scholarly journals Molecular Preadaptation to Antimony Resistance inLeishmania donovanion the Indian Subcontinent

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. e00548-17 ◽  
Author(s):  
F. Dumetz ◽  
B. Cuypers ◽  
H. Imamura ◽  
D. Zander ◽  
E. D’Haenens ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Selection Process ◽  
Indian Subcontinent ◽  
Content Type ◽  
Development Of Resistance ◽  
Direct Exposure ◽  
Main Driver ◽  
Antimony Resistance ◽  
The 19Th Century ◽  
First Time

ABSTRACTAntimonials (Sb) were used for decades for chemotherapy of visceral leishmaniasis (VL). Now abandoned in the Indian subcontinent (ISC) because ofLeishmania donovaniresistance, this drug offers a unique model for understanding drug resistance dynamics. In a previous phylogenomic study, we found two distinct populations ofL. 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 SbIIIsusceptibility of parasites was higher in ISC1 than in the CG, (ii) time to SbIIIresistance 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 SbIIIresistance. 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 beMRPA, a gene involved in SbIIIsequestration 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.IMPORTANCEThe “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-Leishmaniadrug 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 someLeishmaniapopulations of the ISC were preadapted to antimony resistance, likely driven by environmental factors or by drugs used in the 19th century.

scholarly journals MAPK1 of Leishmania donovani Modulates Antimony Susceptibility by Downregulating P-Glycoprotein Efflux Pumps

2015 ◽  
Vol 59 (7) ◽  
pp. 3853-3863 ◽  
Author(s):  
Mansi Garg ◽  
Neena Goyal
Keyword(s):  
Leishmania Donovani ◽  
Drug Sensitivity ◽  
Efflux Pump ◽  
Indian Subcontinent ◽  
Normal Growth ◽  
Efflux Pumps ◽  
Content Type ◽  
P Glycoprotein ◽  
Pump Activity ◽  
Antimony Resistance

ABSTRACTEmergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. InLeishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance inLeishmania donovani. TheL. donovaniMAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug.

scholarly journals Downregulation of Mitogen-Activated Protein Kinase 1 of Leishmania donovani Field Isolates Is Associated with Antimony Resistance

2011 ◽  
Vol 56 (1) ◽  
pp. 518-525 ◽  
Author(s):  
Ashutosh ◽  
Mansi Garg ◽  
Shyam Sundar ◽  
Robert Duncan ◽  
Hira L. Nakhasi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Leishmania Donovani ◽  
Indian Subcontinent ◽  
Field Isolate ◽  
Laboratory Strain ◽  
Mitogen Activated Protein Kinase ◽  
Content Type ◽  
Field Isolates ◽  
Mitogen Activated Protein ◽  
Antimony Resistance

ABSTRACTEmergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoanLeishmania donovani(LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed inEscherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates.

scholarly journals In VivoSelection of Paromomycin and Miltefosine Resistance in Leishmania donovani and L. infantum in a Syrian Hamster Model

2015 ◽  
Vol 59 (8) ◽  
pp. 4714-4718 ◽  
Author(s):  
S. Hendrickx ◽  
A. Mondelaers ◽  
E. Eberhardt ◽  
P. Delputte ◽  
P. Cos ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Indian Subcontinent ◽  
Primary Resistance ◽  
Hamster Model ◽  
Content Type ◽  
Resistance Selection ◽  
Syrian Golden Hamsters ◽  
Resistant Strains ◽  
Selection Of

ABSTRACTIn 2002 and 2006, respectively, miltefosine (MIL) and paromomycin (PMM) were licensed in the Indian subcontinent for treatment of visceral leishmaniasis; however, their future routine use might become jeopardized by the development of drug resistance. Although experimental selection of resistant strainsin vitrohas repeatedly been reported using the less relevant promastigote vector stage, the outcome of resistance selection on intracellular amastigotes was reported to be protocol and species dependent. To corroborate thesein vitrofindings, selection of resistance inLeishmania donovaniandLeishmania infantumwas achieved by successive treatment/relapse cycles in infected Syrian golden hamsters. For PMM, resistant amastigotes were already obtained within 3 treatment/relapse cycles, while their promastigotes retained full susceptibility, thereby sharing the same phenotypic characteristics asin vitro-generated PMM-resistant strains. For MIL, even five treatment/relapse cycles failed to induce significant susceptibility changes in either species, which also corresponds with thein vitroobservations where selection of an MIL-resistant phenotype proved to be quite challenging. In conclusion, these results argue for cautious use of PMM in the field to avoid rapid emergence of primary resistance and highlight the need for additional research on the mechanisms and dynamics of MIL resistance selection.

