A mathematical and biophysical analysis of salmonella macrophage interactions

Lead Research Organisation: University of Cambridge
Department Name: Veterinary Medicine

Abstract

Salmonella enterica causes a wide range of diseases in many animals. Economic losses to the farming industry through Salmonella infection are potentially very high, but also important is the fact that a number of serovars that infect animals can also cause food poisoning and gastroenteritis in humans. Salmonella infect specialised immune cells (macrophages) where the bacteria reside within an intracellular compartment called the salmonella-containing vacuole. Within the macrophage the bacteria may be killed, may hide, may grow and can cause the cell to die. The interaction between macrophages and salmonellae is critically important to the progression of disease. Controlling the numbers of bacteria within the macrophage is an important step in enabling the host to stop the bacteria growing and to survive infection. Many mechanisms involved in the interaction between Salmonella and macrophages are unclear. In particular little is known about how the macrophage physically interacts with the cell. The macrophage detects the presence of bacteria through specialised proteins called Pattern Recognition Receptors (PRRs) which induce macrophage responses to help the host control a salmonella infection. In this project I will use mathematical models to predict how PRR activity effects the process by which Salmonella infect macrophages and then test these theoretical assumptions using biological experiments. I will use physical techniques to investigate whether PRRs influence how Salmonella associate with macrophages. In the final part of the project I will compare how PRR activity affects the global response of macrophages to infection. This project will increase our understanding of how Salmonella infect macrophages and may deliver new targets for developing drugs to treat the diseases caused by these pathogens.

Technical Summary

A crucial aspect of the pathogenesis of invasive Salmonella infections is the ability of the pathogen to invade and survive within phagocytes. Pattern Recognition Receptors (PRRs) on macrophages allow the host to detect pathogens. Many different PRRs detect Salmonella serovars yet the physiological role for most of these proteins in the host response to infection is unclear. Different Salmonella serovars produce bacterial proteins that modulate PRR signaling. The macrophage response to infection involves PRR signaling in concert with the activity of salmonellae factors, but the precise balance of how this occurs is unknown. The question I am therefore asking in this research proposal is what is the contribution of PRRs and bacterial factors to the macrophage control of salmonellosis? This project will use biological, physical and mathematical techniques to study the fundamental mechanisms by which Salmonella enterica serovar Typhimurium (STm) infects macrophages and to determine whether PRRs influence these processes. Iterations of experimental analysis and mathematical models will be used to determine how PRRs affect the process of macrophage infection by STm. Biophysical analysis will be used to determine whether PRRs affect the physical interaction between STm and the macrophage. The final part of this project will be to compare the signaling networks activated by bacterial ligands (such as STm lipopolysaccharide and flagellin) with live STm in macrophages to identify the importance of bacterial factors involved in the cellular infection process.

Planned Impact

The major beneficiaries of this research will be academics in the biological, physical and mathematical sciences. The work will be of particular interest to immunologists and microbiologists. The research will be published in international open access journals to ensure wide access to the data generated by this work. In the long term novel technologies may arise from the physical analysis of Salmonella-macrophage interactions and this will be of benefit to scientists wishing to apply our technologies to other research questions. Should novel technologies arise then they will be developed by Cambridge Enterprise, the University of Cambridge office responsible for commercializing our scientific work. The generation of a macrophage signaling network in response to Salmonella infection may identify novel targets for drug development to prevent salmonellosis which will be on interest to the pharmaceutical industry. Any intellectual property arising from this work will again be developed by Cambridge Enterprise. Our work will generate a number of movies which may be useful teaching material particularly to children explaining how bacteria interact with cells. This material will be used in presentations of the research at the Cambridge Science fair and through the BBSRC media office. I will attend the BBSRC media course in order to develop my communication skills and I plan to run a website where the movies will be available to download.

