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Leishmania parasites cause a broad spectrum of clinical disease. Here we report the sequencing of the genomes of two species of Leishmania : Leishmania infantum and Leishmania braziliensis . The comparison of these sequences with the published genome of Leishmania major reveals marked conservation of synteny and identifies only ∼200 genes with a differential distribution between the three species. L. braziliensis , contrary to Leishmania species examined so far, possesses components of a putative RNA-mediated interference pathway, telomere-associated transposable elements and spliced leader–associated SLACS retrotransposons. We show that pseudogene formation and gene loss are the principal forces shaping the different genomes. Genes that are differentially distributed between the species encode proteins implicated in host-pathogen interactions and parasite survival in the macrophage.

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.

Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. As neither gamete stages nor cell fusion events have been directly observed during parasite development in the vector, we have relied on a classical genetic analysis to determine if Leishmania has a true sexual cycle. Here, we used whole genome sequencing to follow the chromosomal inheritance patterns of experimental hybrids generated within and between different strains of L. major and L. infantum. We also generated and sequenced the first experimental hybrids in L. tropica. We found that in each case the parental somy and allele contributions matched the inheritance patterns expected under meiosis 97-99% of the time. The hybrids were equivalent to F1 progeny, heterozygous throughout most of the genome for the markers that were homozygous and different between the parents. Rare, non-Mendelian patterns of chromosomal inheritance were observed, including a gain or loss of somy, and loss of heterozygosity, that likely arose during meiosis or during mitotic divisions of the progeny clones in the fly or culture. While the interspecies hybrids appeared to be sterile, the intraspecies hybrids were able to produce backcross and outcross progeny. Analysis of 5 backcross and outcross progeny clones generated from an L. major F1 hybrid, as well as 17 progeny clones generated from backcrosses involving a natural hybrid of L. tropica, revealed genome wide patterns of recombination, demonstrating that classical crossing over occurs at meiosis, and allowed us to construct the first physical and genetic maps in Leishmania. Altogether, the findings provide strong evidence for meiosis-like sexual recombination in Leishmania, presenting clear opportunities for forward genetic analysis and positional cloning of important genes.

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.

Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster studies of visceral leishmaniasis.

The surface of Leishmania spp. presents glycoprotein 63 (GP63), a metalloprotease that acts as one of the parasite's major antigens. A vaccine against leishmaniasis has not yet been developed and stationary phase promastigotes have utmost importance in transmitting Leishmania spp. from phlebotomine sand fly to humans or reservoirs. Therefore, this study aimed to analyze GP63 protein in three different Leishmania spp. to determine new vaccine candidate antigen against leishmaniasis using sequencing data of locally detected Leishmania strains and in silico approaches. The GP63 protein sequences of the stationary phase/amastigote form of L. infantum, L. major, and L. tropica were identified and then the gene encoding GP63 protein in Leishmania positive samples (n:59) was amplified and sequenced for variation analysis. According to the results, 4, 6, 19 GP63 variants were found within L. infantum, L. major, and L. tropica isolates, respectively. The most prevalent variants within each species were selected for further analysis using in silico approaches. Accordingly, all selected GP63 proteins were antigenic and the amount of B and T cell epitopes were 23 for L. infantum, 10 for L. major, and 9 for L. tropica. The analysis of each epitope showed that all of them were non-toxic, non-allergen, and soluble but had different antigenicity values. Among these epitopes, EMEDQGSAGSAGS associated with L. major, STHDSGSTTC and AEDILTDEKRDILRK epitopes associated with L. infantum had the highest antigenicity values for B cell, MHC-I, and MHC-II epitopes, respectively. Moreover, conserved epitopes were detected among two or three Leishmania species. This study detected many epitopes that could be used in vaccine studies and the development of serological diagnostic assays.

