Across bacterial, archaeal and eukaryotic kingdoms, heat shock proteins (HSPs) are defined as a class of highly conserved chaperone proteins that are rapidly induced in response to temperature increase through dedicated heat shock transcription factors. While this transcriptional response governs cellular adaptation of fungal, plant and animal cells to thermic shock and other forms of stress, early-branching eukaryotes of the kinetoplastid order, including trypanosomatid parasites, lack classical mechanisms of transcriptional regulation and show largely constitutive expression of HSPs, thus raising important questions on the function of HSPs in the absence of stress and the regulation of their chaperone activity in response to environmental adversity. Understanding parasite-specific mechanisms of stress-response regulation is especially relevant for protozoan parasites of the genus Leishmania that are adapted for survival inside highly toxic phagolysosomes of host macrophages causing the various immuno-pathologies of leishmaniasis. To gain first insight into the role the heat shock repsonse for Leishmania differentiation and pathogenicity, we are studying the evolution and function of members of the HSP70 protein family combining bio-informatics and transgenics apporahces.

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The Sudanese L. donovani strain Ld1SA is the most important experimental reference strain in our field and has been used for various systems level analysis (DNAseq, RNAseq, proteomics). However, all these analyses are strongly compromised by the fact that the current L. donovani genome reference strain LdBPK is from Nepal and very different from the Sudanese isolate (only 60% of DNAseq reads can be mapped).

BioHub LeiSHield project This proposal summarizes the contribution of the BioHub to the LeiSHield action that may be carried out by a single BioHub Leishmania coordinator (Giovanni Bussotti). The coordinator will be implicated in the following actions: 1) Establish the link between LeiSHield members and the BioHub team for all questions regarding data analysis and interpretation. The coordinator will present to the BioHub the bio-informatics needs of the LeiSHield partners. Short (easy) tasks will be answered directly (following the BioHub open door strategy). For more involved tasks i twill be asked to deposit projects via the C3BI web site. 2) Coordinate the setup of an HTseq analysis pipeline, including quality control, read mapping, determination of CNV and SNPs, and data visualization using a combination of tools available at the BioHub, such as SyntView and Listeriomics. A link to Cedric Notredame will be established as scripts for Leishmania have been created there. 3) Oversee the submission of DNA from the different LeiSHield WPs to the IP HTseq facility, follow the progress, store the acquired data, and dispatch the datasets to the corresponding WP leaders. This will be coordinate with the Biomix infrastrcuture. 4) Apply the HTseq analysis pipeline (see point 1) on selected data sets for defined work packages, including WP4 (“Analysis of newly isolated anthroponotic L. donovani s.l. strains from Cyprus and correlation of genotypic profiles to tropism and drug resistance”), WP6 (“Population genetics of Brazilian L. infantum isolates from endemic areas presenting distinct transmission cycle”), WP7 (“Leishmania dovovani genome sequence diversity and disease tropism in the Sudan”), and WP9 (“Systems-wide analysis of Leishmania genomic and transcriptomic adaptation”). 5) Co-organize a course on HTseq data visualization (June 2016) with members of the BioHub team.

Aim : In vitro infection of innate immune cells by L. amazonensis (L.am.) seems to be associated to an increase in resistance to cell death of infected cells. This project aims at deciphering the impact of in vitro L. amazonensis (L.am.) amastigotes infection on cell death processes including apoptosis, autophagy, pyroptosis and necroptosis in different host cells, i.e. Bone-marrow- derived macrophages (BMDMs) and dendritic cells (BMDCs) after one day of infection. Material : RNA samples were obtained from control and infected BMDMs (BALB/c mice) or BMDCs (BALB/c, C57BL/6 and DBA/2 mice) after their sorting by Fluorescence Activated- Cell sorting. For BALB/c BMDCs, amastigotes were also added to cells in presence of immune serum to trigger their opsonization. Samples were analysed a few years ago at the « Plateforme Transcriptome et Epigénome » with the Affymetrix technology. RNAs from BMDMs and BMDCs were analysed with the Affymetrix Mouse430_2 GeneChips and the Affymetrix Mouse Gene ST 1.0 arrays respectively.  

