Survival and dissemination of enteric pathogens through activation and subsequent inhibition of programmed cell death pathways

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

After being ingested on infected food pathogenic bacteria are taken up by cells in the intestine, entering the cells and attempting to grow. An infected cell recognizing the danger undergoes a tightly controlled form of cell suicide known as apoptosis. Thus the cell removes itself and the danger whilst simultaneously sending out warning signals to the immune system. E. coli and Salmonella are two of the most common food poisoning associated pathogens in the U.K. Based on our knowledge of the body's response to infection we would expect cells to undergo apoptosis when they come into contact with E. coli and Salmonella. In the case of these pathogens however the infected cells survive for some time following infection allowing the bacteria to grow within the infected cell. This extra time for growth is crucial, allowing these pathogens time to multiply and cause a more serious prolonged infection. Through this proposal we aim to understand how these pathogens are prolonging the life of infected cells. This work will have relevance for numerous bacterial pathogens, many of which we now know also attempt to interfere with this process of apoptosis during infection.

Our previous work has shed new light on the complex interactions occurring in Salmonella infected cells. During infection the bacteria deliberately target proteins in the cell that respond to the infection by inducing cell suicide. In particular one extremely potent host cell enzyme called caspase-3, or the 'executioner caspase' is targeted. This is the key enzyme in inducing cell suicide, killing the cell quickly but in a controlled fashion. Due to the destructive power of caspase-3 its activation is tightly regulated, making its targeting by bacterial pathogens all the more surprising.

However instead of trying to prevent caspase-3 from working these bacteria initially try to harness its activity bypassing the stringent controls put in place to ensure caspase-3 activity is kept in check. We now know Salmonella infection is activating caspase-3 by a unique mechanism and understanding and exploiting this is a key objective for this proposal.

Activation of an enzyme as destructive as caspase-3 is a risky strategy for a pathogen that only has a limited time to try to grow within an infected cell. Salmonella and E. coli however have developed a mechanism to control the destructive power of the enzyme. Host cells have a natural recycling system called the proteasome that is used to take old or damaged proteins and break them down and use the building blocks to form new proteins. These bacterial pathogens tap into this recycling system, using their own proteins to mimic proteins from the cell that sort host proteins for recycling. Using this tactic the bacteria divert caspase-3 from its normal role, causing the host cell to inadvertently send it for recycling. This delays cell death meaning the bacteria can now multiply within the cell.

This tactic we believe is replicated by numerous other bacterial pathogens during infection, meaning this proposal will have repercussions for the study of numerous microbial infections.

Technical Summary

Bacterial pathogens are now known to interact intimately with apoptotic pathways, promoting their persistence through selective activation and inhibition of these pathways. Here we will characterize these pathogen-apoptosis interactions building on novel findings from my laboratory.

Initial studies will characterize the novel mechanism by which the enzyme is activated by a translocated effector protein in circulating immune cells. Although we have identified the effector responsible, its interaction and that of other effectors with the many pathways regulating apoptosis remain unresolved. We will elucidate the apoptotic response during early stages of infection, generating a comprehensive picture of bacterial interactions with this crucial process.

Induction of apoptosis is a high-risk strategy for a bacterial pathogen but we have identified an innovative bacterial approach to delaying cell death, use of the cell's own ubiquitination machinery. Ubiquitin ligase mimics are well characterized in a number of pathogens and these bacterial proteins are crucial to the unique phenomenon of bacterial induced turnover of caspase-3 that we see during infection. This tagging of caspase-3 with ubiquitin to induce its proteasomal degradation aids in the maintenance of the intracellular replication niche for an increased length of time, enabling bacterial growth and promoting persistence.

Understanding bacterial interactions with these pathways can identify new bacterial protein targets enabling therapeutic intervention or the generation of vaccine candidates to alleviate both acute and persistence infection.

Planned Impact

Many, if not all bacterial pathogens, interact with apoptotic pathways. Our recent discovery of direct activation of caspase-3 by S. Typhimurium in order to promote infection has already had an important impact on the field of infectious disease work. Building on this discovery by elucidating the mechanisms employed by S. Typhimurium to not only activate but also control caspase-3 is the priority in this proposal. Dissemination of the findings will benefit others in the field researching other acute and persistent microbial infections. Activating or preventing cell death is a key part of many types of infection and identifying the mechanisms and bacterial proteins involved will allow for further research into their potential therapeutic targeting. In this regard this proposal will have implications for research into the survival of pathogens within their livestock and human hosts, shedding light on a novel pathogenic mechanism that has far reaching relevance in the field.

