Food-borne Listeria infections: Zinc homeostasis at the host-pathogen interface

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

The bacterium Listeria monocytogenes causes serious food-borne disease in man, with high mortality rates despite antibiotic intervention. The disease can range from stillbirth in infected women, septicaemia (blood poisoning) and meningitis. People with weakened immune systems are particularly susceptible to this disease. A feature of Listeria is its ability to survive a range of harsh conditions and grow at low temperatures routinely used to store food. In order for Listeria to grow, it must acquire the metal zinc. This is true not only during life in the environment, on food and food preparation surfaces, but also during infection of a host. This proposal aims to understand how Listeria acquires zinc especially in conditions inside the host where available zinc may be in short supply. We have already identified two zinc-uptake systems and shown that these are vital for Listeria to be able to survive when there is very little zinc available and also for it to be able to cause infections. Having discovered these systems, the next step is to gain a more detailed understanding of how each one contributes to obtaining zinc and their roles during Listeria infections. We will also seek to find other systems that are needed by Listeria to handle zinc. An important impact of understanding the mechanisms by which Listeria acquires zinc and controls its zinc levels will be the opportunity to exploit this knowledge to design new ways of combating Listeria infections and design new control strategies to reduce contamination of foods by Listeria.

Technical Summary

Obtaining zinc for loading onto zinc-dependent proteins is a vital requirement for bacteria. However, zinc can also be toxic and bacteria must therefore precisely balance the supply and demand for this metal to ensure the correct metal-occupancy of their metalloproteins and avoid toxicity. For bacterial pathogens, such as food-borne Listeria monocytogenes, this challenge becomes exacerbated by host immune defences that can act to restrict access to this metal. We have recently identified two related zinc uptake systems in L. monocytogenes which are required for growth during zinc-limitation and are important for virulence during growth in vitro and in vivo. However, whilst there is some functional redundancy between the two systems, this appears to vary depending upon the environment in which the bacterium is growing. This programme of work will test the hypothesis that the presence of the two specific zinc uptake systems in L. monocytogenes allows access to different sources of zinc within different environments. We will perform a detailed characterisation of these systems and identify their preferred substrates for import. We will also identify other proteins within this organism that we hypothesise also perform a vital role in handling zinc and may contribute to zinc-protein assembly. We will investigate the role of each system in promoting the survival of L. monocytogenes within different host cell types and during infection in vivo. With the incidence of listeriosis on the increase and the requirement for novel antimicrobial strategies, a thorough investigation of the zinc-acquisition systems in L. monocytogenes and their roles in virulence is both timely and necessary.

Planned Impact

The programme of work will focus on Listeria monocytogenes which is an important food-borne pathogen and a target for antimicrobial products in the agri-food industry. L. monocytogenes is responsible for a number of serious clinical syndromes in both humans and animals and accounts for 30% of all human fatalities as a consequence of food-borne infection worldwide. Food-borne pathogens and basic/applied microbiology are outlined within the remit of BBSRC committee B (Plants, Microbes, Food & Sustainability). By providing fundamental knowledge of the mechanisms used to survive within different environments and cause infections, this study will help inform the development of new antimicrobials and control strategies to improve microbial food safety and hence is relevant to the BBSRC strategic research priority 'food security' within the strategic objective 'Microbial food safety'. Elucidation of these systems at the molecular level also overlaps with the scientific areas of biochemistry, biological chemistry, structural biology & molecular biology outlined in the remit of committee D (Molecules, Cells & Industrial Biotechnology).

The main impact from this research will come from the acquisition and transfer of knowledge.

This will include:

1. Informing a broad range of researchers in the bioscience and medical sectors, particularly microbiologists, biochemists and cell biologists engaged in the study of the cell biology of metals and/or bacterial pathogenesis.
2. Providing high quality training for early career scientists.
3. Engaging with the media and the public.
4. Generating and sharing strains and detailed protocols as research resources.
5. Engagement with industrial companies via The University of Manchester Intellectual Property Limited (UMIP), with regard to the development of antimicrobials.
6. Engagement with agencies concerned with the control of food-borne infections.
 