Background Trypanosomatids include the genera Trypanosoma and Leishmania , which are the etiological agents of important human diseases. These pathogens present unique mechanisms of gene expression characterized by functionally unrelated genes positioned in tandem and organized into polycistronic transcription units transcribed in a large pre-mRNA by RNA Polymerase II. Since most of the genome is constitutively transcribed, gene expression is primarily controlled by post-transcriptional processes. As in other organisms, histones in trypanosomatids contain a considerable number of post-translational modifications, highly conserved across evolution, such as the acetylation and methylation of some lysines on histone H3 and H4. These modifications have been mainly studied in Trypanosoma spp. The aim of this work was to elucidate the distribution of histone H3 lysine 4 trimethylation (H3K4me3) over the chromatin landscape of Leishmania infantum , the causative agent of canine and human leishmaniasis in the Mediterranean region. To this end, we investigated by chromatin immunoprecipitation (ChIP)-sequencing either the promastigotes (the flagellated motile form) and the amastigotes (the intracellular form) in an in vitro infection model. Results The chromatin was prepared from THP-1 cells non infected, THP-1 cells infected with L. infantum MHOM/FR/78/LEM75, and THP-1 cells non infected and mixed with L. infantum MHOM/FR/78/LEM75 promastigotes. ChIP was conducted using anti-H3K4me3 or anti-H3K27me3 antibodies and ChIP-seq was performed on an Ion S5 sequencer. We showed that histone H3K4me3 is mainly enriched at transcription start sites (67%) or internally within the polycistronic transcription units (30%), with no differences between L. infantum promastigotes and amastigotes. Moreover, the enriched regions co-localize with another hallmark of transcriptional activation (histone H3 acetylation) in L. major , a species characterized by a high degree of synteny with L. infantum . Conclusions These findings expand our knowledge of the epigenomics of Leishmania parasites, focusing on epigenetic markers associated with transcription in L. infantum , and will contribute to elucidate the transcriptional mechanisms in these pathogens.

Entomological investigations were conducted for the first time in urban forest remnants of Porto Velho, state of Rondônia, Brazil, to explore the transmission dynamics of Leishmania . Sand fly collections were carried out at ten sites, encompassing both canopy and ground strata, from October to December 2021. A total of 1,671 sand flies were collected, representing 42 species within 12 genera. Nyssomyia Antunesi (n = 384) and Psychodopygus davisi (n = 111) were the most abundant species. Molecular analyses targeting the V7V8 region (18S gene) unveiled the presence of sequences 100% identical to Leishmania infantum in females of Bichromomyia flaviscutellata (1), Nyssomyia Antunesi complex (6), Nyssomyia umbratilis (1), Nyssomyia sp. (1), Psychodopygus ayrozai (1), Ps . davisi (3), Psychodopygus paraensis (1), and Sciopemyia sordellii (1). Sequences 100% similar to Trypanosoma minasense were found in two samples of the Nyssomyia Antunesi complex, and two samples of Sc . sordellii presented 100% identity to a Trypanosoma sp. strain, previously identified in this same sand fly in Rondônia. Sequencing of Cytb fragment suggested Homo sapiens , Dasypus novemcinctus and Tamandua tetradactyla as the blood source for distinct sand flies. The identification of sequences similar to L . infantum in sand flies collected in urban forest fragments is noteworthy, correlating with the recent local and regional occurrence of autochthonous cases of human visceral leishmaniasis. However, further studies are imperative to ascertain the presence of hosts/reservoirs and evaluate the risk of L . infantum transmission to humans.

Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis (VL) in the New World (NW) with endemic regions extending from southern USA to northern Argentina. The two hypotheses about the origin of VL in the NW suggest (1) recent importation of L. infantum from the Old World (OW), or (2) an indigenous origin and a distinct taxonomic rank for the NW parasite. Multilocus microsatellite typing was applied in a survey of 98 L. infantum isolates from different NW foci. The microsatellite profiles obtained were compared to those of 308 L. infantum and 20 L. donovani strains from OW countries previously assigned to well-defined populations. Two main populations were identified for both NW and OW L. infantum. Most of the NW strains belonged to population 1, which corresponded to the OW MON-1 population. However, the NW population was much more homogeneous. A second, more heterogeneous, population comprised most Caribbean strains and corresponded to the OW non-MON-1 population. All Brazilian L. infantum strains belonged to population 1, although they represented 61% of the sample and originated from 9 states. Population analysis including the OW L. infantum populations indicated that the NW strains were more similar to MON-1 and non-MON-1 sub-populations of L. infantum from southwest Europe, than to any other OW sub-population. Moreover, similarity between NW and Southwest European L. infantum was higher than between OW L. infantum from distinct parts of the Mediterranean region, Middle East and Central Asia. No correlation was found between NW L. infantum genotypes and clinical picture or host background. This study represents the first continent-wide analysis of NW L. infantum population structure. It confirmed that the agent of VL in the NW is L. infantum and that the parasite has been recently imported multiple times to the NW from southwest Europe.

This study closes important knowledge gaps with respect to Leishmania (L.) infantum genetic heterogeneity in a given endemic country, as exemplified here for Italy, and reveals genetic hybridization as a main cause for re-emerging human leishmaniasis in northern Italy. The observed high diversity of Leishmania parasites on the Italian peninsula suggests different geographical origins, with genomic adaptation to various ecologies affecting both pathogenicity and transmission potential. This is documented by the discovery of a putative L. infantum / L. donovani hybrid strain, which has been shown to preferentially infect humans but not dogs. Our results provide important information to health authorities, which need to consider the public health risk represented by the introduction of new Leishmania species into EU countries due to population displacement or travel from countries where exotic/allochthonous parasite species are endemic.