Molecular bases of the Persister-Host Interactions – MORPHEO

Research addressing the infection process has recurrently overlooked the ability of genetically identical bacteria to develop, constantly and reversibly, subpopulations with distinct functionalities and physiologies. This phenomenon termed phenotypic heterogeneity is of major clinical importance as it implies the formation of persisters. A persister is a nonreplicating individual bacterium, among an otherwise susceptible proliferative population, that transiently evades the bactericidal activity of antibiotics. The persister causes non-heritable antibiotic resistance leading to non-resolving infection and relapse that imposes prolongated and/or repetitive courses of antibiotics. The persisters frequently form within the intracellular environment, even for bacterial pathogens long considered extra-cellular. The very macrophages that typically serve as the initial line of defence to control pathogen burden are emerging as a primary environment for the formation of persisters. For decades, research has sought to elucidate the bacterial molecular mechanisms underlying the formation of intracellular persisters and has established a prominent role for the bcterial stress responses. We have recently established Legionella pneumophila, a facultative intracellular bacterium, as a powerful model organism to study the phenotypic heterogeneity of human pathogens during infection. Legionella is responsible for a life-threatening pneumonia, the Legionnaires’ disease, for which therapeutic failures are clinically documented. In agreement, we demonstrated that Legionella produces nonreplicating antibiotic persisters within phagocytic cells. Our proteomics analysis uncovered the molecular map of the intracellular persisters and unveiled the depth of the differentiation process underlying their formation. However, the role of host factors in persister formation remains unclear. In this project, we propose to comprehensively investigate how the host macrophage contributes to the formation of the intracellular persisters. Studying persisters is challenging, as it is a reversible physiological state occurring in a fraction of the pathogen population. To achieve an unbiased characterisation our consortium will combine methods in microbiology, cell biology, molecular biology to infections assays and cutting-edge high-throughput single-cell acquisition systems, multi-parametric high-resolution microscopy, omics analysis and genome-wide loss of function approaches. MORPHEO will provide the first comprehensively analyse the host cell signalling pathways modulating the intracellular persistence of a bacterial pathogen. Our project focus on the host cell, and its intrinsic heterogeneity, in driving the formation of persisters. Hence, MORPHEO’s outcome should not only bring new insights on the Legionella persister’s biology, but in the persisters field at large. As the formation of persisters is part of a general bacterial strategy to produce multiple phenotypes to foster tissue colonisation and disease progression, this project will foster the paradigm change in ongoing research to better understand the mechanisms underlying cell-to-cell variations during the infection and to eventually identify molecular determinants based on which new medicines may be developed.