scholarly journals Lipase Precursor-Like Protein Promotes Miltefosine Tolerance in Leishmania donovani by Enhancing Parasite Infectivity and Eliciting Anti-inflammatory Responses in Host Macrophages

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Deepak Kumar Deep ◽  
Ruchi Singh ◽  
Arpita Kulshrestha ◽  
Saima Wajid ◽  
Poonam Salotra
Keyword(s):  
Leishmania Donovani ◽  
Inflammatory Responses ◽  
Indian Subcontinent ◽  
Interleukin 10 ◽  
Oral Drug ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Tnf Α ◽  
Antileishmanial Drug

ABSTRACT The oral drug miltefosine (MIL) was introduced in the Indian subcontinent in the year 2002 for the treatment of visceral leishmaniasis (VL). However, recent reports on its declining efficacy and increasing relapse rates pose a serious concern. An understanding of the factors contributing to MIL tolerance in Leishmania parasites is critical. In the present study, we assessed the role of the lipase precursor-like protein (Lip) in conferring tolerance to miltefosine by episomally overexpressing Lip in Leishmania donovani (LdLip++). We observed a significant increase (∼3-fold) in the MIL 50% inhibitory concentration (IC50) at both the promastigote (3.90 ± 0.68 µM; P < 0.05) and intracellular amastigote (9.10 ± 0.60 µM; P < 0.05) stages compared to the wild-type counterpart (LdNeo) (MIL IC50s of 1.49 ± 0.20 µM at the promastigote stage and 3.95 ± 0.45 µM at the amastigote stage). LdLip++ parasites exhibited significantly (P < 0.05) increased infectivity to host macrophages and increased metacyclogenesis and tolerance to MIL-induced oxidative stress. The susceptibility of LdLip++ to other antileishmanial drugs (sodium antimony gluconate and amphotericin B) remained unchanged. In comparison to LdNeo, the LdLip++ parasites elicited high host interleukin-10 (IL-10) cytokine expression levels (1.6-fold; P < 0.05) with reduced expression of the cytokine tumor necrosis factor alpha (TNF-α) (1.5-fold; P < 0.05), leading to a significantly (P < 0.01) increased ratio of IL-10/TNF-α. The above-described findings suggest a role of lipase precursor-like protein in conferring tolerance to the oral antileishmanial drug MIL in L. donovani parasites.

scholarly journals Mutations in an Aquaglyceroporin as a Proven Marker of Antimony Clinical Resistance in the Parasite Leishmania donovani

2020 ◽  
Author(s):  
Jade-Eva Potvin ◽  
Philippe Leprohon ◽  
Marine Queffeulou ◽  
Shyam Sundar ◽  
Marc Ouellette
Keyword(s):  
Leishmania Donovani ◽  
Indian Subcontinent ◽  
Copy Number Variations ◽  
Clinical Isolates ◽  
Gene Copy ◽  
Specific Gene ◽  
Single Nucleotide Variants ◽  
Clinical Resistance ◽  
Antimony Resistance

Abstract Background Antimonial drugs have long been the mainstay to treat visceral leishmaniasis. Their use has been discontinued in the Indian subcontinent because of drug resistance, but they are still clinically useful elsewhere. The goal of this study was to find markers of antimony resistance in Leishmania donovani clinical isolates and validate experimentally their role in resistance. Methods The genomes of sensitive and antimony-resistant clinical isolates were sequenced. The role of a specific gene in contributing to resistance was studied by CRISPR-Cas9–mediated gene editing and intracellular drug sensitivity assays. Results Both gene copy number variations and single nucleotide variants were associated with antimony resistance. A homozygous insertion of 2 nucleotides was found in the gene coding for the aquaglyceroporin AQP1 in both resistant isolates. Restoring the wild-type AQP1 open reading frame re-sensitized the 2 independent resistant isolates to antimonials. Alternatively, editing the genome of a sensitive isolate by incorporating the 2-nucleotide insertion in its AQP1 gene led to antimony-resistant parasites. Conclusions Through genomic analysis and CRISPR-Cas9–mediated genome editing we have proven the role of the AQP1 mutations in antimony clinical resistance in L. donovani.

scholarly journals Cationic Liposomal Sodium Stibogluconate (SSG), a Potent Therapeutic Tool for Treatment of Infection by SSG-Sensitive and -Resistant Leishmania donovani

2014 ◽  
Vol 59 (1) ◽  
pp. 344-355 ◽  
Author(s):  
Roma Sinha ◽  
Jayeeta Roychoudhury ◽  
Partha Palit ◽  
Nahid Ali
Keyword(s):  
Leishmania Donovani ◽  
Toxicity Tests ◽  
Sodium Stibogluconate ◽  
Content Type ◽  
Development Of Resistance ◽  
Protective Environment ◽  
Uptake Studies ◽  
Microscopic Counting