Publications

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Achouri S (2015) The frequency and duration of Salmonella-macrophage adhesion events determines infection efficiency. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Ekpenyong AE (2013) Bacterial infection of macrophages induces decrease in refractive index. in Journal of biophotonics

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Gog JR (2012) Dynamics of Salmonella infection of macrophages at the single cell level. in Journal of the Royal Society, Interface

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Man SM (2014) Actin polymerization as a key innate immune effector mechanism to control Salmonella infection. in Proceedings of the National Academy of Sciences of the United States of America

 
Description The aim of this fellowship project was to use mathematical and biophysical analytical methods to study how the food borne pathogen Salmonella enterica serovar Typhimurium infects macrophages. This is important because after infecting the host in vivo S. Typhimurium hides and grows within macrophages to evade the immune system then kills the cells to spread infection. In particular the project centred around understanding whether the host Pattern Recognition Receptors (PRRs), NOD-Like Receptor C4 (NLRC4) and Toll-like receptor 4 (TLR4), were involved in the physical process of infection.
The work on the fellowship project made several important observations. Using iterations of mathematical models and experiments we found that, despite S. Typhimurium being a parasite of macrophages, very few of these cells became infected by the bacterium. This is a surprise because macrophages readily phagocytose (engulf) particles so would be expected to take up bacteria and S. Typhimurium also produces invasion proteins to promote active invasion of cells. Using live imaging techniques it became clear that the majority of bacteria failed to invade macrophages. Once infected a macrophage could undergo secondary infection events, but this happened relatively rarely. This is because once a macrophage is invaded by the Salmonella proteins produced by the bacterium activates cytosolic NLRC4 which alters the cellular cytoskeletal proteins making the cell stiffer to reduce further uptake of bacteria. This is particularly interesting because it is the bacterial invasion proteins that activate NLRC4 to drive cellular stiffening, but it now appears these proteins also limit the number of infection events that can occur per cell and hence maximising the resources available for the resident bacterium to grow.
Once infected by Salmonella another consequence of this infection-induced stiffness is to arrest cellular movement. This is important because in vivo infected macrophages are the central cells for controlling bacterial growth by forming abscesses which wall off the cells infected by bacteria to prevent further bacterial spread. Stasis of infected macrophages is likely to be a critical early step in driving abscess formation. Mice lacking NLRC4 have dispersed and poorly formed abscesses which supports this hypothesis.
In other work funded by the BBSRC we had observed that infection of macrophages by Salmonella activates two cytosolic NLRs, NLRC4 and also NLRP3. When an NLR is activated it recruits a protein, ASC, to form a massive cytosolic protein complex called the ASC speck. We noted that despite two NLRs being activated only one ASC protein complex forms per cell. Using super-resolution microscopy we have shown that the ASC speck contains both NLRC4 and NLRP3 at the same time contradicting the currently accepted dogma that each NLR will form its own ASC speck.
TLR4 has little effect on the physical process of infection. TLR4 is critical for driving the cellular signalling process that leads to the formation of host protective cytokines and this process differs when driven by live bacteria rather than compounds that activate this receptor.
Exploitation Route Our findings may be used in development of new approaches to immunology research for use in vaccine discovery programs and in education
Sectors Agriculture, Food and Drink,Education,Healthcare

URL http://www.vet.cam.ac.uk/directory/ceb27@cam.ac.uk
 
Description The impact from this work includes several outputs 1. Scientific communications including the publication of several papers and the presentation of several talks to fellow academics 2. Public Communication of Science: I gave the following talk to 3 college open days for children and the general public (Queens', Peterhouse, Downing) and to the Society for Biology in Cambridge. "There are 100 trillion bacteria in your gut: how do we protect ourselves against infection?" I also featured in a 2010 BBC Radio Cambridgeshire "The Naked Scientists" program presenting a talk on our collaborative work with physicists entitled "How do lasers pick up bacteria?". 3. Development of multi-disciplinary research collaborations leading to further funding as well as application of new techniques to immunological and vaccinology research
First Year Of Impact 2010
Sector Agriculture, Food and Drink,Education,Healthcare
Impact Types Societal

 
Description Effects of Nod-like receptor activity on protective immunity against Salmonella infection
Amount £917,208 (GBP)
Funding ID BB/K006436/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2016
 
Description Elion and Black Immunology Catalyst Sabbatical Award
Amount £226,000 (GBP)
Organisation GlaxoSmithKline (GSK) 
Sector Private
Country Global
Start 11/2016 
End 10/2019
 
