PVLSuscept

Staphylococcus aureus is an opportunistic pathogen that causes a wide spectrum of disease in humans. Its virulence is largely associated with the secretion of numerous toxins that are ubiquitously expressed. In contrast, one pore forming toxin, the Panton Valentine Leukocidin (PVL), is only present in a limited number of strains. PVL is epidemiologically associated with primary skin and soft-tissue infections (which are frequent), and highly severe necrotizing pneumonia (mortality 60%) occurring in children and young adults but remaining infrequent (30 cases per year in France). Epidemiological studies clearly highlight a specific role for PVL in exacerbated virulence. Yet, the molecular basis for this role is unclear. While PVL is a pore forming toxin targeting specifically human neutrophils and macrophages leading to their lysis, other ubiquitously expressed hemolysins (e.g. ?-hemolysin) display such an activity in vitro suggesting a redundancy poorly compatible with the epidemiological data. A precise understanding of the redundancy and the specificity of the various hemolysins is hindered by the lack of knowledge of the host receptors (or accessory proteins/lipids which might confer the cell specificity) as well as of the immune responses triggered for most of these toxins. In addition, the extreme rarity of necrotizing pneumonia suggests the existence of a genetic predisposition affecting the host receptor or the innate immune response to PVL. In this project, we aim to identify: the PVL receptor(s), the molecular basis of the susceptibility of certain specific cell types (neutrophils and macrophages), the molecular basis of the species susceptibility (human and rabbits are highly susceptible in contrast to mice and monkeys), the molecular basis of the hyper susceptibility of some individuals to PVL+ S. aureus necrotizing pneumonia. In order to fulfill this goal, three approaches will be used: A-functional screens aimed at identifying cellular susceptibility/resistance factors comprising screening 1-1 : a ShRNA library in a PVL-susceptible cell line 1-2 : a cDNA library from a PVL-susceptible cell line expressed in a PVL-resistant cell line 1-3 : a library of monoclonal antibodies raised against PVL-suceptible cell membrane B-biochemical approaches based on pull-down, immunoprecipitation, cross-link, solid-phase interaction screens to identify host proteins/lipids interacting with Panton Valentine leukocidin C-Immunological and genetic investigations based on a subset of 30 community-acquired PVL+ S. aureus-mediated necrotizing pneumonia pediatric patients who are otherwise healthy and do not present any classical immunodeficiencies. Based on the typical age distribution (affecting children and young adults) and the very low frequency of the disease, we hypothesize that susceptibility to PVL+ S. aureus-mediated necrotizing pneumonia in these patients is associated with monogenic inheritance. We will investigate these patients using both a candidate gene/pathway strategy targeting functional deficiencies in innate immune functions and a genome-wide strategy including linkage and whole exome sequencing approaches in order to discover new defects. The study will benefit from a cohort of 100 PVL+ necrotizing pneumonia patients (whom recruitment has already started) and from the synergistic expertise of: the team Bacterial Pathogenesis and Innate Immunity, INSERM U851, co-directed by F.Vandenesch and T.Henry, who has an expertise in S. aureus-mediated diseases, virulence factors and associated immune responses with a particular focus on PVL, the Laboratory of Human Genetics of Infectious Diseases -INSERM-U980 (C. Picard, L. Abel) who has a leading expertise in investigating the genetic basis of acute infectious diseases, and the Macromolecular Engineering Laboratory (T. Vernet, A.M. Di Gulmi) who has a strong expertise in biochemistry and structural biology of bacterial proteins and their interacting eukaryotic ligands.