The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress.

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

Source Tracing of Leishmania donovani in Emerging Foci of Visceral Leishmaniasis, Western Nepal.

We sequenced Leishmania donovani genomes in blood samples collected in emerging foci of visceral leishmaniasis in western Nepal. We detected lineages very different from the preelimination main parasite population, including a new lineage and a rare one previously reported in eastern Nepal. Our findings underscore the need for genomic surveillance.

Four layer multi-omics reveals molecular responses to aneuploidy in Leishmania.

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.

High throughput single-cell genome sequencing gives insights into the generation and evolution of mosaic aneuploidy in Leishmania donovani.

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.

Genome Analysis of Triploid Hybrid Leishmania Parasite from the Neotropics.

We discovered a hybrid Leishmania parasite in Costa Rica that is genetically similar to hybrids from Panama. Genome analyses demonstrated the hybrid is triploid and identified L. braziliensis and L. guyanensis-related strains as parents. Our findings highlight the existence of poorly sampled Leishmania (Viannia) variants infectious to humans.

Ecological divergence and hybridization of Neotropical Leishmania parasites.

The tropical Andes are an important natural laboratory to understand speciation in many taxa. Here we examined the evolutionary history of parasites of the Leishmania braziliensis species complex based on whole-genome sequencing of 67 isolates from 47 localities in Peru. We first show the origin of Andean Leishmania as a clade of near-clonal lineages that diverged from admixed Amazonian ancestors, accompanied by a significant reduction in genome diversity and large structural variations implicated in host-parasite interactions. Within the Andean species, patterns of population structure were strongly associated with biogeographical origin. Molecular clock and ecological niche modeling suggested that the history of diversification of the Andean lineages is limited to the Late Pleistocene and intimately associated with habitat contractions driven by climate change. These results suggest that changes in forestation over the past 150,000 y have influenced speciation and diversity of these Neotropical parasites. Second, genome-scale analyses provided evidence of meiotic-like recombination between Andean and Amazonian Leishmania species, resulting in full-genome hybrids. The mitochondrial genome of these hybrids consisted of homogeneous uniparental maxicircles, but minicircles originated from both parental species. We further show that mitochondrial minicircles-but not maxicircles-show a similar evolutionary pattern to the nuclear genome, suggesting that compatibility between nuclear-encoded mitochondrial genes and minicircle-encoded guide RNA genes is essential to maintain efficient respiration. By comparing full nuclear and mitochondrial genome ancestries, our data expand our appreciation on the genetic consequences of diversification and hybridization in parasitic protozoa.

Visceral Leishmaniasis, Northern Somalia, 2013-2019.

We identified visceral leishmaniasis caused by Leishmania donovani in a previously unknown focus in northern Somalia. Clinical and epidemiologic characteristics of 118 cases during 2013-2019 in Bosaso, the region's commercial capital, have raised suspicion of visceral leishmaniasis endemicity status there.

Viruses of protozoan parasites and viral therapy: Is the time now right?

Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases.

Mitonuclear genomics challenges the theory of clonality in Trypanosoma congolense: Reply to Tibayrenc and Ayala.

We recently published the first genomic diversity study of Trypanosoma congolense, a major aetiological agent of Animal African Trypanosomiasis. We demonstrated striking levels of SNP and indel diversity in the Eastern province of Zambia as a consequence of hybridization between divergent trypanosome lineages. We concluded that these and earlier findings in T. congolense challenge the predominant clonal evolution (PCE) model. In a recent comment, Tibayrenc and Ayala claim that there are many features in T. congolense supporting their theory of clonality. While we can follow the reasoning of the authors, we also identify major limitations in their theory and interpretations that resulted in incorrect conclusions. First, we argue that each T. congolense subgroup should be analysed independently as they may represent different (sub)species rather than "near-clades". Second, the authors neglect major findings of two robust population genetic studies on Savannah T. congolense that provide clear evidence of frequent recombination. Third, we reveal additional events of introgressive hybridization in T. congolense by analysing the maxicircle coding region using next-generation sequencing analyses. At last, we pinpoint two important misinterpretations by the authors and show that there are no spatially and temporally widespread clones in T. congolense. We stand by our earlier conclusions that the clonal framework is unlikely to accurately model the population structure of T. congolense. Other theoretical frameworks such as Maynard Smith's epidemic model may better represent the complex ancestry seen in T. congolense, where clones delimited in space and time arise against a background of recombination.