Description Listeria can cause serious food-borne disease in humans which is typified by high mortality rates (20-30%) despite antibiotic intervention. Disease mainly occurs due to the consumption of contaminated food, with Listeria being associated with raw vegetables, dairy, meat and fish products. Following ingestion, disease is started by the bacteria being able to survive within the intestinal tract and invade the epithelial cells that line the gut lumen, whilst entry into immune cells (phagocytes) permits their spread throughout the body. The most vulnerable groups are pregnant women and people with weakened immunity with the clinical manifestations including stillbirth in infected women, septicaemia and meningitis. Listeria is the most commonly encountered form of bacterial meningitis amongst the immunocompromised with mortality rates as high as 60%. In order for Listeria to grow, it must acquire the metal zinc. This is true not only during life in the environment, on food and food preparation surfaces, but also during infection of a host. We have established that Listeria has two zinc uptake systems (ZinABC and ZurLAM) and that both contribute to zinc acquisition inside and outside of host cells. We have shown that both systems are regulated in response to zinc by the zinc-sensing repressor Zur and that Zur binds to the corresponding gene operator-promoter regions in a zinc-dependent manner. Importantly, we found that the two systems show key differences in their expression profiles in response to changing zinc levels and this coincides with differences in the number of Zur binding sites within their gene promoter regions as well as in how tightly Zur binds to these regulatory sequences. It is likely that these differences in regulation therefore contribute to the varying requirements for each system for bacterial growth in different environmental niches. However, we were unable to detect differences in the rate of zinc uptake by each system when cells were grown on different zinc substrates and zinc uptake by neither system was inhibited by high levels of other metals (iron and manganese) that are thought to be present within a host cell cytosol. It is possible therefore that these factors are not associated with the requirement for the two separate high affinity zinc-uptake systems in Listeria as originally hypothesised. Notably, within this study we have also identified a system responsible for Listeria tolerance to high zinc and confirmed that this is regulated in response to zinc by a DNA-binding, zinc-responsive repressor. This is the first report of a listerial system that supports growth in high levels of zinc and highlights the requirement for tightly controlled intracellular zinc levels in this pathogen.
Exploitation Route The discovery that bacterial zinc handling systems contribute to their ability to cause infections is being exploited in the development of antimicrobials.

We have identified zinc-homeostatic proteins in Listeria that represent targets for antimicrobial drug-development.

Knowledge of bacterial metal handling systems may be applied to improving industrial biotechnology and bioprocessing applications.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description In public engagement activities. Findings from this award have been: Used as an example to support the A-level syllabus via an article in the 'Biological Sciences Review' magazine for sixth formers (2014) Presented in a lecture to year 12 school pupils - Discover Life Sciences (2016).
First Year Of Impact 2014
Sector Education
Impact Types Societal

 
Description Responsive mode
Amount £713,334 (GBP)
Funding ID BB/R010412/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 03/2021
 
Description Collaboration - Poole 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Research collaboration
Collaborator Contribution Research collaboration
Impact Development of laboratory techniques
Start Year 2014
 
Description Collaboration Leicester 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Research collaboration leading to publications and future funding
Collaborator Contribution Research collaboration leading to publications and future funding
Impact Publications in research journals (Corbett et al., 2011 & 2012) and future funding (BB/K016881/1)
Start Year 2010
 
Description 1st Midlands Molecular Microbiology Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentations to disseminate knowledge of our research
Year(s) Of Engagement Activity 2014
 
Description Article for Biological Sciences Review 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Article for Biological Sciences Review (2014) Salmonella: A bacterial pathogen that invades our gut. Aimed at 6th formers to support A level syllabus and has resulted in requests for visits/placements from 6th formers.