Gene ontology analysis of RNAseq data from uninfected and Leishmania-infected mouse macrophages.  Scientific context During the course of cutaneous or visceral disease in humans or experimental animal models, the resolution of leishmanial infections or the control of parasite growth is dependent on appropriate innate and adaptive immune responses developed by the parasitized host. Leishmania largely evades and subverts these responses by its intracellular life style inside the mammalian host, where the parasites develop into amastigotes mainly within macrophages (BMDMs). We have focused our interest in the BMDM inflammasome and the way Leishmania amastigotes interfere or subvert BMDM inflammatory responses. Our recent data are in favor of an absence of stimulation, even a down-regulation of the inflammasome in BMDMs harboring replicating amastigotes at the gene and protein expression levels. To go further on this, we have performed RNAseq experiments on uninfected and infected BMDMs. This project was done at the “Transcriptome and Epigenome” platform and in close collaboration with the C3BI for normalization and statistical analysis procedures. Objective In the present proposal we will perform a deep analysis of the repartition of modulated genes between the different conditions using these RNAseq data. Using C3Bi expertise we will classify known functions of modulated genes into GO aspects i.e. molecular function, cellular component and biological process, visualize gene annotations and perform statistical analyses for the distribution of the annotated genes over the GO hierarchy for the different gene lists analyzed. Hopefully, these analyses will bring us a better understanding of the mechanisms underlying the subversion of BMDM functions in the innate and adaptive immune response to Leishmania infection which is a prerequisite to design novel anti-parasitic intervention strategies targeting the infected host cell rather than the parasite.

I would like to retrieve all HMT and HDM in Leishmania

Looking for DNMT (DNA methyltransferase) orthologs in Leishmania which could be potential targets of epigenetic inhibitors active against the parasite.

We tested DNMT and RNMT inhibitors on Leishmania parasites survival and would like to know which could be the potential targets.

African trypanosomes are transmitted by the bite of the tsetse fly and cause the debilitating, and often fatal, neglected tropical disease sleeping sickness, or Human African Trypanosomiasis (HAT). Trypanosoma brucei gambiense, the parasites responsible for 98% of human cases, first reside in the patient blood and skin for months to years before invading the central nervous system, where they cause the neurological symptoms of the disease. HAT is approaching elimination, with the number of cases reported in 2017 dropping to approximately 1,442 from only a dozen African countries. In this context, HAT was included in the WHO roadmap on neglected tropical diseases, with 2020 set as target date for elimination as a public health problem. A secondary goal of zero transmission by 2030 has also been set. These targets have, in part, been encouraged by the success of surveillance efforts that rely on detecting extracellular trypanosomes in human blood. Nevertheless, the reduction in case numbers brings about other challenges. For example, the sensitivity of any diagnostic test diminishes as the disease burden drops, and this is being seen with the serological tests available for HAT. In this context, new highly sensitive and specific diagnostic tools will be required to accurately monitor the occurrence of new cases and the possible emergence of drug-resistant trypanosomes during the elimination phase. Most diagnostic tests currently under development are based on optimization of existing methods that may not combine all the requirements to stand up to the harsh constraints imposed by the elimination phase requirements, especially in terms of sensitivity and specificity. We propose that adapting the recently developed Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) technology that combines a CRISPR-Cas system and lateral flow test to trypanosomes will provide the sensitivity and specificity required for a diagnostic test in the elimination and post elimination phases. The SHERLOCK system relies on the collateral effect of Cas13a promiscuous RNA cleavage activity upon target recognition. Combining the collateral effect with pre-amplification of RNAs resulted in rapid RNA detection with attomolar (10^-18 moles/l) sensitivity and single-base mismatch specificity, in a diagnostic setting. This technology has been used to detect specific strains of Zika and Dengue viruses and distinguish pathogenic bacteria in a mixed sample. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications. A lateral flow test for a simple and rapid readout can be easily implemented after a reaction that does not exceed two hours from the sampling step. The first step of this project is to identify promising RNA targets in silico by data mining and multiple alignements of all available transcriptomic data on bloodstream African trypanosomes.