ABSTRACTPentavalent antimonials have been the first-line treatment for leishmaniasis for decades. However, the development of resistance to sodium stibogluconate (SSG) has limited its use, especially for treating visceral leishmaniasis (VL). The present work aims to optimize a cationic liposomal formulation of SSG for the treatment of both SSG-sensitive (AG83) and SSG-resistant (GE1F8R and CK1R)Leishmania donovaniinfections. Parasite killing was determined by the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic counting of Giemsa-stained macrophages. Macrophage uptake studies were carried out by confocal microscopic imaging. Parasite-liposome interactions were visualized through transmission electron microscopy. Toxicity tests were performed using assay kits. Organ parasite burdens were determined by microscopic counting and limiting dilution assays. Cytokines were measured by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry. Although all cationic liposomes studied demonstrated leishmanicidal activity, phosphatidylcholine (PC)-dimethyldioctadecylammonium bromide (DDAB) vesicles were most effective, followed by PC-stearylamine (SA) liposomes. Since entrapment of SSG in PC-DDAB liposomes demonstrated enhanced ultrastructural alterations in promastigotes, PC-DDAB-SSG vesicles were further investigatedin vitroandin vivo. PC-DDAB-SSG could effectively alleviate SSG-sensitive and SSG-resistantL. donovaniinfections in the liver, spleen, and bone marrow of BALB/c mice at a dose of SSG (3 mg/kg body weight) not reported previously. The parasiticidal activity of these vesicles was attributed to better interactions with the parasite membranes, resulting in direct killing, and generation of a strong host-protective environment, necessitating a very low dose of SSG for effective cures.

scholarly journals Relapse after Treatment with Miltefosine for Visceral Leishmaniasis Is Associated with Increased Infectivity of the Infecting Leishmania donovani Strain

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Keshav Rai ◽  
Bart Cuypers ◽  
Narayan Raj Bhattarai ◽  
Surendra Uranw ◽  
Maya Berg ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Treatment Failure ◽  
Relapse Rate ◽  
Leishmania Donovani ◽  
Indian Subcontinent ◽  
Drug Susceptibility ◽  
Current View ◽  
Content Type ◽  
Control Programs ◽  
Kala Azar

ABSTRACTLeishmania donovaniis an intracellular protozoan parasite that causes leishmaniasis, which can range from a self-healing cutaneous disease to a fatal visceral disease depending on the infecting species. Miltefosine is currently the latest and only oral antileishmanial that came out of drug discovery pipelines in the past few decades, but recent reports indicate a significant decline in its efficacy against visceral leishmaniasis (also known as kala-azar) in the Indian subcontinent. This relapse rate of up to 20% within 12 months after treatment was shown not to be related to reinfection, drug quality, drug exposure, or drug-resistant parasites. We therefore aimed to assess other phenotypes of the parasite that may affect treatment outcome and found a significant association between the number of metacyclic parasites, parasite infectivity, and patient treatment outcome in the Indian subcontinent. Together with previous studies on resistance ofL. donovaniagainst pentavalent antimonials, these data suggest that the infectivity of the parasite, or related phenotypes, might be a more determinant factor for treatment failure in visceral leishmaniasis than drug susceptibility, warranting a reassessment of our current view on treatment failure and drug resistance in leishmaniasis and beyond.IMPORTANCEThe high miltefosine relapse rate poses a major challenge for the current Kala-Azar Elimination Program in the Indian subcontinent and other leishmaniasis control programs worldwide. This relapse rate could not be related to reinfection, drug-resistant parasites, or reduced treatment quality. Here we report that an increased infectivity of the parasite is associated with miltefosine relapse of visceral leishmaniasis (VL) patients. These results supplement those obtained with antimonial-resistantL. donovaniwhere an increased infectivity was also observed. This challenges the current view ofLeishmaniadrug susceptibility being the biggest parasitic factor that contributes to treatment failure in leishmaniasis. These selected more infectious parasites may pose an additional burden to leishmaniasis control programs, highlighting the importance of multifaceted control measures to achieve leishmaniasis elimination in the Indian subcontinent and other regions where leishmaniasis is endemic.

scholarly journals Antimony-Resistant Clinical Isolates of Leishmania donovani Are Susceptible to Paromomycin and Sitamaquine

2011 ◽  
Vol 55 (6) ◽  
pp. 2916-2921 ◽  
Author(s):  
Arpita Kulshrestha ◽  
Ruchi Singh ◽  
Dhiraj Kumar ◽  
Narender Singh Negi ◽  
Poonam Salotra
Keyword(s):  
Leishmania Donovani ◽  
Content Type ◽  
Field Isolates ◽  
Development Of Resistance ◽  
No Release ◽  
Variable Sensitivity ◽  
The Right ◽  
Treatment Policies ◽  
Intracellular Amastigote