None so far
Year(s) Of Engagement Activity 2014
 
Description Biometals meeting 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at an International conference focussing on biometals to disseminate knowledge from this research (title: Two Zinc Uptake Systems Contribute to the Full Virulence of Listeria monocytogenes during Growth In Vitro and In Vivo)
Year(s) Of Engagement Activity 2014
 
Description Ernst Strüngmann Scientific Forum 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited rappateur and participator in a discussion group for the Ernst Strüngmann Scientific Forum on 'Metals and Infectious Disease' (Frankfurt, Germany) - a discussion forum for international scientists to discuss themes that transcend classic disciplinary boundaries. The goal of this forum was to understand the linkage between heavy metals and the pathogenesis of infectious diseases and to address the underlying mechanisms that moderate the outcome of infection. As an outcome of this forum, a book was published to disseminate information from these discussions.
Year(s) Of Engagement Activity 2014
 
Description International zinc meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker at International Society for Zinc Biology (ISZB) meeting, Cyprus. Involved discussion of all aspects of zinc biology with others in the field.
Year(s) Of Engagement Activity 2017
URL http://iszb.org/meetings/
 
Description Invited Seminar (University of Leicester) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited to give a seminar at the University of Leicester as part of their seminar series. Sparked questions and discussion afterwards and initiated new collaborations.
Year(s) Of Engagement Activity 2018
 
Description Lecture to year 12 school pupils 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Research findings were presented to ~20 year 12 school pupils who visited the research organisation
Year(s) Of Engagement Activity 2014
 
Description NIBB (Metals in Bioprocessing Scoping Workshop) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Core member (Dr JS Cavet) of a BBSRC network in industrial biotechnology and bioenergy (NIBB) - Metals in Biology: The elements of Biotechnology and Bioenergy.
Participant and speaker at a scoping workshop regarding 'Metals in Bioprocessing' which was attended by academics and industrialists with the purpose of initiating new collaborations.
Year(s) Of Engagement Activity 2014
URL http://prospect.rsc.org/MiB_NIBB/
 
Description NIBB (metal-related antimicrobials event) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Dr JS Cavet is a core member of a BBSRC network in industrial biotechnology and bioenergy (NIBB) - Metals in Biology: The elements of Biotechnology and Bioenergy.
Co-organised, attended, chaired and speaker at a metals-related antimicrobials event (co-organised by NIBB and BBSRC) which included plenary lectures and a scoping workshop. The event was attended by academics, postgraduate students, industrialists and stakeholders and included presentations of on-going research involving Academia & Industry with the purpose of the event being to identify new opportunities for Innovation, initiate collaborations and understand potential implications of policy regulations.

As a follow on from the success of the event, JSC encouraged by the President of the Microbiology Society to propose a biometals focused meeting for the Society's Annual meeting with the intention of promoting networking between academics and industrialists.
Year(s) Of Engagement Activity 2015
URL http://prospect.rsc.org/MiB_NIBB/wp-content/uploads/2015/11/Final-programme.pdf
 
Description NorthWest Microbiology 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Poster and oral presentations at North West Microbiology meeting aimed at young researchers - presentations sparked questions and possible collaborations
Year(s) Of Engagement Activity 2014
 
Description Oral Presentation at The Microbiology Society Annual Conference 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker at The Microbiology Society Annual Conference 2018. Sparked questions and discussion afterwards and initiated new collaborations.
Year(s) Of Engagement Activity 2018
URL https://microbiologysociety.org/event/annual-conference/annual-conference-2018.html
 
Description Oral presentation at the Microbiology Society Annual Conference 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker at The Microbiology Society Annual Conference 2016 (Liverpool, UK)
Year(s) Of Engagement Activity 2016
URL http://www.microbiologysociety.org/events/annual-conferences/index.cfm/annual-conference-2016
 
Description Placement for school 6th former 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Placement encouraged 6th former to pursue further education in biological sciences

After this placement there have been enquiries from others at the same school regarding future placements
Year(s) Of Engagement Activity 2014