Leishmania causes devastating human diseases – leishmaniases - representing an important public health problem in the Mediterranean basin and declared as emerging diseases in the EU due to climate change and population displacement. The LeiSHield-MATI consortium will for the first time investigate in an integrative fashion the complex parasite-vector-host interplay in cutaneous leishmaniasis affecting Morocco, Algeria, Tunisia, and Iran (MATI), using field isolates and human clinical samples. The ultimate goal of our project is to identify genetic factors selected during natural infection and to understand how the complex parasite-vector-animal interaction impacts clinical outcome in infected patients. This goal will be achieved through a highly ambitious secondment plan between all partners, and the organization of courses and workshops to train the next generation of scientists generating a long-term impact on the research capacities in endemic areas. Capitalizing on complementary infrastructures of its EU, African and Asian partners and their expertise in molecular parasitology, epidemiology, systems level analyses, bioinformatics, computational biology, immunology, dermatology, field studies, and public health, our project will drive important innovation in clinical research, strengthen capacities in disease endemic regions, inform authorities on control measures, and raise awareness in all partner countries on this emerging EU public health problem. The highly inter-disciplinary and inter-sectorial structure of LeiSHield-MATI, and its powerful integrative and comparative approach is novel in parasitic systems and will drive a unique bio-marker discovery pipeline for the future development of new prognostic and diagnostic tools, as well as novel preventive and therapeutic measures that will ensure long-term collaboration, promote scientific and commercial self-sustainability of its partners, and will have an important impact to improve public health.

This project resonates with our LeiSHield effort and expands our genomic analysis of Leishmania field isolates from its current focus on the MATI countries (Morocco, Algeria, Tunisia, Iran) to the bordering EU country Italy. We have prime access to short read genome data produced by the National Italian Reference centre of Leishmaniasis and have established a collaboration to apply our computation al pipeline (Späth and Bussotti, NAR 2021) for the comparative analyses of these samples. The scope of our collaboration is to shed new light in the genetic diversity of L. infantum across different regions in Italy.

Aim : When L. amazonensis (L.am.) amastigotes infect BMDMs, they induce multiple strategies to allow their survival and multiplication. This project aims at deciphering the transcriptional modulations induced after three days of in vitro infection of C57BL/6 BMDMs with L. amazonensis (L.am.) amastigotes, in unstimulated conditions, or conditions that induce NLRP3 inflammasome activation.

The aim of the project is to create a viewer that will help visualisation and correlation between genomic, transcriptomic, proteomic and metabolomic data generated by the comparison of amastigote and promastigote stages of the Leishmania donovani parasite.

Chromosome amplification is commonly used by Leishmania during adaptation to environment.  In this context it is challenging to look for genes relevant for parasite virulence/attenuation/drug resistance... To restrict this chromosomal amplification, a cosmid approach (CoSeq) has been chosen to select for genes that provide fitness gain to  Leishmania donovani parasites in culture and in the animal. Therefore, a cosmid library has been generated with genomic DNA from the parasites which needs to be sequenced to control for genome coverage before transfection to the parasites. The transfected parasites will then be injected to animals or submitted to different culture conditions. Only those transfected with cosmids providing advantage under the studied conditions will be selected and will replicate. These cosmids will be extracted from the parasites and will be sequenced to reveal genes relevant for the parasite survival. The C3Bi would be implicated in the analyses of the sequencing data obtained from the PF1 (retrieve the data, mapping of the reads to Leishmania genome, estimation of the genome coverage, listing of genes selected for a given condition...).

We are generating massive amounts of omics data for Leishmania donovani. Anna Zukhova enabled to use the BiNGO module of Cytoskape to perform and visualize our GO enrichment analyses. We would like to continue our collaboration and ask Anna's help to assess the current state of GO annotation of the Leishmania genome and if possible to complete it using domain searches or ortholog mapping from other genomes, including L. major, T. bruce or model organisms such as yeast. We expect that improvement of GO annotation will also us to better reveal enriched GO terms in our datasets and generate testable hypotheses.

Leishmania genomic adaptation during sand fly infection is only poorly understood. In particular, the possibility of allelic selection in a given parasite population inside the insect vector has not been investigated, even though sexual recombination and the possibly of self-sex (selfing) can change allele frequencies and thus may be relevant for parasite transmission and virulence. We investigate this important open question conducting experimental sand fly infection with bona fide amastigotes isolated from infected hamster spleen, and derived promastigotes with different karyotypic profiles. Applying our genome instability pipeline (GIP) on sand fly-recovered parasites revealed a novel form of Leishmania genome instability inside the insect vector relying on gene conversion, which causes haplotype shuffling and likely allows for the selection of beneficial alleles.

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