Periodic Reporting for period 4 - DecodingInfection (Decoding the host-pathogen interspecies crosstalk at a multiparametric single-cell level) Reporting period: 2022-04-01 to 2023-03-31 Summary of the context and overall objectives of the project Management of many bacterial infections is becoming increasingly difficult due to new and rapidly evolving pathogens with increased virulence and drug resistance. Promising alternative approaches to targeting pathogens are to harness the host’s own response to infection or to target virulent processes of the pathogen. To achieve this, we need a much better understanding of the dynamic host-pathogen interactions. One of the least-understood periods in the course of infectious disease is the early stages from the initial inoculation with a pathogen to the appearance of the first symptoms of disease. To realize these intriguing alternatives and bridge the current knowledge gap, our DECODINGINFECTION proposal is aimed towards a comprehensive and systematic analysis of the complex dynamics between host and pathogen, to understand how the initial inoculation with a small number of bacteria can establish clinical disease even years later, and in only a small minority of individuals. Our working hypothesis in this proposal was that infection outcome may in fact be determined already at early stages, by individual encounters between host and pathogen cells, and that instead of a general inability to control infection, differences in the outcome of infection between individuals could be driven by small subpopulations. To test this hypothesis, we developed single cell RNA-seq multi-parametric approach that allows quantitative, unbiased tools for characterizing the molecular details underlying distinct infection outcomes of individual host-pathogen encounters: from the tissue culture setting to whole organism infection, and finally to understanding variation within the human population. This comprehensive strategy, from the dynamics of a single host and a single pathogen to that of the intact immune system, enables us to determine what is needed to mount an efficient immune response against invading agents, and when immunity fails. Characterization of these critical junctions of infection will provide tools for early detection of infection and targets as potential therapy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far After 60 months from the start of the project (since 1/10/2017), we achieved important scientific results, working in 3 parallel and complementing routes: (1) Developed an approach to simultaneously profile, at the single cell level, both host and pathogen during encounters; 2) Characterized the molecular details that underlie the formation of subpopulations during macrophage infection; and (3) Determined how host and pathogen encounters in vivo result in emergence of specialized subpopulations that underlie divergent phenotypic outcomes. These are completely in line with our initial proposal, and the aims of the grant to achieve our goal of a comprehensive understanding of infection at early stage. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The fulfilment of DECODINGINFECTION main goal is (as indicated in the proposal): “…to reconstruct the repertoire of host and pathogen strategies that prevail at critical stages of early infection.”. This is well beyond the current state of the art in the field. We expect to fundamentally shift the paradigms of infectious disease pathogenesis and lay the groundwork for the development of a new generation of therapeutic agents targeting the specific host-pathogen interactions ultimately driving disease.