Trans-signalling: A novel mechanism of Leishmania host cell immune evasion through the release of parasite signalling proteins – TranSig

To achieve our objectives, we use a multidisciplinary approach including microscopy, biochemistry, genetics and transcriptomics. The use of microscopy will allow us to follow our proteins of interest to know where they are located. These observations will be in the parasite and the parasite within the host cell. The biochemical approach will allow us to identify the proteins that form a complex with our proteins of interest, it will allow us to better understand their function. The genetic approach is to delete the genes encoding CK1 and Hsp90. The goal is to see if the parasite can still survive in the host cell in the absence of these proteins. The last approach is to identify the genes that are activated in the host cell in the presence of CK1 and Hsp90. Our goal is to understand what processes of the host cell are controlled by the two parasitic proteins.

First, we confirmed the presence of the CK1.2 of Leishmania (LmCK1.2) in exosomes suggesting that the protein is exported into the host cell. We then confirmed the interaction of LmCK1.2 with certain proteins of the host cell and identified pathways that could be modified by the kinase. Thus our analysis revealed that LmCK1.2 could play a role in regulating the immune system and the response to a stimulus from the host cell. We are also interested in the functions of LmCK1.2 in the the amastigote parasite and showed that this protein was mainly involved in protein trafficking, protein translation or regulation of certain metabolic pathways. We also showed that LmCK1.2 could specifically modify HSP90 phosphorylation (addition of phosphate on HSP90). We searched for the amino acids modified by LmCK1.2 and identified several possible sites that we will study in more detail. Next, we studied the impact of these phosphorylation sites on the growth of the parasite. We have shown that some of these sites, especially those located in the ATP binding pocket of HSP90 are very important for the growth of promastigotes while others are essential to the intracellular growth of amastigotes. This work has allowed us to write the first publication that will be submitted in the coming months. The data presented here are the result of a very close collaboration between the French laboratory and the German laboratory, with a constant exchange of technology between the two laboratory.

By increasing our knowledge about casein kinase 1 and Hsp90, two very important proteins for the survival of intracellular parasites, we can improve the development of new anti-parasitic drugs. These proteins are, in fact, very good therapeutic targets. More broadly, we want to show that targeting proteins that are exported by the parasite in the host cell could make treatments more effective because it would prevent the parasites to develop defense mechanisms against these treatments.

Participation to an international conference in France and a publication close to submission.

Intracellular parasitism is a major hallmark of the most successful and deadly human pathogens. This microbial survival strategy is especially devastating if immune cells are exploited as hosts. Here we propose to use the protozoan parasite Leishmania (L.) donovani as model system to investigate the impact of pathogen-derived signalling molecules on parasite viability, virulence, and host cell expression profile. L. donovani is the causative agent of visceral leishmaniasis that represents a major public health problem worldwide and has been declared as the most significant emerging parasitic disease in Europe due to global warming. Despite the relevance of intracellular Leishmania infection in global mortality and morbidity, surprisingly little is known on how these microbes reprogram their host cell to establish permissive conditions for survival.

The TranSig consortium is focused on secreted Leishmania signalling proteins that may act in “trans” to modulate the host cell phenotype. Our project emerges from a series of previously published observations showing (i) inactivation of macrophage immune signalling and anti-microbial activities by intracellular Leishmania, (ii) release of the parasite casein kinase homolog CK1.2 and the chaperone HSP90 into the host cell cytoplasm, and (iii) direct interaction of CK1.2 with host immune proteins. We hypothesize that CK1.2 is released through exosomes into the host cell cytoplasm in a HSP90-dependent manner, where it modulates signalling by phosphorylation of host proteins in order to establish permissive conditions for intracellular parasite survival. TranSig investigates this innovative working hypothesis through three complementary and multi-disciplinary tasks by applying genetic and microscopic approaches to gain insight into the function and localization of the Leishmania ecto-kinase CK1.2 (Task 1), by using a chemical-genetics approach to functionally analyse the role of HSP90 phoshorylation on chaperone function and localization (Task 2), and by investigating the regulatory relationship between CK1.2 and Hsp90 and their interaction partners, and the impact of both kinase and chaperone activities on the host cell phenotype by transcript profiling using RNAseq technology (Task 3). Our ultimate goal is to translate our research findings into novel potent anti-leishmanial therapies by interfering with parasite protein release thus restoring the host cell anti-microbial potential.

The TranSig consortium mobilizes and synergizes two world-renowned centers in infectious diseases and parasitology, the Institut Pasteur in France (Partner 1) and the Bernhard Nocht Institute for Tropical Medicine in Germany (Partner 2). Significantly, both partners have a common interest in Leishmania stress signaling, with the German partner being an expert in parasite heat shock protein and chaperone biology, and the French partner providing expertise in parasite kinase biology and stress-induced protein phosphorylation. This complementary interest and expertise in parasite stress response is documented through a recent joint high-impact publication in PNAS. TranSig thus provides a unique opportunity to (for the first time) financially support this validated collaboration and to establish a powerful platform driving scientific excellence across European borders. TranSig will deliver considerable progress beyond the state-of-the-art with respect to (i) our very limited knowledge in the fields of parasite protein kinase and heat shock protein biology, and Leishmania-host cell interaction, with relevance to other intracellular parasites, such as Trypanosoma cruzi and Plasmodium falciparum, and (ii) the identification of novel drug targets that directly feed into the drug development pipeline established by Partner 1 through the LEISHDRUG (www.leishdrug.org) and TRANSLEISH (www.transleish.org) consortia.