Our research program is focused on the analysis of parasite and host regulatory pathways implicated in intracellular parasite development and subversion of host cell functions that qualify as novel drug targets. The largely constitutive expression of the Leishmania genome at both transcript and protein levels raises the question on how these parasites can maintain different life cycle stages 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 functional genetics, genomics, systems-level analyses. The research program of our Unit is animated through three research axes on complementary aspects of host/parasite interaction that allowed us to make a series of important discoveries: Axis 1 investigates how Leishmania adapts to its hosts using two strategies: signal-induced stage differentiation and genomic adaptation. Systems analyses at the transcript, protein, and phosphoprotein levels of bona fide L. donovani amastigotes purified from the spleen of infected hamsters and derived promastigotes uncovered an unexpected reciprocal regulatory relationship between protein kinase and proteasome activities essential for parasite differentiation, thus defining both biological processes as fertile source for the discovery of novel drug targets (Pescher et al., in preparation). Comparative genomics analysis of Leishmania field isolates and conducting in vitro evolutionary studies using hamster-derived parasites revealed the highly dynamic and complex nature of Leishmania evolutionary adaptation that draws from a vast genetic landscape of spontaneous karyotypic fluctuations, stochastic gene amplifications, and nucleotide polymorphisms. We showed that these genetic fluctuations generate genotypically and phenotypically diverse mosaic populations that are substrate for Leishmania evolutionary adaptation and fitness gain in response to environmental change. Surprisingly, genomic adaptation occurred in a polyclonal fashion resulting in co-existing sub-populations that preserve the original genetic diversity. Our results define aneuploidies and gene copy number variations as a major source for Leishmania biomarker discovery, and challenge current, parasite-directed drug discovery strategies as these will drive the evolution of drug resistant phenotypes.
Axis 2 applies pharmacologic, genetic and systems level approaches to investigates Leishmania signaling pathways underlying adaptive stage differentiation and revealed essential roles for the Leishmania MAP kinases MPK4, 7, and 10 as well as the chaperones CyP40 and HRP4 in parasite viability and infectivity. Together with a series of functional proteomics studies using immobilized staurosporine or ATP our studies discovered and validated parasite ATP-binding proteins as important targets for anti-leishmanial therapy. Finally, we genetically and pharmacologically validated the parasite ecto-kinase LmCK1.2 as drug target with essential intra-parasitic functions, but also extracellular functions that modulate host cell signaling through direct phosphorylation of numerous host chaperones and transcription factors.
Axis 3 finally employs immunological, pharmacological and systems-level investigations to study the impact of intracellular Leishmania infection on macrophage phenotype and functions. Our data delivered the first demonstration that Leishmania establishes permissive conditions for persistent, intracellular survival by remodelling the host cell chromatin during infection, causing massive changes in host cell gene expression with important consequences on the macrophage metabolome and immune functions (Lecoeur et al., in revision). The discovery of a series of host-directed hit compounds in our phenotypic screening assay opens exciting new venues to target the host cell epigenome for anti-leishmanial intervention – a novel strategy that may be more refractory for the development of drug resistant Leishmania.
Our current and future research program will finalize the thematic transition toward systems-level analysis of Leishmania/host interaction through the coordination of the two major international projects that animate our three complementary Research Axes in the future: the EU-funded LeiSHield project (Axis 1) and our IPIN-funded International Mixed Unit (IMU) (Axes 2 and 3). Our major aims are (i) to reveal novel mechanisms of parasites genomic adaptation using our L. donovani LD1S experimental system that will directly inform our LeiSHield partner teams and their epidemiological field studies, and (ii) to assess the macrophage response to intracellular Leishmania infection and uncover mechanisms of parasite immune-subversion that will inform our IMU on parasite- and host-directed drug targets. The ultimate goal of our research project is to establish – through the LeiSHield and IMU collaborations – the genomic and immunological read outs as well as the experimental, computational, and clinical infrastructure to approach in the future one of the major open question in clinical Leishmania infection: How parasite genetic heterogeneity affects anti-leishmanial immunity and immuno-pathology, and vice versa how the genetic diversity of the host affects these responses and shapes the parasite phenotypic landscape and its pathogenic potential.