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

Leishmania is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected in vitro. Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, L. mexicana amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT10-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.

• Keywords
• Leishmania (L) mexicana, amastigote, axenic, lesion, macrophage, proteome, tandem mass tags labeling,
• MeSH
• Chromatography, Liquid MeSH
• Leishmania mexicana * metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Proteome metabolism   MeSH
• Proteomics MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Proteome MeSH