Bacterial aggregation facilitates internalin-mediated invasion of .

Dissemination of food-borne in the host relies on internalin-mediated invasion, but the underlying invasion strategies remain elusive. Here we use live-cell microscopy to follow single cell interactions between individual human cells and and elucidate mechanisms associated with internalin B (InlB)-mediated invasion. We demonstrate that whilst a replicative invasion of nonphagocytic cells is a rare event even at high multiplicities of invasion, overcomes this by utilising a strategy relaying on PrfA-mediated ActA-based aggregation. We show that forms aggregates in extracellular host cell environment, which promote approximately 5-fold more host cell adhesions than the non-aggregating actA-C mutant (which lacks the C-terminus coding region), with the adhering bacteria inducing 3-fold more intracellular invasions. Aggregation is associated with robust MET tyrosine kinase receptor clustering in the host cells, a hallmark of InlB-mediated invasion, something not observed with the mutant. Finally, we show via RNA-seq analyses that aggregation involves a global adaptive response to host cell environment (including iron depletion), resulting in metabolic changes in and upregulation of the PrfA virulence regulon. Overall, our analyses provide new mechanistic insights into internalin-mediated host-pathogen interactions of .