A novel pooled mutant screen approach to analyze bacterial secretion effectors with single-cell resolution

The key for pathogenic success of intracellular pathogen is based on the bacterium abilities to invade and survive inside its host. For that, intracellular pathogens developed various strategies to evade the host immune system surveillance and succeed in multiplying inside the cell. Central to this, many intracellular pathogens use secretion systems that promote injection of effectors proteins into the host cells and manipulate cellular processes. Salmonella typhimurium is one of the well-studied pathogens that its pathogenicity is secretion system-depended. Salmonella uses the Salmonella Pathogenicity Island (spi)- 1 & 2 secretion systems to inject 28 effector proteins from within the phagosome into the macrophage cytoplasm. While biochemical studies uncovered the activities of several of these secreted proteins, the function of other effectors is still unknown, and a systematic understanding of their cooperation is still lacking. Here, we developed a novel method combining single cell RNA sequencing of host cells together with unique bacterial barcoding that allows detection of the identity of the invading bacterial mutant. To demonstrate this method, we generated a tagged barcoded library of spi-2 effectors mutants. We aim to accurately identify individual host targets, gene signatures, and cell states affected by individual spi-2 mutants and their genetic interactions.