Description GSK Varsity Award
Amount £300,000 (GBP)
Organisation GlaxoSmithKline (GSK) 
Sector Private
Country Global
Start 05/2016 
End 04/2018
 
Description Interdisciplinary Research Grant
Amount £204,000 (GBP)
Funding ID ARUK-IRG2014-13 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2015 
End 01/2018
 
Description Royal Society Wolfson Refurbishment Scheme
Amount £267,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 02/2018
 
Description Wellcome Trust Investigator Award
Amount £2,200,000 (GBP)
Organisation Wellcome Trust 
Department Wellcome Trust Senior Investigator Award
Sector Private
Country United Kingdom
Start 01/2016 
End 12/2021
 
Description Collaboration with GSK 
Organisation GlaxoSmithKline (GSK)
Department Respiratory Biology GSK
Country United Kingdom 
Sector Private 
PI Contribution We hold a GSK-Varsity grant to understand why bacteria stick to epithelial cells from patients. We are developing assays for increasing the scale of screening for bacterial adherance to cells, generating bacterial mutants to prevent bacterial adhesion and looking at novel human molecules that may be important for bacterial adherance
Collaborator Contribution Provision of expertise, novel knock out mice, CRISPR screens in human cells, epithelia from patients
Impact New collaboration
Start Year 2015
 
Description Doug Golenbock, University of Massachusetts Medical School 
Organisation University of Massachusetts
Department University of Massachusetts Medical School
Country United States 
Sector Academic/University 
PI Contribution I went on sabbatical to the Golenock laboratory for 2 months in 2008
Start Year 2008
 
Description GSK Immunology Catalyst Sabbatical Award 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution GSK have bought out my teaching and administration duties for 3 years. I spend 3 days a week at GSK and have 2 postdoctoral researchers at GSK. I am working on inflammasome biology and Pattern Recognition Receptor research. I intellectually input into several of the GSK therapeutic units.
Collaborator Contribution GSK bought out my teaching and administration duties for 3 years and fund 2 postdoctoral researchers to work with me at GSK
Impact None yet
Start Year 2016
 
Description Genentech Visiting Professorship 
Organisation Genetech, Inc
Country United States 
Sector Private 
PI Contribution I visited Genentech South San Francisco site and performed research in the laboratory there for 9 months. Two of my PhD students also had 3 month internships working with myself and my Genentech collaborators. We learnt new techniques and gained access to new reagents. I intellectually contributed to their inflammasome research program.
Collaborator Contribution My collaborators gave me novel reagents and supported my research during my time in the USA
Impact None yet as the work is still in progress
Start Year 2016
 
Description International and national collaboration with David Underhill 
Organisation University of California, Los Angeles (UCLA)
Country United States 
Sector Academic/University 
PI Contribution Collaboration
Start Year 2000
 
Description International and national collaboration with Eicke Laetz 
Organisation University of Massachusetts
Department University of Massachusetts Medical School
Country United States 
Sector Academic/University 
PI Contribution University of Massachusetts Medical School
Start Year 2008
 
Description International and national collaboration with Shizuo Akira, Osaka University 
Organisation Osaka University
Country Japan 
Sector Academic/University 
PI Contribution Collaboration
Start Year 1999
 
Description International and national collaborations with Kate Fitzgerald 
Organisation University of Massachusetts
Department University of Massachusetts Medical School
Country United States 
Sector Academic/University 
PI Contribution Intellectual input and collaborative discussions about common research interests.
Collaborator Contribution Provision of reagents, cell lines and intellectual input to our research.
Impact Publications linked to our research grants have been facilitated by this collaboration.
Start Year 2006
 
Description Mike White 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Technology transfer and collaborative discussions
Collaborator Contribution Technology transfer and collaborative discussions
Impact Development of new freeware computer software to track single cell movement and translocation of nuclear factor kappa B
Start Year 2010
 
Title Image analysis software for tracking cells 
Description Image analysis software for tracking moving cells and for analysis of fluorescence levels in these cells 
Type Of Technology Software 
Year Produced 2016 
Impact Allows analysis of many cells for single cell signaling assays 
URL http://dx.doi.org/10.17863/CAM.6018
 