Discovery and genomic analyses of hybridization between divergent lineages of Trypanosoma congolense, causative agent of Animal African Trypanosomiasis.

Hybrid populations and introgressive hybridization remain poorly documented in pathogenic micro-organisms, as such that genetic exchange has been argued to play a minor role in their evolution. Recent work demonstrated the existence of hybrid microsatellite profiles in Trypanosoma congolense, a parasitic protozoan with detrimental effects on livestock productivity in sub-Saharan Africa. Here, we present the first population genomic study of T. congolense, revealing a remarkable number of single nucleotide polymorphisms (SNPs), small insertions/deletions (indels) and gene deletions among 56 parasite genomes from ten African countries. One group of parasites from Zambia was particularly diverse, displaying a substantial number of heterozygous SNP and indel sites compared to T. congolense parasites from the nine other sub-Saharan countries. Genomewide 5-kb phylogenetic analyses based on phased SNP data revealed that these parasites were the product of hybridization between phylogenetically distinct T. congolense lineages. Other parasites within the same region in Zambia presented a mosaic of haplotypic ancestry and genetic variability, indicating that hybrid parasites persisted and recombined beyond the initial hybridization event. Our observations challenge traditional views of trypanosome population biology and encourage future research on the role of hybridization in spreading genes for drug resistance, pathogenicity and virulence.

Antimony-Resistant Leishmania donovani Exploits miR-466i To Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection.

Infection with antimony-resistant Leishmania donovani (Sb(R)LD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (Sb(S)LD) infection. Sb(R)LD, but not Sb(S)LD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mϕs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mϕs from MyD88(-/-) mice with Sb(R)LD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mϕs. Τhese attributes were not seen with Sb(S)LD in similar experiments. Further, Sb(R)LD infection upregulated miR-466i, which binds with 3'-untranslated region, leading to the downregulation of MyD88. Infection of MyD88(-/-) Mϕ or IL-12(-/-) Mϕ with Sb(R)LD induced IL-10 surge at 4 h, whereas the same in WT Mϕ started from 12 h. Thus, absence of IL-12 in MyD88(-/-) mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD88(-/-) mice with Sb(R)LD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that Sb(R)LD-infected MyD88(-/-) mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that Sb(R)LD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage.

Species typing in dermal leishmaniasis.

Leishmania is an infectious protozoan parasite related to African and American trypanosomes. All Leishmania species that are pathogenic to humans can cause dermal disease. When one is confronted with cutaneous leishmaniasis, identification of the causative species is relevant in both clinical and epidemiological studies, case management, and control. This review gives an overview of the currently existing and most used assays for species discrimination, with a critical appraisal of the limitations of each technique. The consensus taxonomy for the genus is outlined, including debatable species designations. Finally, a numerical literature analysis is presented that describes which methods are most used in various countries and regions in the world, and for which purposes.

Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

Imipramine exploits histone deacetylase 11 to increase the IL-12/IL-10 ratio in macrophages infected with antimony-resistant Leishmania donovani and clears organ parasites in experimental infection.

The efflux of antimony through multidrug resistance protein (MDR)-1 is the key factor in the failure of metalloid treatment in kala-azar patients infected with antimony-resistant Leishmania donovani (Sb(R)LD). Previously we showed that MDR-1 upregulation in Sb(R)LD infection is IL-10-dependent. Imipramine, a drug in use for the treatment of depression and nocturnal enuresis in children, inhibits IL-10 production from Sb(R)LD-infected macrophages (Sb(R)LD-Mϕs) and favors accumulation of surrogates of antimonials. It inhibits IL-10-driven nuclear translocation of c-Fos/c-Jun, critical for enhanced MDR-1 expression. The drug upregulates histone deacetylase 11, which inhibits acetylation of IL-10 promoter, leading to a decrease in IL-10 production from Sb(R)LD-Mϕs. It abrogates Sb(R)LD-mediated p50/c-Rel binding to IL-10 promoter and preferentially recruits p65/RelB to IL-12 p35 and p40 promoters, causing a decrease in IL-10 and overproduction of IL-12 in Sb(R)LD-Mϕs. Histone deacetylase 11 per se does not influence IL-12 promoter activity. Instead, a imipramine-mediated decreased IL-10 level allows optimal IL-12 production in Sb(R)LD-Mϕs. Furthermore, exogenous rIL-12 inhibits intracellular Sb(R)LD replication, which can be mimicked by the presence of Ab to IL-10. This observation indicated that reciprocity exists between IL-10 and IL-12 and that imipramine tips the balance toward an increased IL-12/IL-10 ratio in Sb(R)LD-Mϕs. Oral treatment of infected BALB/c mice with imipramine in combination with sodium stibogluconate cleared organ Sb(R)LD parasites and caused an expansion of the antileishmanial T cell repertoire where sodium stibogluconate alone had no effect. Our study deciphers a detailed molecular mechanism of imipramine-mediated regulation of IL-10/IL-12 reciprocity and its impact on Sb(R)LD clearance from infected hosts.

Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1.

The molecular mechanism of antimony-resistant Leishmania donovani (Sb(R)LD)-driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Ms) has been investigated. This study showed that both promastigote and amastigote forms of Sb(R)LD, but not the antimony-sensitive form of LD, express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in Sb(R)LD (KD Sb(R)LD), compromises the ability to induce the above effects. Infection of Ms with KD Sb(R)LD enhanced the sensitivity toward antimonials compared with infection with Sb(R)LD, and infection of BALB/c mice with KD Sb(R)LD caused significantly less organ parasite burden compared with infection induced by Sb(R)LD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by Sb(R)LD to activate ERK and nuclear translocation of NF-κB involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway. Interestingly both recombinant IL-10 and Sb(R)LD up-regulate MDR1 in M with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Ms derived from IL-10(-/-) mice are unable to show MDR1 up-regulation on infection with Sb(R)LD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10-driven MDR1 up-regulation is c-Fos/c-Jun and not NF-κB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs.

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.

Experimental resistance to drug combinations in Leishmania donovani: metabolic and phenotypic adaptations.

Together with vector control, chemotherapy is an essential tool for the control of visceral leishmaniasis (VL), but its efficacy is jeopardized by growing resistance and treatment failure against first-line drugs. To delay the emergence of resistance, the use of drug combinations of existing antileishmanial agents has been tested systematically in clinical trials for the treatment of visceral leishmaniasis (VL). In vitro, Leishmania donovani promastigotes are able to develop experimental resistance to several combinations of different antileishmanial drugs after 10 weeks of drug pressure. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics approach, we identified metabolic changes in lines that were experimentally resistant to drug combinations and their respective single-resistant lines. This highlighted both collective metabolic changes (found in all combination therapy-resistant [CTR] lines) and specific ones (found in certain CTR lines). We demonstrated that single-resistant and CTR parasite cell lines show distinct metabolic adaptations, which all converge on the same defensive mechanisms that were experimentally validated: protection against drug-induced and external oxidative stress and changes in membrane fluidity. The membrane fluidity changes were accompanied by changes in drug uptake only in the lines that were resistant against drug combinations with antimonials, and surprisingly, drug accumulation was higher in these lines. Together, these results highlight the importance and the central role of protection against oxidative stress in the different resistant lines. Ultimately, these phenotypic changes might interfere with the mode of action of all drugs that are currently used for the treatment of VL and should be taken into account in drug development.

Failure of miltefosine in visceral leishmaniasis is associated with low drug exposure.

BACKGROUND: Recent reports indicated high miltefosine treatment failure rates for visceral leishmaniasis (VL) on the Indian subcontinent. To further explore the pharmacological factors associated with these treatment failures, a population pharmacokinetic-pharmacodynamic study was performed to examine the relationship between miltefosine drug exposure and treatment failure in a cohort of Nepalese patients with VL. METHODS: Miltefosine steady-state blood concentrations at the end of treatment were analyzed using liquid chromatography tandem mass spectrometry. A population pharmacokinetic-pharmacodynamic analysis was performed using nonlinear mixed-effects modeling and a logistic regression model. Individual estimates of miltefosine exposure were explored for their relationship with treatment failure. RESULTS: The overall probability of treatment failure was 21%. The time that the blood concentration was >10 times the half maximal effective concentration of miltefosine (median, 30.2 days) was significantly associated with treatment failure: each 1-day decrease in miltefosine exposure was associated with a 1.08-fold (95% confidence interval, 1.01-1.17) increased odds of treatment failure. CONCLUSIONS: Achieving a sufficient exposure to miltefosine is a significant and critical factor for VL treatment success, suggesting an urgent need to evaluate the recently proposed optimal allometric miltefosine dosing regimen. This study establishes the first evidence for a drug exposure-effect relationship for miltefosine in the treatment of VL.