The overarching goal of this research is to gain an understanding of bacterial pathogens' ability to undermine the apoptotic process. The potential for exploitation of this finding for use in therapy is great as this project studies two novel discoveries in caspase-3 control, direct activation and its inhibition through ubiquitination. Elucidating how bacteria undermine caspase-3 through the use of bacterial effectors will potentially allow for vaccine mediated intervention. This will result in potential vaccine candidates that can be tested, an area that the mentor for this project Professor Mark Roberts has many years experience and success in.

So while the field of infectious disease research has much to gain from this proposal in the longer term this research will also I believe prove invaluable to understanding the basic cellular biology of apoptosis. Bacteria in this proposal are being used by us to highlight potential weak points in the regulation and control of apoptosis, weak points which without the use of bacteria would remain undiscovered. Elucidating the mechanisms employed by Salmonella to undermine caspase-3, specifically in regard to SipA which directly activates the enzyme, could lead to the development of therapies for directly stimulating apoptosis in cells where it is defective. In this regard collaborations have been put in place with to share our findings with researchers at the Beatson Institute for Cancer Research in Glasgow with a view to testing selected bacterial proteins in appropriate animal models in the future. Understanding the mechanism behind direct activation of caspase-3 is of great significance and potentially has a great many therapeutic uses but this activation may be very complex and must be understood completely.

Understanding apoptosis and in particular defective apoptosis has become an important part of my research in the past number of years. Presently I collaborate with the Medical Genetics Unit at Yorkhill Children's Hospital in Glasgow where I am analyzing samples from patients that are heterozygous for caspase-3 expression in an attempt to better understand caspase-3 regulation. The work undertaken in my laboratory has positively benefitted these medical professionals and patients and aided in the diagnosis of a highly unusual genetic condition. In addition we have recently instigated collaborations with researchers studying veterinary pathogenesis at the University of Glasgow and at the Moredun Research Institute. We hope that through these collaborations we can apply our work to reduce the carriage of pathogenic organisms in livestock.

Publications

10 25 50
 
Description We have identified short chain fatty acids (SCFAs) as key mediators in allowing certain bacterial pathogens to thrive in the inflamed human intestine. In the case of Crohn's Disease these SCFAs are perturbed and these bacteria use this as a signal to grow and survive under conditions that are hostile to most other bacteria. These bacteria called adherent and invasive E. coli (AIEC) are found in increased numbers in these patients for reasons which to date were unknown. We believe our work, which we are preparing for publication and which has been further funded by the BBSRC, will be a major step forward in understanding the role these bacteria play in instigating or exacerbating the disease.
This work has also linked with other projects in our lab whereby we are looking at SCFAs produced by bacteria in the intestine that then cross the blood brain barrier. This may in the longer term help better understand the links between gastrointestinal and neurological disorders which are often inexplicably interwoven.
A number of further publications we are working on will describe this work in detail over the coming months.
Exploitation Route Our findings regards SCFAs are becoming increasingly significant given heightened concerns over antimicrobial resistance. In order to reduce antibiotic use, antimicrobials such as SCFAs, are increasingly used. As these SCFAs are naturally present at high levels in the human intestine, we believe excessive use in the environment and agriculture is increasing the risk of bacteria in particular becoming resistant to their antimicrobial effects in the human intestine. The longer term plan to increase the impact of our work is to inform those involved in use of these SCFAs as to their potential side effects. Also given these SCFAs are primarily used in the developed world we intend to disseminate our findings to developing countries to increase awareness as to the risk of excessive use of SCFAs.
Sectors Agriculture, Food and Drink,Chemicals,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.gla.ac.uk/researchinstitutes/iii/staff/danielwall/
 
Description Presently we are collaborating with consultants in the NHS to expand our findings to clinical bacterial isolates. This would allow us to not only confirm our findings clinically but it is also of interest to clinicians who wish to understand both the origins of bacteria associated with IBD and also their effect during IBD inflammatory episodes.
First Year Of Impact 2015
Sector Education,Healthcare
Impact Types Societal

 
Description Appointment to BBSRC Pool of Experts
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Cystic Fibrosis Trust Scientific Advisory Board
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Wellcome Trust 4 year PhD studentships - Molecular Functions of Disease
Amount £130,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description Wellcome Trust 4 year PhD studentships - Molecular Functions of Disease (2nd studentship)
Amount £130,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description mass spectrometry imaging in both infections already supported by the BBSRC New Investigator Grant
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2016
 
