BACKGROUND: The present study investigates implications of a sub-chromosomal deletion in Leishmania infantum strains, the causative agent of American Visceral Leishmaniasis (AVL). Primarily found in New World strains, the deletion leads to the absence of the ecto-3'-nucleotidase/nuclease enzyme, impacting parasite virulence, pathogenicity, and drug susceptibility. The factors favoring prevalence and the widespread geographic distribution of these deleted mutant parasites (DEL) in the NW (NW) are discussed under the generated data. METHODS: We conducted phenotypic assessments of the sub-chromosomal deletion through in vitro assays with axenic parasites and experimental infections in both in vitro and in vivo models of vertebrate and invertebrate hosts using geographically diverse mutant field isolates. RESULTS: Despite reduced pathogenicity, the DEL strains efficiently infect vertebrate hosts and exhibit relevant differences, including enhanced metacyclogenesis and colonization rates in sand flies, potentially facilitating transmission. This combination may represent a more effective way to maintain and disperse the transmission cycle of DEL strains. CONCLUSIONS: Phenotypic assessments reveal altered parasite fitness, with potential enhanced transmissibility at the population level. Reduced susceptibility of DEL strains to miltefosine, a key drug in VL treatment, further complicates control efforts. The study underscores the importance of typing parasite genomes for surveillance and control, advocating for the sub-chromosomal deletion as a molecular marker in AVL management.

• MeSH
• Gene Deletion * MeSH
• Leishmania infantum * genetics   pathogenicity   MeSH
• Leishmaniasis, Visceral * parasitology   transmission   epidemiology   MeSH
• Humans MeSH
• Mice MeSH
• Psychodidae parasitology   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

• MeSH
• Genomics MeSH
• Crosses, Genetic MeSH
• Leishmania donovani * genetics   MeSH
• Leishmaniasis, Cutaneous * MeSH
• Leishmaniasis, Visceral * diagnosis   prevention & control   epidemiology   MeSH
• Phlebotomus * genetics   MeSH
• Psychodidae * genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Instability is an intriguing characteristic of many protist genomes, and trypanosomatids are not an exception in this respect. Some regions of trypanosomatid genomes evolve fast. For instance, the trypanosomatid mitochondrial (kinetoplast) genome consists of fairly conserved maxicircle and minicircle molecules that can, nevertheless, possess high nucleotide substitution rates between closely related strains. Recent experiments have demonstrated that rapid laboratory evolution can result in the non-functionality of multiple genes of kinetoplast genomes due to the accumulation of mutations or loss of critical genomic components. An example of a loss of critical components is the reported loss of entire minicircle classes in Leishmania tarentolae during laboratory cultivation, which results in an inability to generate some correctly encoded genes. In the current work, we estimated the evolutionary rates of mitochondrial and nuclear genome regions of multiple natural Leishmania spp. We analyzed synonymous and non-synonymous substitutions and, rather unexpectedly, found that the coding regions of kinetoplast maxicircles are among the most variable regions of both genomes. In addition, we demonstrate that synonymous substitutions greatly predominate among maxicircle coding regions and that most maxicircle genes show signs of purifying selection. These results imply that maxicircles in natural Leishmania populations remain functional despite their high mutation rate.

• Keywords
• L. infantum, L. major, L. turanica, SNP, genome instability, leishmania donovani,
• Publication type
• Journal Article MeSH

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.

• Keywords
• Leishmania, allelic selection, sand fly infection,
• MeSH
• Leishmania donovani * MeSH
• Humans MeSH
• Mutation MeSH
• DNA Repair genetics   MeSH
• Oxidative Stress genetics   MeSH
• Psychodidae * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The evolution in Leishmania is governed by the opposite forces of clonality and sexual reproduction, with vicariance being an important factor. As such, Leishmania spp. populations may be monospecific or mixed. Leishmania turanica in Central Asia is a good model to compare these two types. In most areas, populations of L. turanica are mixed with L. gerbilli and L. major. Notably, co-infection with L. turanica in great gerbils helps L. major to withstand a break in the transmission cycle. Conversely, the populations of L. turanica in Mongolia are monospecific and geographically isolated. In this work, we compare genomes of several well-characterized strains of L. turanica originated from monospecific and mixed populations in Central Asia in order to shed light on genetic factors, which may drive evolution of these parasites in different settings. Our results illustrate that evolutionary differences between mixed and monospecific populations of L. turanica are not dramatic. On the level of large-scale genomic rearrangements, we confirmed that different genomic loci and different types of rearrangements may differentiate strains originated from mixed and monospecific populations, with genome translocations being the most prominent example. Our data suggests that L. turanica has a significantly higher level of chromosomal copy number variation between the strains compared to its sister species L. major with only one supernumerary chromosome. This suggests that L. turanica (in contrast to L. major) is in the active phase of evolutionary adaptation.

• MeSH
• Genomics MeSH
• Gerbillinae parasitology   MeSH
• Leishmania * genetics   MeSH
• DNA Copy Number Variations MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Mongolia MeSH

The protozoan parasite Leishmania donovani causes fatal human visceral leishmaniasis in absence of treatment. Genome instability has been recognized as a driver in Leishmania fitness gain in response to environmental change or chemotherapy. How genome instability generates beneficial phenotypes despite potential deleterious gene dosage effects is unknown. Here we address this important open question applying experimental evolution and integrative systems approaches on parasites adapting to in vitro culture. Phenotypic analyses of parasites from early and late stages of culture adaptation revealed an important fitness tradeoff, with selection for accelerated growth in promastigote culture (fitness gain) impairing infectivity (fitness costs). Comparative genomics, transcriptomics and proteomics analyses revealed a complex regulatory network associated with parasite fitness gain, with genome instability causing highly reproducible, gene dosage-independent and -dependent changes. Reduction of flagellar transcripts and increase in coding and non-coding RNAs implicated in ribosomal biogenesis and protein translation were not correlated to dosage changes of the corresponding genes, revealing a gene dosage-independent, post-transcriptional mechanism of regulation. In contrast, abundance of gene products implicated in post-transcriptional regulation itself correlated to corresponding gene dosage changes. Thus, RNA abundance during parasite adaptation is controled by direct and indirect gene dosage changes. We correlated differential expression of small nucleolar RNAs (snoRNAs) with changes in rRNA modification, providing first evidence that Leishmania fitness gain in culture may be controlled by post-transcriptional and epitranscriptomic regulation. Our findings propose a novel model for Leishmania fitness gain in culture, where differential regulation of mRNA stability and the generation of modified ribosomes may potentially filter deleterious from beneficial gene dosage effects and provide proteomic robustness to genetically heterogenous, adapting parasite populations. This model challenges the current, genome-centric approach to Leishmania epidemiology and identifies the Leishmania transcriptome and non-coding small RNome as potential novel sources for the discovery of biomarkers that may be associated with parasite phenotypic adaptation in clinical settings.

• MeSH
• Leishmania donovani * genetics   MeSH
• Leishmaniasis, Visceral * parasitology   MeSH
• Humans MeSH
• Genomic Instability MeSH
• Proteomics MeSH
• Gene Expression Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and the evolutionary history of admixture that produced these hybrid populations remain unclear. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterized as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids based on whole-genome sequences. We propose that these hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of data that shows how recombination, and gene flow, shape natural populations of Leishmania.

• MeSH
• Chimera * MeSH
• Genotype MeSH
• Leishmania donovani genetics   MeSH
• Leishmaniasis, Visceral parasitology   MeSH
• Humans MeSH
• Recombination, Genetic MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Ethiopia MeSH