Titel Deelnemers "Korte inhoud" "Diversity and dissemination of viruses in pathogenic protozoa" "Senne Heeren, Ilse Maes, Mandy Sanders, Lon-Fye Lye, Vanessa Adaui, Jorge Arevalo, Alejandro Llanos-Cuentas, Lineth Garcia, Philippe Lemey, Stephen M. Beverley, James A. Cotton, Jean-Claude Dujardin, Frederik Van den Broeck" "Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-virus symbioses, a process that may change the epidemiology of leishmaniasis in the region. Heeren et al study the evolutionary genomics of leishmaniasis in Peru and Bolivia to show that parasite hybridization increases the prevalence, diversity and spread of viruses that have been previously associated with disease severity and treatment failure." "High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani" "Gabriel H. Negreira, Pieter Monsieurs, Hideo Imamura, Ilse Maes, Nada Kuk, Akila Yagoubat, Frederik Van den Broeck, Yvon Sterkers, Jean-Claude Dujardin, Malgorzata A. Domagalska" "Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies. [GRAPHICS] ." "Four layer multi-omics reveals molecular responses to aneuploidy in Leishmania" "Bart Cuypers, Pieter Meysman, Ionas Erb, Wout Bittremieux, Dirk Valkenborg, Geert Baggerman, Inge Mertens, Shyam Sundar, Basudha Khanal, Cedric Notredame, Jean-Claude Dujardin, Malgorzata Anna Domagalska, Kris Laukens" "Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between closely related aneuploid strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, as in other Eukaryotes, we observed attenuation of dosage effects for protein complex subunits and in addition, non-cytoplasmic proteins. Differentially expressed transcripts and proteins between aneuploid Leishmania strains also originated from non-aneuploid chromosomes. At protein level, these were enriched for proteins involved in protein metabolism, such as chaperones and chaperonins, peptidases, and heat-shock proteins. In conclusion, our results further support the view that aneuploidy in Leishmania can be adaptive. Additionally, we believe that the high karyotype diversity in vitro and absence of classical transcriptional regulation make Leishmania an attractive model to study processes of protein homeostasis in the context of aneuploidy and beyond." "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 antileishmanial compound" "Aya Hefnawy, Gabriel Negreira, Marlene Jara, James A. Cotton, Ilse Maes, Erika D’Haenens, Hideo Imamura, Bart Cuypers, Pieter Monsieurs, Christina Mouchtoglou, Hans De Winter, Isabel Pintelon, Jean-Pierre Timmermans, Matt Berriman, Mandy Sanders, Julio Martin, Geraldine de Muylder, Jean-Claude Dujardin, Yann Sterckx, Malgorzata Anna Domagalska" "The implementation of prospective drug resistance (DR) studies in the research-and-development (R&D) pipeline is a common practice for many infectious diseases but not for neglected tropical diseases (NTDs). 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 GlaxoSmithKline (GSK) ""Leishbox"" to treat VL. We experimentally selected resistance to TCMDC-143345 in vitro and characterized resistant parasites at the 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 the 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/reduced susceptibility of L. donovani to TCMDC143345. 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 Leishbox 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." "Transcriptional shift and metabolic adaptations during Leishmania quiescence using stationary phase and drug pressure as models" "Marlene Jara, Michael Barrett, Ilse Maes, Clement Regnault, Hideo Imamura, Malgorzata Anna Domagalska, Jean-Claude Dujardin" "Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavorable conditions. Quiescent cells are characterized by slow or non-proliferation and a deep downregulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but the molecular and metabolic features enabling its maintenance are unknown. Here, we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either, through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components, such as amastins and GP63, or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. It is noteworthy that among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers afford novel insight into cell regulation and show commonly modulated features across stimuli and stages." "A novel bioimpedance-based detection of miltefosine susceptibility among clinical leishmania donovani isolates of the Indian subcontinent exhibiting resistance to multiple drugs" "Souradeepa Ghosh, Souvik Biswas, Sandip Mukherjee, Arijit Pal, Aaditya Saxena, Shyam Sundar, Jean-Claude Dujardin, Soumen Das, Syamal Roy, Rupkatha Mukhopadhyay, Budhaditya Mukherjee" "The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model." "A case-control study on the association between intestinal helminth infections and treatment failure in patients with cutaneous Leishmaniasis" "Dalila Y. Martinez, Alejandro Llanos-Cuentas, Jean-Claude Dujardin, Katja Polman, Vanessa Adaui, Marleen Boelaert, Kristien Verdonck" "Background. Endemic regions of cutaneous leishmaniasis (CL) and intestinal helminthiasis overlap. CL treatment with systemic pentavalent antimonial drugs (Sb5+) fails in 10%-30% of patients. The study objective was to assess the etiological role of intestinal helminthiasis in CL treatment failure. Methods. An unmatched case-control study was done in 4 CL treatment sites in Peru in 2012-2015. Cases were CL patients with Sb5+ treatment failure; controls were CL patients with Sb5+ treatment success. Patients with a parasitologically confirmed CL diagnosis who had received supervised Sb5+ treatment and could be classified as cases or controls were eligible. The main exposure variables were intestinal helminthiasis and strongyloidiasis, diagnosed through direct examination, rapid sedimentation, Baermann, Kato-Katz, or agar culture of stool samples. Additional exposure variables were other infections (HIV, human T-lymphotropic virus 1, tuberculosis, hepatitis B, intestinal protozoa) and noninfectious conditions (diabetes, renal insufficiency, and immunosuppressive medication). Age, gender, CL history, probable exposure place, and Leishmania species were treated as potential confounders in multiple logistic regression. Results. There were 94 case and 122 control subjects. Overall, infectious and noninfectious comorbidities were frequent both among cases (64%) and controls (71%). The adjusted odds ratio (OR) for the association between any intestinal helminth infection and CL treatment failure was 0.65 (95% confidence interval [CI], 0.30-1.38), and the adjusted OR for the association between strongyloidiasis and CL treatment failure was 0.34 (95% CI, 0.11-0.92). Conclusions. In the Peruvian setting, high Sb5+ treatment failure rates are not explained by intestinal helminthiasis. On the contrary, strongyloidiasis had a protective effect against treatment failure." "Blackfly ecology and **Onchocerca volvulus** transmission in three formerly hyperendemic foci in Uganda, Tanzania and Cameroon" "Adam Hendy, Jean-Claude Dujardin, Dirk Berkvens" "The concept of fitness in Leishmania" "Manu Vanaerschot, Franck Dumetz, Marlene Jara, Jean-Claude Dujardin, Alicia Ponte-Sucre" "Epidemiology of Leishmaniasis in the time of drug resistance (the Miltefosine Era)" "Jean-Claude Dujardin"