Single-cell imaging reveals non-cooperative and cooperative infection strategies of Listeria monocytogenes in macrophages

Pathogens have developed intricate strategies to overcome the host's innate immune responses. In this paper we use live-cell microscopy with a single bacterium resolution to follow in real time interactions between the food-borne pathogen L. monocytogenes and host macrophages, a key event controlling the infection in vivo. We demonstrate that infection results in heterogeneous outcomes, with only a subset of bacteria able to establish a replicative invasion of macrophages. The fate of individual bacteria in the same host cell was independent from each other and non-cooperative, but a higher multiplicity of infection resulted in reduced probability of replication. Using internalisation assays and conditional probabilities to mathematically describe the multi-stage invasion process, we demonstrate that the secreted Listeriolysin toxin (LLO) of the PrfA regulon regulates replication probability by compromising ability to phagocytose bacteria. Using strains expressing fluorescent reporters to follow transcription of either the LLO-encoding hly or actA genes, we show that replicative bacteria exhibited higher PrfA regulon expression in comparison to those bacteria that did not replicate, however elevated PrfA expression per se was not sufficient to increase probability of replication. Overall, this demonstrates a new role for the population-level, but not single cell PrfA-mediated cooperativity to regulate outcomes of host pathogen interactions. ### Competing Interest Statement The authors have declared no competing interest.