year. Treating lesions with a cream—instead of with injections as currently done—would greatly improve the well-being of affected patients. No cream formulation that would be efficient and would not create important skin irritation has been identified yet. Here, we tested a new cream formulation (WR279,396) containing paromomycin and gentamicin, two members of a well-known family of antibacterial antibiotics (aminoglycosides). Injectable paromomycin is efficient in other forms of the disease (visceral leishmaniasis). This was a carefully monitored study (phase 2) involving mainly children in Tunisia and France. The cream was applied twice a day for 20 days. The proportion of patients treated with the paromomycin-containing cream (active formulation) that cured (94%) was higher than that observed (71%) in patients treated with a cream that did not contain the active product (placebo formulation). Local irritation affected less than one-third of the patients and was usually mild. This new cream formulation was safe and effective in treating cutaneous leishmaniasis, thereby providing a new, simple, easily applicable, and inexpensive treatment for this neglected disease

Collagen based magnetic nanocomposites for oil removal applications

A stable magnetic nanocomposite of collagen and superparamagnetic iron oxide nanoparticles (SPIONs) is prepared by a simple process utilizing protein wastes from leather industry. Molecular interaction between helical collagen fibers and spherical SPIONs is proven through calorimetric, microscopic and spectroscopic techniques. This nanocomposite exhibited selective oil absorption and magnetic tracking ability, allowing it to be used in oil removal applications. The environmental sustainability of the oil adsorbed nanobiocomposite is also demonstrated here through its conversion into a bi-functional graphitic nanocarbon material via heat treatment. The approach highlights new avenues for converting bio-wastes into useful nanomaterials in scalable and inexpensive ways

Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani

Multiple Mutations in Heterogeneous Miltefosine-Resistant Leishmania major Population as Determined by Whole Genome Sequencing

Leishmania spp. are parasitic protozoa responsible for a spectrum of diseases known as leishmaniasis. There are few drugs available for the treatment of these diseases, and miltefosine is the first oral drug used in treatment of visceral leishmaniasis, a form of the disease that can be lethal if not treated. In this study, we seek to understand the mechanism of action and identify targets of the drug by generating promastigote mutants highly resistant to miltefosine. Two independent mutants were submitted to short read whole genome sequencing. Genome analysis of these mutants has permitted us to identify point mutations in three genes (P-type ATPase, pyridoxal kinase and α-adaptin like protein) that were also present in other independent miltefosine resistant mutants. Some of the new genes identified here could be useful as potential markers for miltefosine resistance in Leishmania. Moreover, our approach has permitted us to highlight that resistance can be highly heterogeneous at the population level with individual clones derived from this population differing both in terms of genotypes but also susceptibility phenotypes. This may have practical applications while studying resistance