Pairwise screening of viral-bacterial co-infections in vitro reveals molecular interaction points that affect pathogen proliferation and host cell viability

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Abstract The broadening of accessible methodologies has enabled mechanistic insights into single-pathogen infections, yet the molecular mechanisms underlying co-infections remain largely elusive, despite their clinical frequency and relevance, generally exacerbating symptom severity and fatality. Here, we describe an unbiased in vitro screening of pairwise co-infections in a murine macrophage model, quantifying pathogen proliferation and host cell death in parallel over time. The screen revealed that the majority of interactions are antagonistic for both metrics, highlighting general patterns depending on the pathogen virulence strategy. We subsequently decipher two distinct molecular interaction points: Firstly, murine Adenovirus 3 modifies ASC-dependent inflammasome responses in murine macrophages, altering host cell death and cytokine production, thereby impacting secondary Salmonella infection. Secondly, murine Adenovirus 2 infection triggers upregulation of Mprip, a crucial mediator of phagocytosis, which in turn causes increased Yersinia uptake, specifically in virus pre-infected bone-marrow-derived macrophages. This work therefore encompasses both a first-of-its-kind systematic assessment of host-pathogen-pathogen interactions, and mechanistic insight into molecular mediators during co-infection. Content of the Repository Raw screen runs -- Folder containing the source data (excel-format) for each plate-reader-based replicate of the screen mAdV2_microscopy -- Folder containing the source images (.czi-format) for the siRNA-knockdown of Mprip and subsequent mAdV2-coinfection, inculding the quantification and analysis mAdV3_microscopy -- Folder containing the source images (.czi-format) for ASC-speck counting after mAdV3-infection and inflammasome stimulation Calculation_template.xlsx -- excel-based analysis sheet to calculate cell death, bacterial growth and viral growth from the plate reader data Quality_Control.xlsx -- summary of replicate Pearson correlations and paired p-value testing to ensure reproducibility or decide for the necessity of a third replicate Screen_overall_results.xlsx -- Summary of the results of the co-infection screen Screen_static_summary.xlsx -- Quantification of the dynamic metrics for each of the screening runs (indicated by number and name) Validation_Bliss_Scores.xlsx -- Summary of the validation of a subset of idetified interactions mAdV2_FACS_quantification.xlsx -- quantified cell populations (uninfected, singly infected, doubly infected) after mAdV2-co-infection mAdV2_SILAC.xlsx -- analyzed proteomics data for SILAC labeling during mAdV2 infection (for source files, see proteomeXchange) mAdV3_SILAC.xlsx -- analyzed proteomics data for SILAC labeling during mAdV3 infection (for source files, see proteomeXchange) mAdV3_inflammasome_activation.xlsx -- quantification of cell death, cytokine release and ASC-speck-positive cells upon mAdV3-infection and inflammasome stimulation

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