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

Pathogens have developed intricate strategies to overcome the hosts 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 a 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 the 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 the 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. Key pointsO_LIL. monocytogenes invasion of innate immune macrophages results in heterogeneous infection outcomes at the single cell level C_LIO_LIFate of individual bacteria in the same host cell is independent from each other and non-cooperative C_LIO_LIBacterial populations coordinate host cell uptake via the rate of phagocytosis to reduce internalization at high MOI C_LIO_LIThe PrfA regulon system is necessary but not sufficient for L. monocytogenes replication, but population-level PrfA virulence regulates single cell outcome probability C_LI