Company Name Polypharmakos 
Description Spin out company with Monique Simmonds (Kew), Mark Holmes (Cambridge), Duncan Maskell (Cambridge) to screen natural products for antimicrobial and innate immune activity. 
Year Established 2016 
Impact None Yet
Website http://www.polypharmakos.com/
 
Description ANU, Canberra, Australia, "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk to faculty and students at ANU with associated questions
Year(s) Of Engagement Activity 2018
 
Description Bacterial recognition by PRRs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Toll2011, Garda, Italy: "Bacterial recognition by PRRs"

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Cambridge Science Fair Panel Discussion 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact I chaired a session and talks and a question and answer session on receptors at the Cambridge Science Fair
Year(s) Of Engagement Activity 2018
 
Description Discussion slot on the Naked Scientists Radio Show 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A media interview where different aspects of immunology were reviewed and discussed
Year(s) Of Engagement Activity 2018
 
Description Hudson Innate Immunity Institute, Melbourne, Australia "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk to faculty and students at the Hudson Institute and associated questions
Year(s) Of Engagement Activity 2018
 
Description Institute for Child Heath, London (2011):Pattern recognition receptors: the key to host recognition of infection or a load of old PAMPs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Institute for Child Heath, London

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description International Cytokine Meeting, Boston, USA "Bacterial recognition by Pattern Recognition Receptors" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk at the international cytokine meeting and questions after.
Year(s) Of Engagement Activity 2018
 
Description Lafferty Debate participant 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Participated in a public debate on "Adaptive Immunity is innately redundant"
Year(s) Of Engagement Activity 2016
 
Description NIH, Bethesda, USA "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk to staff and researcher at the NIH and responded to the associated questions
Year(s) Of Engagement Activity 2018
 
Description Pattern recognition receptors: the key to host recognition of infection or a load of old PAMPs 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Talk to the University of Maryland, Baltimore, USA

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Pattern recognition receptors: therapeutic targets for allergic as well as infectious diseases 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at Cambridge Institute for Medical Research, Cambridge.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Recognition of bacterial infection by Pattern recognition receptors 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Talk to University of Massachusetts Medical School, Worcester, USA

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Recognition of bacterial infection by Pattern recognition receptors 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact NIH NIAID, Bethesda, USA

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Talk Young Microbiologists Belfast 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Talk to young microbiologists (post graduate and post doctoral researchers) and associated questions
Year(s) Of Engagement Activity 2018
 
Description The Lorne Innate Immunity Meeting, Lorne, Australia "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk to the iInnate Immunity meeting and the associated questions
Year(s) Of Engagement Activity 2018
 
Description The University of Brisbane, Australia "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk to faculty and students at the The University of Brisbane with associated questions
Year(s) Of Engagement Activity 2018
 
Description There are 100 trillion Bacteria in Your Gut: How do we protect ourselves against infection? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public Talk Queens' College, Cambridge

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Trinity College Dublin "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk to the faculty members and students at Trinity College Dublin plus associated questions
Year(s) Of Engagement Activity 2018
 
Description University of Baltimore "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk to faculty and students at the University of Baltimore and responded to associated questions
Year(s) Of Engagement Activity 2018
 
Description University of Strasbourg, France "Bacterial recognition by Pattern Recognition Receptors" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk to postgraduate students at the University of Strasberg
Year(s) Of Engagement Activity 2018
 
Description University of Trondheim, Norway "Bacterial recognition by Pattern Recognition Receptors" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk (and answered questions in response to talk) to students and researchers at the University of Trondheim in Norway.
Year(s) Of Engagement Activity 2018
 
Description Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia "Pattern Recognition Receptor Signalling in response to Infection" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Talk to faculty members and students at Walter and Eliza Hall Institute for Medical Research with associated questions
Year(s) Of Engagement Activity 2018
 
Description What can maths and physics do for infection biology? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at Cardiff University School of Biosciences

no actual impacts realised to date
Year(s) Of Engagement Activity 2012