Live-cell imaging reveals single-cell and population-level infection strategies of Listeria monocytogenes in macrophages

Abstract

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 the host cell and non-cooperative, being independent from co-infecting bacteria. A higher multiplicity of infection resulted in a reduced probability of replication of the overall bacterial population. By use of internalisation assays and conditional probabilities to mathematically describe the two-stage invasion process, we demonstrate that the higher MOI compromises the ability of macrophages to phagocytose bacteria. We found that the rate of phagocytosis is mediated via the secreted Listeriolysin toxin (LLO), while the probability of replication of intracellular bacteria remained constant. 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 activity to regulate outcomes of host pathogen interactions.

Bacterial aggregation facilitates internalin-mediated invasion of Listeria monocytogenes

“…Given that we already estimated the overall probability of host cell harbouring replicative invasion ( Figure 2A ), we applied conditional probabilities to quantify if internalised wildtype and actA-ΔC bacteria had different abilities to replicate in the host. Using this approach, the conditional probability of harbouring a replicative invasion given that the bacteria are internalised (probability of replication of intracellular bacteria) P(R/I)=P(R)/P(I) is defined by the ratio of the overall replication event probability P(R) and the probability of internalisation P(I) ( Moran et al., 2023 ). We found that the probability of a host cell harbouring replicative infection of intracellular wildtype L. monocytogenes was 0.53 ±0.14, while the equivalent probability for the actA-ΔC strain was 0.66 + 0.4 (based on error propagation).…”

“…Whether E-cadherin-mediated host cell entry also relies on aggregation was not investigated in this manuscript and remains to be seen. In general, probabilistic outcomes of the single cell host pathogen interactions may be the consequence of heterogeneity of host and pathogen at the single cell level, involving stochastic gene expression or signalling events in the host or pathogen or both, which together control infection outcomes ( Avraham et al., 2015 ; Guldimann et al., 2017 ; Bagnall et al., 2020 ; Rengarajan and Theriot, 2020 ; Moran et al., 2023 ). Here we demonstrate that L. monocytogenes overcomes these low invasion rates by employing a simple, yet effective strategy relying on extracellular aggregation, enabling more robust adhesion and host cell entry.…”

“…In turn, robust expression of PrfA-mediated InlB/A by bacteria is likely required to facilitate a functional interface with the host receptors ( Niemann et al., 2007 ; Abdulkadieva et al., 2023 ). Previous analyses suggest that PrfA (and presumably InlB/A) activation is heterogenous ( Guldimann et al., 2017 ; Moran et al., 2023 ), which might be a limiting factor for permissive host-cell interactions. Our data show that L. monocytogenes aggregates induce 5-fold more host cell interactions than the non-aggregating actA-ΔC strain.…”

“…PrfA expression and activation is tightly regulated through multiple feedback mechanisms involving transcriptional and post-transcriptional regulation as well as adaptation to environmental conditions such as pH and temperature ( Johansson et al., 2002 ; Reniere et al., 2015 ; Radoshevich and Cossart, 2018 ; Krypotou et al., 2019 ). The PrfA regulon is expressed robustly upon intracellular invasion, in particular in infected blood samples ( Toledo-Arana et al., 2009 ), however PrfA activation (at least in a subset of bacteria) also occurs without presence of host cells ( Guldimann et al., 2017 ; Moran et al., 2023 ). A consequence of the latter is PrfA and ActA-mediated aggregation and biofilm formation to promote colonisation and persistence of bacteria in the host ( Travier et al., 2013 ).…”