DS0403 - Exploration des systèmes et organes leur fonctionnement normal et pathologique : physiologie, physiopathologie, vieillissement

Novel common host targets during bacterial invasion of humans – StopBugEntry

Submission summary

Infectious diseases are a major public health problem, and identification of novel potential therapeutic candidates is mandatory in a context of growing antibiotic resistance. As classical infection biology approaches are nearly exhausted in their current capacity to address the emergence of multi-drug resistance, new conceptual approaches are required. Targeting common cellular pathways hijacked by pathogens during their encounter with host cells represents a novel strategy to inhibit infection. The implementation of comprehensive system biology approaches, together with robust assays from front edge technologies, would be the most efficient method for identifying critical cellular hubs to be targeted as novel therapeutic candidates. The objective of our proposal is to identify such cellular hubs by obtaining unprecedented details of the infection strategies of major model bacterial pathogen. Taking advantage of high-end super-resolutive/correlative approaches, we will functionally characterize these hubs in the context of infection in a rational strategy for anti-bacterial drug development. We will therefore focus our investigation on the study of four relevant human pathogens - Francisella, Listeria Shigella and Yersinia - and we will determine which are the signaling cascades activated by these bacteria at the plasma membrane of host cells or within intracellular vacuolar membrane-bound compartments. We will build our project on complex data sets generated with a subset of pathogens from previous unbiased high-content siRNA screens, which have identified potential host factors involved in the control of cell invasion, vacuolar escape or intracellular proliferation. These data sets provide the basis for a proof-of-principle study to demonstrate the feasibility of our proposal. To address the biological questions focused in this proposal, we will take advantage of unique technological developments achieved by several consortium partners which allow: a) to control adhesion of bacteria at freely-chosen cell entry sites and to perform atomic force microscopy (AFM) measurements of plasma membrane biophysical parameters together with super-resolution imaging; and b) to establish 3-dimensional large volume correlative light electron microscopy (CLEM) via focused-ion beam scanning electron microscopy (FIB-SEM) which allow to monitor the early steps of invasion and particularly vacuolar disruption by cytoplasmic replicative bacteria. Taking advantage of our unique data sets and of these technological innovations, we will integrate them within the context of this proposal to decipher major common host signaling hubs subverted by our group of four model pathogens, and which could be targeted for development of novel anti-bacterial drug activities. Specifically, we will: 1) Identify host signaling hubs activated during the entry steps of invasive bacteria by cross-correlating results from previous screens; through the application of high-end microscopy techniques, we will determine their contribution to adhesion, entry, vesicular trafficking and vacuolar rupture in our set of model pathogens; 2) Decipher signaling cascades triggered at the cell surface by bacterial pathogens by combining AFM measurements with super-resolution imaging during the first contact between bacteria and host cells; 3) Determine signals emanating from pathogens or targeted compartments during the internalization steps, using super-resolution imaging approaches together with functional inactivation of candidate cellular effectors using drugs, siRNAs, dominant negative constructs or CRISPR/Cas9-engineered cells; and 4) Perform dynamic functional ultra-structural imaging of the successive entry steps by using large volume CLEM/FIB-SEM approaches to correlate local ultra-structural changes in vacuolar integrity and induced signaling events.

Project coordination

Javier PIZARRO-CERDA (Unité Interactions Bactéries-Cellules - Institut Pasteur)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IP Unité de Dynamique des Interactions Hôte-Pathogène - Institut Pasteur
CIIL Centre d'Infection et d'Immunité de Lille
INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
IP Unité Interactions Bactéries-Cellules - Institut Pasteur

Help of the ANR 548,525 euros
Beginning and duration of the scientific project: September 2015 - 48 Months

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