ABSTRACTWidespread antimonial resistance in anthroponotic visceral leishmaniasis (VL) makes it critical to monitor the susceptibility of prevailing field isolates to upcoming antileishmanials in order to frame the right treatment policies to protect these drugs against development of resistance. We aimed to generate the baseline data on naturalin vitrosusceptibility to paromomycin and sitamaquine inLeishmania donovanifield isolates from VL patients (n= 20) coming from zones of varying sodium antimony gluconate (SAG) resistance. We further monitored nitric oxide (NO) release in infected macrophages treated with these drugs. Field isolates exhibited variable sensitivity to paromomycin and sitamaquine with respective mean 50% effective dose (ED50) values ± standard error of the mean (SEM) of 3.9 ± 0.3 μM and 2.1 ± 0.2 μM at the intracellular amastigote stage and 29.8 ± 2.5 μM and 17.7 ± 1.0 μM at the promastigote stage. Susceptibilities at the two parasite stages did not correlate for either drug. Isolates from high SAG resistance zones exhibited significantly lower susceptibility to sitamaquine than those from low SAG resistance zones, while isolates from different zones showed similar susceptibilities to paromomycin. NO release was promoted inL. donovani-infected macrophages upon treatment with paromomycin/sitamaquine. NO inhibitors significantly compromised amastigote killing by sitamaquine, but not by paromomycin. In conclusion, SAG-resistant/sensitive VL isolates were susceptible to both paromomycin and sitamaquine. Paromomycin, exhibiting higher efficacy against SAG-resistant parasites and having a distinct mechanism of action, appears to be a promising drug for combination therapy.

Bonobos

2018 ◽  
Keyword(s):  
19Th Century ◽  
Historical Data ◽  
Line Drawings ◽  
History Of ◽  
The 19Th Century ◽  
First Time

This is a comprehensive, illustrated catalogue of the 200+ marine chronometers in the collections of Royal Museums Greenwich. Every chronometer has been completely dismantled, studied and recorded, and illustrations include especially commissioned line drawings as well as photographs. The collection is also used to illustrate a newly researched and up-to-date chapter describing the history of the marine chronometer, so the book is much more than simply a catalogue. The history chapter naturally includes the story of John Harrison’s pioneering work in creating the first practical marine timekeepers, all four of which are included in the catalogue, newly photographed and described in minute detail for the first time. In fact full technical and historical data are provided for all of the marine chronometers in the collection, to an extent never before attempted, including biographical details of every maker represented. A chapter describes how the 19th century English chronometer was manufactured, and another provides comprehensive and logically arranged information on how to assess and date a given marine chronometer, something collectors and dealers find particularly difficult. For further help in identification of chronometers, appendices include a pictorial record of the number punches used by specific makers to number their movements, and the maker’s punches used by the rough movement makers. There is also a close-up pictorial guide to the various compensation balances used in chronometers in the collection, a technical Glossary of terms used in the catalogue text and a concordance of the various inventory numbers used in the collection over the years.

scholarly journals Catalogue of the primatological collection of the Torino University

2016 ◽  
Vol 3 (2) ◽  
pp. 3 ◽  
Author(s):  
Mara Calvini ◽  
Maria Stella Siori ◽  
Spartaco Gippoliti ◽  
Marco Pavia
Keyword(s):  
20Th Century ◽  
19Th Century ◽  
Great Part ◽  
Type Specimen ◽  
Zoological Garden ◽  
Historical Material ◽  
The 19Th Century ◽  
First Time

The revised catalogue of primatological material stored in the Museo Regionale di Scienze Naturali of Torino and in the Dipartimento di Scienze della Vita e Biologia dei Sistemi of the Università degli Studi di Torino and belonging to the historical material of the Torino University is introduced. The material, 494 specimens belonging to 399 individuals of 122 taxa, is of particular importance since specimens were mainly obtained during the 19th Century and the beginning of the 20th Century. A relevant part of the collection was created by the collaborators of the Museum, among which it is worth to mention F. De Filippi, A. Borelli and E. Festa, while other material came from purchases and donations from private people or the Royal Zoological Garden of Torino. Great part of the specimens is stuffed but also the osteological materials are of particular importance, as many of them derived from the specimens before being prepared and consisting of skulls or more or less complete skeletons. After this revision, the Lectotype and Paralectotypes of <em>Alouatta</em> <em>palliata</em> <em>aequatorialis</em> have been selected, and the type-specimen of the <em>brunnea</em> variety of <em>Cebus</em> <em>albifrons</em> <em>cuscinus</em> has been recognized. In addition, some specimens of particular historical-scientific importance have also been identified and here presented for the first time.

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