@article{Hefnawy2021.01.05.425522,

title = {Genomic and phenotypic characterization of experimentally selected resistant Leishmania donovani reveals a role for dynamin-1 like protein in the mechanism of resistance to a novel anti-leishmanial compound},

author = {Aya Hefnawy and Gabriel Negreira and Marlene Jara and James A Cotton and Ilse Maes and Erika D{textquoteright} Haenens and Hideo Imamura and Bart Cuypers and Pieter Monsieurs and Christina Mouchtoglou and Hans De Winter and Matt Berriman and Mandy Sanders and Julio Martin and Geraldine de Muylder and Jean-Claude Dujardin and Yann G -J Sterckx and Malgorzata Anna Domagalska},

url = {https://www.biorxiv.org/content/early/2021/01/06/2021.01.05.425522},

doi = {10.1101/2021.01.05.425522},

year  = {2021},

date = {2021-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

abstract = {The implementation of prospective drug resistance (DR) studies in the R\&D pipelines is a common practice for many infectious diseases, but not for Neglected Tropical Diseases. Here, we explored and demonstrated the importance of this approach, using as paradigms Leishmania donovani, the etiological agent of Visceral Leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK textquoteleftLeishboxtextquoteright to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at genomic and phenotypic levels. We found that it took more time to develop resistance to TCMDC-143345 than to other drugs in clinical use and that there was no cross resistance to these drugs, suggesting a new and unique mechanism. By whole genome sequencing, we found two mutations in the gene encoding the L. donovani dynamin-1-like protein (LdoDLP1) that were fixed at highest drug pressure. Through phylogenetic analysis, we identified LdoDLP1 as a family member of the dynamin-related proteins, a group of proteins that impacts the shapes of biological membranes by mediating fusion and fission events, with a putative role in mitochondrial fission. We found that L. donovani lines genetically engineered to harbor the two identified LdoDLP1 mutations were resistant to TCMDC-143345 and displayed altered mitochondrial properties. By homology modeling, we showed how the two LdoDLP1 mutations may influence protein structure and function. Taken together, our data reveal a clear involvement of LdoDLP1 in the adaptation/resistance of L. donovani to TCMDC-143345.Importance Humans and their pathogens are continuously locked in a molecular arms race during which the eventual emergence of pathogen drug resistance (DR) seems inevitable. For neglected tropical diseases (NTDs), DR is generally studied retrospectively, once it has already been established in clinical settings. We previously recommended to keep one step ahead in the host-pathogen arms race and implement prospective DR studies in the R\&D pipeline, a common practice for many infectious diseases, but not for NTDs. Here, using Leishmania donovani, the etiological agent of Visceral Leishmaniasis (VL), and TCMDC-143345, a promising compound of the GSK textquoteleftLeishboxtextquoteright to treat VL, as paradigms, we experimentally selected resistance to the compound and proceeded to genomic and phenotypic characterization of DR parasites. The results gathered in the present study suggest a new DR mechanism involving the L. donovani dynamin-1 like protein (LdoDLP1) and demonstrate the practical relevance of prospective DR studies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Deben2020,

title = {Characterization of acquired nutlin-3 resistant non-small cell lung cancer cells},

author = {Christophe Deben and Laurie Freire Boullosa and Andreas Domen and An Wouters and Bart Cuypers and Kris Laukens and Filip Lardon and Patrick Pauwels},

