AbstractL. monocytogenes is a Gram-positive bacterium that is found ubiquitously throughout the world. It is a major foodborne intracellular pathogen and can cause a range of human diseases. It invades the cells of mammalian hosts, escapes the intracellular vacuole, replicates in the cytosol and spreads to neighbouring cells using actin-based motility. After breaching the intestinal barrier L. monocytogenes causes systemic infections in immunocompromised people. The intracellular lifecycle of L. monocytogenes is under the control of the regulator PrfA which regulates major virulence genes. It is widely characterised and well-studied. However, many studies on L. monocytogenes have used population level snapshot data which masks the kinetics of single cell interactions. Individual host cells and individual L. monocytogenes cells are heterogenous and infection outcomes depend on these heterogenous interactions. By designing a live cell imaging model, designed to specifically study single-cell interactions between bacteria and host cells, this study revealed novel host-pathogen interactions during infection. Successful replicative invasions are a rare event, only 9.1% (+-1.2%) of host cells are susceptible to infection and only 0.4% (+-0.05%) of the bacterial population can successfully form replicative invasions. L. monocytogenes forms aggregates in response to a proteinaceous host factor >10 kDa with the aggregates being PrfA regulated and ActA mediated. This is a novel strategy the bacteria use to increase invasion success rate through multiple invasion events. Aggregating bacteria were 3.5-fold more invasive than a non-aggregating mutant, formed 4-fold more replicative invasions in HeLa and 10-fold more in primary HUVEC cells. Additionally, in a competition assay the wildtype outcompeted the non-aggregating mutant by 10-fold. Aggregates were also shown to preferentially interact with and invade cells in the G2/M phase and this was due to InlB-Met interactions, as Met is upregulated during the G2/M phase. Host cells depleted of Met showed a 2.5-fold reduction in the number of associated bacteria. RNA-Seq analysis of L. monocytogenes treated with the causative host factor upregulated PrfA regulated virulence genes, and genes involved in iron uptake and glycerol metabolism. Analysis of gene expression between the wildtype and the non-aggregating mutant showed that aggregation downregulates genes involved in cationic peptide resistance. However, the process of aggregation did not upregulate the PrfA virulence regulon. Overall, this study has demonstrated the value of live imaging single cell interactions between human cells and pathogens and how this can uncover novel interactions that lead to a greater understanding of the biology of infection.
|Date of Award||1 Aug 2023|
|Supervisor||Mark Muldoon (Supervisor), Ian Roberts (Supervisor) & Pawel Paszek (Supervisor)|
- Live cell imaging
- Listeria monocytogenes
- Host-pathogen interactions