The ParSig Unit studies the molecular basis of virulence of Leishmania, an important human pathogen that infects host macrophages producing severe and even fatal diseases worldwide. Our research program is focused on the analysis of Leishmania signal transduction pathways implicated in intracellular parasite development and subversion of host cell functions that qualify as novel targets for anti-parasitic drug development. The largely constitutive expression of the Leishmania genome at both transcript and protein levels raises the question on how these parasites regulate the developmental transition between insect-stage promastigotes and vertebrate-stage amastigotes, and how they can evolve intra-species divergence in drug susceptibility, tropism, and infectivity. Furthermore, the exploitation of immune sentinel cells (macrophages, dendritic cells) as niche to escape and subvert host immunity provides an interesting model system to gain insight into how intracellular pathogens co-evolve with their hosts and modulate host cell immune and metabolic functions to establish infection. We address these questions by investigating parasite-specific molecular mechanisms that govern Leishmania environmental adaptation using systems-wide analyses. We then apply functional genetics approaches on candidate molecular players to reveal their biological relevance in parasite viability and infectivity. Finally we translate our basic research findings on Leishmania-specific biology into preclinical applications through drug target validation and anti-leishmanial compound screens, which feed back on our basic research projects through drug target deconvolution studies (Fig. 1). Today, this scientific strategy to develop and apply innovative approaches to gain insight into parasite-specific biological processes and mechanisms of host/pathogen interaction relevant for therapy is at the core of four major research axes that animate our International Mixed Unit (IMU) with partners at IP Korea and IP Shanghai.
Axis 1: Adaptive differentiation
This research axis has been funded through the FP7 LeishDrug and the ANR LabEx IBEID projects and is focused on the analysis of Leishmania signal transduction pathways that are relevant for intracellular parasite differentiation and qualify as novel targets for anti-parasitic drug discovery. Genetics and phospho-proteomics assessment of Leishmania protein kinase function and regulation, and of stage-specific phosphorylation events uncovered highly parasite-specific mechanisms of stress response regulation through phosphorylation of HSPs and chaperones on parasite-specific residues by stress kinases (Figure 2). Combining gain- and loss-of-function approaches with pharmacological, structural, and functional analyses we (i) established the requirement of the casein kinase CK1.2 for intracellular parasite survival, (ii) revealed essential roles of the chaperones STI1 and CyP40 in parasite viability and infectivity, and (iii) linked the MAP kinases LmaMPK4, 7, and 10 to pH-regulated metacyclic differentiation, sensing of environmental stress at stationary phase culture, and axenic amastigote differentiation, respectively. We will further refine our working model by investigating interaction and kinase/substrate relationships exploiting our expertise in proteomics and kinase biology. We are expanding our interest on signal transduction from its current focus on intracellular protein kinases and their role in Leishmania biology to secreted Leishmania protein kinases that may act in “trans” to modulate the host cell phenotype (Axis 2).
Figure 2: Model of post-translational regulation of the Leishmania stress response. HSP, heat shock protein; HSF, heat shock transcription factor; SK, stress kinase vs HSKs?, TFs, transcription factors.
Axis 2: Host subversion
Funded by the international ANR project TranSig, this axis investigates the impact of Leishmania-derived signaling molecules on parasite viability, virulence, and host cell expression profile with the major aim to understand how these pathogens reprogram their host cell to establish permissive conditions for survival. We focus on secreted Leishmania signaling proteins that may act in “trans” to modulate the host cell phenotype and currently investigate the impact of the parasite ecto-kinase CK1.2 on parasite survival and host signal transduction. We will dissect the domains and residues important for CK1.2 activity, release, and interaction with parasite and host molecular partners by generating a series of CK1.2 regulatory and structural mutants, which will be analyzed by combining biochemical, cell biological and genetic approaches. This investigation will set the stage for experimental assessment of other ecto-kinases that we will identify by proteomics analysis of the parasite secretome and ecto-proteome. The molecular investigation of parasite protein kinase functions inside the parasite and the infected host cell, and their interaction with the parasite stress pathway, will directly feed into our drug discovery effort (Axis 4) and may deliver the proof that ecto-kinases are interesting (if not superior) targets for chemotherapeutic intervention as they will act outside the parasite and in interaction with host molecules, thus being refractory for the development of parasite drug resistance (see Axis 3).
Figure 3: Overview of screening results. 4028 compounds from various libraries were screened on both recombinant L. major LmCK1.2 and mammalian SsCK1 to identify 128 compounds potent on LmCK1.2. Only 37 of these compounds had the same potency on both CK1s. The histogram represents the repartition of the compounds that inhibit LmCK1.2 and/or SsCK1 by more than 90%, according to their specificity toward either CK1s.