url = {https://doi.org/10.20517/cdr.2020.91},

doi = {10.20517/cdr.2020.91},

issn = {2578-532X},

year  = {2020},

date = {2020-01-01},

journal = {Cancer Drug Resistance},

volume = {3},

pages = {[Online First]},

abstract = {Aim: The purpose of this manuscript is to study the potential characteristics of acquired nutlin-3 resistant non-small cell lung cancer cells (NSCLC). Nutlin-3 is an inhibitor of the murine-double minute 2 protein, the main negative regulator of wild type p53, of which several derivatives are currently in clinical development.Methods: A549 NSCLC cells were exposed to increasing concentrations of nutlin-3 for a period of 18 weeks. Monoclonal derivates were cultured, and the most resistance subclone was selected for whole transcriptome analysis. Gene set enrichment analysis was performed on differentially expressed genes between A549 nutlin-3 resistant cancer cells and the parental A549 p53 wild type cancer cells. Relevant findings were validated at the gene, protein and/or functional level.Results: All nutlin-3 resistant subclones acquired mutations in the TP53 gene, resulting in overexpression of the mutant p53 protein. The most resistant subclone was enriched for genes related to epithelial to mesenchymal transition (EMT), resulting in increased migratory and invasive potential. Furthermore, these cells were enriched in genes related to inflammation, tissue remodelling, and angiogenesis. Importantly, expression of several immune checkpoints, including PD-L1 and PD-L2, was significantly upregulated, and cisplatin-induced cell death was reduced.Conclusion: Transcriptome analysis of a highly nutlin-3 resistant A549 subclone shows the relevance of studying (1) resistance to standard of care chemotherapy; (2) secretion of immunomodulating chemo- and cytokines; (3) immune checkpoint expression; and (4) EMT and invasion in nutlin-3 resistant cancer cells in addition to acquired mutations in the TP53 gene.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{10.7554/eLife.12613,

title = {Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent},

author = {Hideo Imamura and Tim Downing and Frederik Van den Broeck and Mandy J Sanders and Suman Rijal and Shyam Sundar and An Mannaert and Manu Vanaerschot and Maya Berg and G\'{e}raldine De Muylder and Franck Dumetz and Bart Cuypers and Ilse Maes and Malgorzata Domagalska and Saskia Decuypere and Keshav Rai and Surendra Uranw and Narayan Raj Bhattarai and Basudha Khanal and Vijay Kumar Prajapati and Smriti Sharma and Olivia Stark and Gabriele Sch\"{o}nian and Harry P De Koning and Luca Settimo and Benoit Vanhollebeke and Syamal Roy and Bart Ostyn and Marleen Boelaert and Louis Maes and Matthew Berriman and Jean-Claude Dujardin and James A Cotton},

editor = {Dominique Soldati-Favre},

url = {https://doi.org/10.7554/eLife.12613},

doi = {10.7554/eLife.12613},

issn = {2050-084X},

year  = {2016},

date = {2016-03-01},

journal = {eLife},

volume = {5},

pages = {e12613},

publisher = {eLife Sciences Publications, Ltd},

abstract = {textitLeishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of textitL. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of textitL. donovani evolution in the ISC in response to drug treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Raie00611-13b,

title = {Relapse after Treatment with Miltefosine for Visceral Leishmaniasis Is Associated with Increased Infectivity of the Infecting Leishmania donovani Strain},

author = {Keshav Rai and Bart Cuypers and Narayan Raj Bhattarai and Surendra Uranw and Maya Berg and Bart Ostyn and Jean-Claude Dujardin and Suman Rijal and Manu Vanaerschot},

editor = {Louis M Weiss},

url = {https://mbio.asm.org/content/4/5/e00611-13},

doi = {10.1128/mBio.00611-13},

year  = {2013},

date = {2013-01-01},

journal = {mBio},

volume = {4},

number = {5},

publisher = {American Society for Microbiology},

abstract = {Leishmania donovani is 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 of L. donovani against 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.IMPORTANCE The 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-resistant L. donovani where an increased infectivity was also observed. This challenges the current view of Leishmania drug 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{doi:10.1111/mmi.12374,

title = {Metabolic adaptations of Leishmania donovani in relation to differentiation, drug resistance, and drug pressure},

author = {Maya Berg and Manu Vanaerschot and Andris Jankevics and Bart Cuypers and Ilse Maes and Sandip Mukherjee and Basudha Khanal and Suman Rijal and Syamal Roy and Fred Opperdoes and Rainer Breitling and Jean-Claude Dujardin},

url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/mmi.12374},

doi = {10.1111/mmi.12374},

year  = {2013},

date = {2013-01-01},

journal = {Molecular Microbiology},

volume = {90},

number = {2},

pages = {428-442},

abstract = {Summary Antimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG-resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC-MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSG-susceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG-resistant and SSG-sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG-resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG-resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG-resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under SbIII drug pressure.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}