DECODING VIRULENCE: A NOVEL FUNCTIONAL APPROACH FOR POOLED MUTANT SCREENS WITH TAGGED BACTERIA AND SINGLE CELL RESOLUTION OF HOST TRANSCRIPTS

The interaction between a pathogen and a host is a highly dynamic process in which host factors restrict bacterial growth, while the pathogens protect themselves from host attack. We have recently observed significant variation in expression from cell to cell in the host response to bacterial invasion, and similarly, variation in bacterial virulence programs. We proposed that this implies that infection outcome may, in fact, be driven by small subpopulations of cells. What is now needed is a functional approach to link gene expression variation to cellular phenotypes of infection outcome. Here, we developed a novel method for pooled mutant screening, combining single cell RNA sequencing of infected host cells together with unique bacterial barcoding, that allows detection of the identity of the invading bacterial mutant within the same infected cell. To demonstrate this method, we generated a tagged barcoded library of all Salmonella Pathogenicity Island (spi)-2 secretion system effectors mutants. We used the pooled library to infect bone marrow derived macrophages, and de-convoluted by single cell analysis the host gene expression that is associated with the unique invading effector mutants. We were able to accurately identify individual host targets, gene signatures, and cell states affected by individual spi-2 mutants and their genetic interactions. Importantly, this method allows functional analysis of bacterial mutants at the single cell level, and can now be adapted to perform genome-wide bacterial mutant analysis also in vivo, in any bacterial strain and any host pathogen infection model.