Axis 3: Adaptive evolution
Initiated by PTR392 with partners at IP Tunis and now continued as part of the LeiSHield consortium, we applied systems-wide analyses at proteomics, transcriptomics, and genomics levels on cultured, animal-derived or human parasite isolates to investigate the impact of the host/culture environment on parasite genotype and phenotype. Using culture adaptation of hamster-derived parasites as a proxy to study parasite evolution in response to environmental change, we revealed dramatic genomic, transcriptomic and proteomic changes as soon as two passages (or 20 generations) after in vitro culture, largely caused by chromosomal amplification. Quantification of the chromosome ploidy state in spleen- and liver-derived amastigotes ex vivo at the single cell level by DNA-FISH analysis confirmed the presence of mosaic aneuploidies in situ revealing a bet hedging strategy adopted by Leishmania. We are currently developing protocols for single cell sequencing to study the dynamics of genome evolution, and for direct tissue sequencing to bypass culture adaptation for epidemiological investigations. Our results suggest that Leishmania drug resistance is likely governed by direct selection of parasite genetic variants. To avoid this selection pressure and limit the emergence of drug resistance we are now targeting parasite-released ecto-proteins required for Leishmania intracellular survival through their interaction with host pathways (Axis 2 and 4). Furthermore we continue to apply our systems level approaches to investigate genotype-genotype and environment-genotype interactions by analyzing the impact of drug selection, immune selection, and vector/host-specific selection on the parasite genotype and phenotype. These analyses will uncover amplifications and transcripts (coding and non-coding) that correlate with observed phenotypic changes and will allow us to prioritize genes for subsequent functional genetic analysis using transgenic and null mutant approaches as descried in Axis 1.
Figure 4: (Left panel) Circos representation of gene copy number variation (outer ring) and differential transcipt abundance (inner ring) in splenic (green) and axenic amastigotes (red). (Right panel) Circos representation of total genomic reads of splenic (green) and axenic amastigotes (red) for protein coding genes (inner circle) and genes coding for long non-coding RNAs (outer circle).
Axis 4: Target/hit discovery
Feeding on our expertise in recombinant expression, kinase activity analysis (Axis 1), and live imaging, we developed and applied target-based and phenotypic screening strategies as part of the FP7 LeishDrug consortium, with the major aim to translate our basic research results into application for pre-clinical drug discovery. Screening kinase-biased inhibitor libraries against recombinant CK1.2 and primary macrophages infected with mCherry transgenic L. amazonensis amastigotes, we identified a series of novel hit compounds representing different chemical scaffolds with anti-leishmanial activity. The best hits are currently subject of structure/activity relationship analyses (SAR) that are based on iterative cycles between chemical synthesis of compound derivatives and assessment of anti-leishmanial activity in our assays. We continue to apply our very successful screening strategies for the pharmacological assessment of parasite signaling and epigenetic regulation using dedicated compound libraries though the FP7 A-ParaDDise and ANR PathoMethylome projects, and for the discovery of novel anti-leishmanial hits in collaboration with the Chinese National Compound Library in Shanghai (CNCL), accessible through our IMU. As part of the ANR TansLeish project we have developed novel approaches for drug target deconvolution that will allow us to reveal the cellular targets of our hit compounds and study off-target effects. Validated target kinases will be functionally characterized applying transgenic and facilitated null mutant analysis to gain insight into their biological functions, validate their essential nature, and further test their druggability potential by structure/function analysis using the genetic approaches described under Axis 1.
Figure 5: (Left panels) Merged phase contrast and fluorescence images of living primary mouse macrophages infected with mCherry transgenic L. amazonensis amastigotes (red colour). Parasites are developing within giant acidic parasitophorous vacuoles that are stained with LysoTracker Green (green colour) as main readout for anti-leishmanial activity. In parallel, the health status of host macrophages is assessed by nuclear staining with Hoechst 33342 (blue). (Right panel) Bi-parametric dot plot for compound biological activity. Normalized anti-leishmanial activity (PV readout, horizontal axis) and toxic activity on host macrophages (nuclear readout, vertical axis) are displayed for every compound of a representative 384-well plate (yellow diamonds). Controls include infected macrophages treated with amphotericin (blue circle), cycloheximide (red triangle) and DMSO (green circle).
The International Mixed Unit (IMU)
The major goal of our IMU is to coordinate a highly ambitious, translational research program on novel anti-parasitic intervention strategies by interfacing scientific expertise and local infrastructure available at IP Paris, IP Korea and IP Shanghai. Our IMU combines complementary expertise in molecular parasitology, macrophage immunology, and phenotypic screening with the objectives (i) to assess the impact of Leishmania-released proteins on the host macrophage phenotype and exploit the parasite ecto-proteome for the identification of novel drug targets, (ii) to discover proteins and compounds that can regulate the functions of the macrophage inflammasome and assess their potential to control intracellular Leishmania infection, and (iii) to identify and pharmacologically target host cell factors required for Leishmania infection. Our IMU will coordinate institutional infrastructure, teaching capacities, and private partnerships across France, China, and Korea with the final goal to exploit host macrophage/Leishmania interaction for anti-parasitic intervention. The IMU project benefits from two PTRs, (1) PTR496 with G. Meng from IPS that investigates the reciprocal regulatory relationship between macrophage inflammasome activation and intracellular Leishmania survival, and (2) PTR539 with J.H. No (coordinator) from IPK that applies systems-wide analyses to elucidate the molecular mechanisms underlying the metabolic relationship between parasite and macrophage. The IMU will structure and further develop our international research program that relies (i) on genetic validation of pathways of Leishmania/host cell interaction as novel anti-leishmanial drug targets (Axis 1), (ii) on investigating the impact of parasite ecto-proteins on host cell immune status and metabolism (Axis 2), and (iii) exploitation of these proteins for novel treatment options that are refractory to the genetic selection of parasite resistance (Axis 3) using phenotypic and target-based screening campaigns (Axis 4) .
Figure 5: Overview of the IMU projects.