Biogenesis, trafficking and roles of Legionella pneumophila extracllular vesicles during infection – BioEV

We analyse the functional role(s) of selected EV-contained-RNAs in the host cell using molecular and cell biology approaches. In parallel, we analysed the proteome and plan to analyse the metabolome, lipid and DNA content of the Lp-EVs to define their overall content, with the particular aim to identify proteins (or other molecules) that might be implicated in RNA loading, targeting and release. Furthermore, how Lp-EVs form, how and where they are released and which structure they have, has not been characterized. Thus, to define the exact structure in vitro and in cellulo we employ Cryo EM of Lp-EVs and cryo-CLEM at the Institut Pasteur. Additionally, we use 3D super-resolution microscopy in living human cells (hMDMs) at the Institut Pasteur Imaging platform to answer these questions. We also use on our new assays where EV containing a cargo tagged with a split luciferase (HiBiT) is loaded on to acceptor cells expressing cytosolic complementary split luciferase (LgBiT). Luciferase complementation occurs upon EV and acceptor cell content mixing. This allows to discriminate EV uptake and content delivery to fully characterise the mode of transport between Lp and hcells. The results will pave the way for a new field of investigation, analysing EV-mediated transport between bacteria and eukaryotic cells and the impact on both partners. Finally, to detect a putative EV-dedicated core machinery conserved during evolution we are setting up a CRISPR-dCas9 knock down library of Lp, equipped with an EV-cargo tagged with luciferase and identify genes that inhibit Lp-derived secretion and delivery to human cells. The results obtained from the genome-wide screen in Lp, will allow to search for mammalian homologues.

- We showed for the first time that L. pneumophila uses EVs to translocate bacterial small RNAs (sRNAs) into host cells that act on host defence signalling pathways
- We have identified additional sRNAs that might act on the host response
- We have identified promising proteins that might participate in RNA loading in the EVs
- We have identified proteins that may participate in membrane fusion processes
- We can measure the uptake of bacterial vesicles in mammalian cells

The main future aims are to continue analyzing the functional role(s) of selected sRNAs that we have identified via RNAseq of L. pneumophila as enriched within the extracellular vesicles (EVs) and to analyze specific proteins that we identified via a proteomics analyses of L. pneumophila EVs. We are setting up and are refining the cargo delivery assay to study cargo release of bacterial EVs and human EVs and the impact of bacterial EVs on human EVs. We have obtained the proof of principal that CRISPRi is working in L. pneumophila and we will now construct the guide library for the L. pneumophila genome.
Based on our results already obtained and the tools set up to obtain new, original knowledge on EV biogenesis, trafficking and their functional roles our project will impact the field of EV biogenesis and trafficking in general and the field of host-pathogen interactions in particular. We will gain in-depth knowledge of how pathogen EVs are able to subvert host functions by delivering RNAs and how pathogen end eukaryotic EVs form, are structured, traffic and may influence each other. Thus we will reveal at the cellular and molecular level how EV-mediated transport impact infection of human host cells by L. pneumophila, a mechanism that might be generalizable to other intracellular, Gram-negative pathogens. Our project results will impact fundamental cell and infection biology by revealing new, general mechanisms of EV biogenesis and new strategies of how pathogens counteract the host defenses. In addition, many of the new tools and reagents that we will generate will be available to the broad research community.

Sahr T, Escoll P, Rusniok C, Bui S, Pehau-Arnaudet G, Lavieu G, Buchrieser C (2022) Translocated Legionella pneumophila small RNAs mimic eukaryotic microRNAs to dampen the host immune response. Nature Communications, 13(1):762 doi: 10.1038/s41467-022-28454-x

Extracellular vesicles (EVs), produced by eukaryotes, bacteria and archaea are increasingly recognized as important mediators of intercellular communication via transfer of a wide variety of molecular cargoes. Recently EVs of several intracellular pathogens have been implicated in pathogenicity and communication. Legionella pneumophila (Lp) is such an intracellular, bacterial pathogen that is responsible for Legionnaires’ disease, a severe pneumonia that can be fatal. To replicate inside cells and to cause disease this pathogen secretes over 300 protein effectors in the host cell. However, these bacteria also produce EVs implicated in virulence. We hypothesise that bacterial EVs traffic in eukaryotic host cells and deliver their content similar, as do eukaryotic EVs. Thus, we propose to use Lp EVs as a model to compare and understand the biogenesis, content delivery and trafficking of bacterial and eukaryotic EVs in eukaryotic cells.