Publications by authors named "Franck Dumetz"

8 Publications

  • Page 1 of 1

The Absence of C-5 DNA Methylation in Allows DNA Enrichment from Complex Samples.

Microorganisms 2020 Aug 18;8(8). Epub 2020 Aug 18.

Molecular Parasitology, Institute of Tropical Medicine, 2000 Antwerp, Belgium.

Cytosine C5 methylation is an important epigenetic control mechanism in a wide array of eukaryotic organisms and generally carried out by proteins of the C-5 DNA methyltransferase family (DNMTs). In several protozoans, the status of this mechanism remains elusive, such as in , the causative agent of the disease leishmaniasis in humans and a wide array of vertebrate animals. In this work, we showed that the genome contains a C-5 DNA methyltransferase () from the subfamily, whose function is still unclear, and verified its expression at the RNA level. We created viable overexpressor and knock-out lines of this enzyme and characterized their genome-wide methylation patterns using whole-genome bisulfite sequencing, together with promastigote and amastigote control lines. Interestingly, despite the DNMT6 presence, we found that methylation levels were equal to or lower than 0.0003% at CpG sites, 0.0005% at CHG sites, and 0.0126% at CHH sites at the genomic scale. As none of the methylated sites were retained after manual verification, we conclude that there is no evidence for DNA methylation in this species. We demonstrated that this difference in DNA methylation between the parasite (no detectable DNA methylation) and the vertebrate host (DNA methylation) allowed enrichment of parasite vs. host DNA using methyl-CpG-binding domain columns, readily available in commercial kits. As such, we depleted methylated DNA from mixes of promastigote and amastigote DNA with human DNA, resulting in average human enrichments from 62× up to 263×. These results open a promising avenue for unmethylated DNA enrichment as a pre-enrichment step before sequencing clinical samples.
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http://dx.doi.org/10.3390/microorganisms8081252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463849PMC
August 2020

Parasitic Protozoa: Unusual Roles for G-Quadruplexes in Early-Diverging Eukaryotes.

Molecules 2019 Apr 5;24(7). Epub 2019 Apr 5.

Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.

Guanine-quadruplex (G4) motifs, at both the DNA and RNA levels, have assumed an important place in our understanding of the biology of eukaryotes, bacteria and viruses. However, it is generally little known that their very first description, as well as the foundational work on G4s, was performed on protozoans: unicellular life forms that are often parasitic. In this review, we provide a historical perspective on the discovery of G4s, intertwined with their biological significance across the protozoan kingdom. This is a history in three parts: first, a period of discovery including the first characterisation of a G4 motif at the DNA level in ciliates (environmental protozoa); second, a period less dense in publications concerning protozoa, during which DNA G4s were discovered in both humans and viruses; and third, a period of renewed interest in protozoa, including more mechanistic work in ciliates but also in pathogenic protozoa. This last period has opened an exciting prospect of finding new anti-parasitic drugs to interfere with parasite biology, thus adding new compounds to the therapeutic arsenal.
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http://dx.doi.org/10.3390/molecules24071339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480360PMC
April 2019

Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

mBio 2018 11 6;9(6). Epub 2018 Nov 6.

Unité de Parasitologiemoléculaire et Signalisation, Institut Pasteur, Paris, France

Protozoan parasites of the genus adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast growth. Together our data draw a complex picture of genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. Protozoan parasites of the genus cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the , , and complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.
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http://dx.doi.org/10.1128/mBio.01399-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222132PMC
November 2018

Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent.

Infect Genet Evol 2018 08 19;62:170-178. Epub 2018 Apr 19.

Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium. Electronic address:

Leishmania donovani is the responsible agent for visceral leishmaniasis (VL) in the Indian subcontinent (ISC). The disease is lethal without treatment and causes 0.2 to 0.4 million cases each year. Recently, reports of VL in Nepalese hilly districts have increased as well as VL cases caused by L. donovani from the ISC1 genetic group, a new and emerging genotype. In this study, we perform for the first time an integrated, untargeted genomics and metabolomics approach to characterize ISC1, in comparison with the Core Group (CG), main population that drove the most recent outbreak of VL in the ISC. We show that the ISC1 population is very different from the CG, both at genome and metabolome levels. The genomic differences include SNPs, CNV and small indels in genes coding for known virulence factors, immunogens and surface proteins. Both genomic and metabolic approaches highlighted dissimilarities related to membrane lipids, the nucleotide salvage pathway and the urea cycle in ISC1 versus CG. Many of these pathways and molecules are important for the interaction with the host/extracellular environment. Altogether, our data predict major functional differences in ISC1 versus CG parasites, including virulence. Therefore, particular attention is required to monitor the fate of this emerging ISC1 population in the ISC, especially in a post-VL elimination context.
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http://dx.doi.org/10.1016/j.meegid.2018.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6261844PMC
August 2018

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani.

Nat Ecol Evol 2017 Dec 6;1(12):1961-1969. Epub 2017 Nov 6.

Institut Pasteur, INSERM U1201, Unité de Parasitologie Moléculaire et Signalisation, 75015, Paris, France.

The parasite Leishmania  donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.
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http://dx.doi.org/10.1038/s41559-017-0361-xDOI Listing
December 2017

Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids.

Sci Rep 2017 06 16;7(1):3725. Epub 2017 Jun 16.

Molecular Parasitology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

High throughput sequencing techniques are poorly adapted for in vivo studies of parasites, which require prior in vitro culturing and purification. Trypanosomatids, a group of kinetoplastid protozoans, possess a distinctive feature in their transcriptional mechanism whereby a specific Spliced Leader (SL) sequence is added to the 5'end of each mRNA by trans-splicing. This allows to discriminate Trypansomatid RNA from mammalian RNA and forms the basis of our new multiplexed protocol for high-throughput, selective RNA-sequencing called SL-seq. We provided a proof-of-concept of SL-seq in Leishmania donovani, the main causative agent of visceral leishmaniasis in humans, and successfully applied the method to sequence Leishmania mRNA directly from infected macrophages and from highly diluted mixes with human RNA. mRNA profiles obtained with SL-seq corresponded largely to those obtained from conventional poly-A tail purification methods, indicating both enumerate the same mRNA pool. However, SL-seq offers additional advantages, including lower sequencing depth requirements, fast and simple library prep and high resolution splice site detection. SL-seq is therefore ideal for fast and massive parallel sequencing of parasite transcriptomes directly from host tissues. Since SLs are also present in Nematodes, Cnidaria and primitive chordates, this method could also have high potential for transcriptomics studies in other organisms.
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http://dx.doi.org/10.1038/s41598-017-03987-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473914PMC
June 2017

Evolutionary genomics of epidemic visceral leishmaniasis in the Indian subcontinent.

Elife 2016 Mar 22;5. Epub 2016 Mar 22.

Department of Biomedical Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium.

Leishmania 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 L. 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 L. donovani evolution in the ISC in response to drug treatment.
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http://dx.doi.org/10.7554/eLife.12613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811772PMC
March 2016

Treatment failure in leishmaniasis: drug-resistance or another (epi-) phenotype?

Expert Rev Anti Infect Ther 2014 Aug 6;12(8):937-46. Epub 2014 May 6.

Molecular Parasitology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerpen, Belgium.

Two major leishmaniasis treatments have shown a significant decrease in effectiveness in the last few decades, mostly in the Indian subcontinent but also in other endemic areas. Drug resistance of Leishmania correlated only partially to treatment failure (TF) of pentavalent antimonials, and has so far proved not to be important for the increased miltefosine relapse rates observed in the Indian subcontinent. While other patient- or drug-related factors could also have played a role, recent studies identified several parasite features such as infectivity and host manipulation skills that might contribute to TF. This perspective aims to discuss how different parasitic features other than drug resistance can contribute to TF of leishmaniasis and how this may vary between different epidemiological contexts.
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http://dx.doi.org/10.1586/14787210.2014.916614DOI Listing
August 2014
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