Understanding infection strategies of the food borne pathogen Listeria monocytogenes at the single cell level

Mammals have central cellular defence systems that resist infection by a range of pathogens. These involve the signalling systems, which are critical regulators of the antibacterial immune response. To date studying the interactions between these cells and bacterial pathogens have been done at the population level. We have now developed novel tools to study the direct interaction of individual host immune cells with bacterial pathogens at the single cell level thereby generating a greater understanding of the events involved in infection. We have focussed on the food-borne pathogen Listeria monocytogenes responsible for a number of serious infections of man and animals. L. monocytogenes invades host cells in which it then grows before spreading to adjacent uninfected cells thereby propagating the infection. Our preliminary data suggests that the outcome of the infection with L. monocytogenes, actually depends on a very small subset of bacteria, which can establish successful infections after invasion of host immune cells, We show that in doing so, L. monocytogenes is able to inhibit antibacterial host defence mechanisms, including the production of protective signalling molecules critically in enlisting an effective immune response. These events, which control the overall outcome of infection, have been masked in previous population-level analyses, which simply considered average cell behaviour. Therefore, our single cell data raises fundamental questions about the mechanisms employed by the very few successful pathogens to overwhelm the antibacterial defence mechanisms of the host. Whether this is a consequence of changes in gene expression in L. monocytogenes or changes in the host cells that make them permissive for replication, or both, is unclear and this is being addressed in this application.

In this project we aim to understand the interactions between individual L. monocytogenes bacteria and host cells and decipher what leads to a successful infection. To achieve this we have established state-of-the-art and uniquely quantifiable real time single cell imaging and gene expression approaches. This will allow us for the first time to follow under the microscope the behaviour of hundreds of individual host and bacterial cells and characterise their patterns of behaviour. We will monitor by microscopy in individual infected host cells the expression of both virulence factors by L. monocytogenes, required for growth inside host cells and the response of individual host cells to infection. As a consequence, this project will discover the detailed real time interactions between this important food-borne pathogen and host cells, revealing critical stages in the development of infection inside host immune cells thereby leading to a greater understanding of the overall infection outcome.

In the longer term, the discoveries made in this project in understanding of immune cells responses to pathogen infection, will inform future novel treatment-strategies, less dependent on the use of antibiotics. This study will establish a paradigm for analysis of host pathogen interactions in single cells, and will be applicable to understand generic cell and tissue responses to diverse biological stimuli.

The dynamic and stochastic nature of immune cell and pathogen interactions ultimately decide the fate of the whole organism, however the underlying mechanisms are masked in typical population-level analyses. In this project, we will combine cutting-edge single cell approaches (live-cell imaging and gene expression studies) to mechanistically understand strategies employed by Listeria monocytogenes, an important food-borne pathogen of man during infection of macrophages, a critical step controlling the overall infection outcome. Our data show that only ~1 in 100 of L. monocytogenes establishes sucessful infection after invasion of macrophages, and in this proposal we investigate the underlying regulatory mechanisms in host and pathogen cells. We will use live-cell time-lapse microscopy including single molecule Fluorescent Correlation Spectroscopy, and monitor activation of a critical PrfA virulence system in single infected cells to quantitatively understand its regulation upon infection of macrophages, phagosome escape and replication. To understand how L. monocytogenes overcomes the antibacterial host responses and spread to neighbouring macrophages we will visualise (and perturb) the activation of the NF-kappaB and STAT signalling in host cells during the course of infection. Will also use single cell RNA sequencing to globally reconstruct key molecular systems in the pathogen and the host (beyond PrFA and NF-kappaB/STAT systems) that underlie the outcomes of L. monocytogenes and macrophage interactions. Together, these cutting-edge single cell approaches will for the first time provide real insights into how a small subset of "successful" L. monocytogenes overcomes the antibacterial host responses. Understanding the strategy of this very small subset of L. monocutogenes will important for predicting immune response to L. monocytogenes (and other pathogens). Tools developed in this project will have wide applicability to other tissue systems and pathogens.

This project offers a great potential to better understand pathogen infection strategies and antibacterial defence mechanisms. We focus on a quantitative understanding of infections with Listeria monocytogenes an important food-borne pathogen of man (~30% mortality rate, affecting the most vulnerable groups of the population) and farm animals, a major burden on our society. L. monocytogenes is also a well-established model system for other pathogens. This creates the opportunity for the step change in our understanding of infection biology, and is relevant to healthcare and to the pharmaceutical industry.

By its very nature this project will be of specific interest to microbiologists, immunologists, biomedical scientists and clinicians who are working in the field of infection disease and inflammation. The novel single cell approaches will benefit the wider scientific community since they will be applicable to studies of other processes or disease, e.g., cancer, developmental biology, interactions between plant cells and saprophytic/pathogenic microorganisms and biofilm formation by bacteria. We will ensure the best use of the results by the community. We will engage with academics through high impact publications and talks at major conferences. We will make data and tools available, on publication, in established public depositories (e.g. the Dryad, Array Express, Omero), where suitable databases exist. Genetic tools (plasmids) will be made available to other researchers (via the Addgene). We have a vast experience in interdisciplinary training (Paszek is a theoretician who runs an experimental lab as a key BBSRC David Phillips Fellowship output). We will closely work with the project's PDRA to transfer our expertise in interdisciplinary research and develop his career in infection biology. We will contribute to the depth of systems biology training in Manchester and in the UK.

Better understanding of host-pathogen interactions could provide novel immune-based drug targets (that do not rely on antibiotics and thereby avoid issues of AMR). This has a potential of mirroring recent success of immune-based anti-cancer therapies. We will use existing links via Manchester Collaborative Centre for Inflammation Research, focusing on translation to clinical science and pharmaceutical industry (co-funded by AZ and GSK) would allow rapid assessment of target drugability.

We will continue to strongly engage with instrumentation companies who are interested in the utilization of the quantitative dynamic imaging technology and genomics capabilities. We have close relationships with bio-imaging instrumentation companies via Systems Microscopy Centre (SMC), in particular with Carl Zeiss and Hamamatsu Photonics. This involves direct funding for meetings and training that is already in place. We will seek opportunities to work with next-generation sequencing instrumentation companies (e.g., Fluidigm) to improve current microfluidic design (e.g. future combination of time-lapse imaging with a genomics experiments will be critical).

The use of microscopy generates movies and images that are colourful and visual. They represent an excellent resource for the development of public understanding of science. In 2006 a group from SMC presented an exhibit entitled "The Language of Cells" at the Royal Society exhibitions in London, Glasgow and at Science Day at Buckingham Palace. We will use similar opportunities; talk to schools and other groups to specifically raise the issue of antibiotic resistance. Members of SMC have participated in several public engagement activities including "The Worm Wagon", "Fabulous Physics" and FLS Community Open Day. When the publicity of outcomes from this project are important, we will engage with the University of Manchester Press Office to coordinate this. We have good experience of media publicity and have previously worked with the BBSRC Press office in publicising high impact publications.