Description BBSRC Case PhD studentship Heather Hulme 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution We provide infection models to try to improve the use of the technique of Mass Spectrometry Imaging in following the resolution of infectious disease. Our team has trained the student in established infection models, tissue preparation, tissue culture and pathway/metabolite analysis.
Collaborator Contribution Our collaborators at both the University of Glasgow and AstraZeneca provide both expertise and high tech equipment to ensure success of the mass spec imaging aspects of the project. Further collaborations have also been developed through our collaborators by the PhD student travelling to both the USA and Sweden to be trained further.
Impact Collaboration is multidisciplinary: Infectious disease from our laboratory Polyomics from our collaborators
Start Year 2013
 
Description BBSRC Case PhD studentship Heather Hulme 
Organisation University of Glasgow
Department Centre for Cognitive Neuroimaging
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide infection models to try to improve the use of the technique of Mass Spectrometry Imaging in following the resolution of infectious disease. Our team has trained the student in established infection models, tissue preparation, tissue culture and pathway/metabolite analysis.
Collaborator Contribution Our collaborators at both the University of Glasgow and AstraZeneca provide both expertise and high tech equipment to ensure success of the mass spec imaging aspects of the project. Further collaborations have also been developed through our collaborators by the PhD student travelling to both the USA and Sweden to be trained further.
Impact Collaboration is multidisciplinary: Infectious disease from our laboratory Polyomics from our collaborators
Start Year 2013
 
Description Phylogenetics of adherent and invasive Escherichia coli 
Organisation Heriot-Watt University
Department Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Working on collaborative publications exploring the origin of adherent and invasive E. coli strains associated with inflammatory bowel disease. My laboratory has been leading the research with our colleagues at Heriot-Watt providing bioinformatic input. Our work has primarily focused on classifying differences between the isolated strains from IBD patients with regular commensal or non-pathogenic E. coli. This has all been through in vitro analysis in immune and intestinal epithelial cells.
Collaborator Contribution Our partners have attempted to phylogenetically translate our laboratory findings bioinformatically to see if our data shows a greater overall trend in these bacterial strains.
Impact Presently working towards publication
Start Year 2015
 
Description SCFA detection 
Organisation Glasgow Royal Infirmary
Country United Kingdom 
Sector Hospitals 
PI Contribution Providing samples from animal models of Crohn's disease for comparison to those of humans as regards their short chain fatty acid content. We believe this may be indicative of perturbations in gastrointestinal metabolism and disease.
Collaborator Contribution Short chain fatty acid detection by LC-MS.
Impact Paper under review with respect to this work.
Start Year 2017
 
Description Wellcome Trust Studentship Hannah Wessel 
Organisation University of Glasgow
Department Institute of Infection, Immunity and Inflammation
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint studentship studying host-pathogen interactions with specific emphasis on persistent bacterial infections and their contribution to Inflammatory Bowel Disease (IBD). Our group contributes relevant infection models, both in vitro and in vivo, along with training, expertise, bacterial strains and equipment.
Collaborator Contribution Our collaborators offer immunology experience along with associated training and expertise. IBD type models are also to be used in which our collaborators have experience.
Impact Collaboration crosses immunology and infection and has led to one publication (PMID: 23861899) and also a new Wellcome Trust PhD studentship which began approximately 6 weeks ago.
Start Year 2013
 
Description Wellcome Trust Studentship Sile Johnson 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We are using knowledge gained through our infection research to utilise bacteria to destabilise and ultimately destroy tumours. Our group is contributing extensive knowledge, training and expertise in the area of tissue culture models, in vivo work and also bacterial manipulation and infection analysis.
Collaborator Contribution In depth knowledge of tumour formation and cancer biology is provided by our collaborators. Also they provide access to training in in vitro and in vivo techniques and extensive collections of useful materials for cancer pathway analysis and tumour destruction.
Impact No outputs yet as collaboration is in very early stages. Collaboration is multidisciplinary: Infectious disease from our laboratory Cancer biology from the BICR
Start Year 2014
 
Description Annual Meeting Society for General Microbiology - Birmingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Third sector organisations
Results and Impact Five members of the laboratory presented work at the Annual meeting of the biggest Microbiology Society in the UK.
Year(s) Of Engagement Activity 2015
 
Description Human Microbiome Project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This art installation is due to be established within the next few months and be in place over the Summer months at the Eden project.

To be seen what impact this will have. To date we have hosted the student in our laboratory and she is presently putting together her work.
Year(s) Of Engagement Activity 2014
 
Description Society for General Microbiology meeting - Galway, Ireland 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Third sector organisations
Results and Impact Data was presented by two members of the group at this conference.
Year(s) Of Engagement Activity 2015