Identification and functional analysis of surface factors that enable human pathogens to adhere to and colonise plants.

Lead Research Organisation: James Hutton Institute
Department Name: Cell & Molecular Sciences

Abstract

Foodborne illness is one of the main burdens of infectious disease in the developed world. Microbes such as viruses, bacteria and parasites are all associated with food and many of them can be found in farm animals, crop plants and water. However, there is an important distinction between those microbes that can use plants or animals as hosts, i.e. are able to proliferate on or within the host, and those that are simply transported through the food chain by them. Just a few bacteria that are foodborne pathogens (including E. coli O157 and Salmonella) account for a large proportion of all foodborne illness, largely because of their extraordinary ability to adapt to a wide range of environments and to proliferate on hosts of any biological kingdom. These pathogens have a strong association with animal hosts, in particular farm animals. From this source, the bacteria can be transmitted into water, by flies or onto growing crops. Although we traditionally associate these bacteria with animal hosts, we have good evidence to show that they can also proliferate on and within plant hosts. This is important because the number of foodborne outbreaks from contaminated fresh produce, in the form of ready-to-eat or minimally processed fruit and vegetables has increased over the past two decades. The increase can only be partly accounted for by better surveillance and detection methods, and there is a possibility that it is also linked to climatic change. Consumption of fresh produce is rightly promoted as part of a healthy life-style, which increases the need to fully understand the basis of colonisation of crop plants by foodborne bacteria. Appreciation of this area is on the rise, although our knowledge of the biological basis to bacteria-plant interactions is in its infancy. Therefore, it is important to build up a solid foundation from which we can make informed decisions that affect food safety practices and government policy. This project aims to determine some of the basic information about how foodborne bacteria colonise crop plants. There are likely to be a large number of genes involved which will fall into different functional families. This project will focus on the genes that encode structures present on the bacterial cell surface, i.e. those most likely to interact directly with plant cells. Preliminary work has already indicated a role for an E. coli O157:H7 adherence factor in bacteria-plant interactions and it will be investigated in much greater detail to fully characterise its role. Structures on the bacteria cell surface seldom work in isolation and when they do, they are extremely tightly controlled to ensure that they are only produced at the most appropriate time. Therefore, additional surface factors of E. coli O157:H7 will be identified, which can then be tested to determine their role in the bacteria-plant interactions. A proportion of the bacterial population are able to enter the internal tissues of plants, where they cannot be removed by conventional sanitation techniques used in food production. Whether any of the bacterial surface factors play a role in bacterial internalisation of plant tissue will also be assessed. The other side of the bacteria-plant relationship will also be examined, to determine whether the plant can sense any of the bacterial surface factors specifically. The approaches used will provide a clearer picture as to the nature of the relationship between plants host and bacteria. The information will contribute to a wider wealth of knowledge, with a common goal to reduce the incidence of foodborne illness.

Technical Summary

The hypothesis to be tested is that bacterial surface-expressed factors mediate interactions between human pathogenic enterobacteria and plants. The project will focus on enterohaemorrhagic E. coli (EHEC) and will characterise functional surface factors the play a role in colonisation and internalisation of plants. Preliminary work has shown several surface factors are induced in planta and indicated a role for the F9 fimbrial cluster. Objectives: 1 - Functional analysis of EHEC F9 fimbriae in bacteria-plant interactions. 2 - Identification of additional surface-expressed factors from EHEC O-island deletion mutants and an EHEC BAC library 3 - Functional analysis of additional EHEC surface expressed factors in bacteria-plant interactions 4 - Determine whether any EHEC surface factors affect the plant host response Libraries of EHEC O-island mutants and BAC clones will be screened to identify surface factors that are functional in planta (available from Prof. Gally, Edinburgh). Functional analysis of F9 fimbriae and additional surface factors will determine their expression in planta, whether they confer specificity to any plant tissue type or species and whether they induce a host response. Surface factors from other EHEC with sequence variation will be tested to determine whether they confer any advantage in plant colonisation. Candidate genes will identified from initial in planta and bioinformatics screens and assessed by microbiology and microscopy. Light and electron microscopy will also be used in functional analysis. Plant infection assays will be routinely carried out on plants associated with fresh produce outbreaks and different time points will distinguish between initial adherence and establishment of colonisation. In addition, the internalised population will be investigated. Infection of edible tissue will be initiated on the roots of growing plants since the rhizosphere is colonised to a far greater extent than the phyllosphere.

Planned Impact

The aim of the work is to identify and characterise surface structures that enable human pathogenic enterobacteria to colonise fresh produce plants, with the long term aim of reducing illness from foodborne bacteria. The project is relevant to government and policy makers involved in food safety, infectious diseases and public health. The work fits within the remit of Food Security, a major priority publically funded by multiple agencies, including RERAD and the BBSRC. Theme four of the global food security programme aims to reduce the incidence of foodborne illness. It is relevant to the 'Healthier' strategic objective of the Scottish Government and fits within the BBSRC priority on Animal Health, which includes 'foodborne or other zoonotic diseases with implications for public health that are carried by farmed animals'. The work also fits into the priority area on living with environmental change (LWEC) since foodborne outbreaks from fresh produce is considered an emerging threat that has links to changing climatic conditions. National agencies interested in this area include the Food Standards Agency (national and Scottish) and the Health Protection Agency. Private sector beneficiaries in food safety that are funded by the food industry, include the Chilled Food Association and the Agriculture and Horticulture Development Board levy body (previously the Horticultural Development Agency). The main impact of the work is in scientific advancement of a relatively new area of research, which has important implications for food safety. As it stands the evidence to show colonisation and especially internalisation has been mainly visual, based on microscopic images. Questions remain as to the likelihood and relevance of internalisation of plant tissue by zoonoses. This project will unambiguously demonstrate firstly that internalisation is a bona fide phenotype of zoonotic pathogens, and secondly identify surface factors required for the phenotype. The project also aims to characterise any sequence differences in surface factors that may confer a plant colonisation advantage for different EHEC isolates. The knowledge will be of use to policy makers to better understand the biology that underpins colonisation of plant hosts by foodborne bacteria. As it stands, this area is still so novel that it does not fit into current keyword classifications or generic descriptions. Interactions are either considered for foodborne pathogens with animal hosts or microbial pathogens that cause plant disease. Whilst some effort has gone into understanding the basis to bacterial interactions on cut leaf surfaces, these describe a different aspect of food safety, most relevant to cross-contamination of produce post-harvest, during the production process. This is quite distinct from the two-way interactions that occur between bacterial pathogens and growing crop plants. By getting a fundamental understanding of bacteria-plant interactions under these conditions, we are in a better position to inform policy-makers and industry stakeholders of the underpinning biology and impress on them the impact of such interactions. An important aspect of the work is in the training a highly skilled scientist who will be able to cross boundaries between plant and animal microbiology. Traditionally, these two areas have remained distinct and it is still the case that the work falls outwith established fields of medical or veterinary microbiology and plant microbiology, but rather includes aspects of both areas. Policy-makers and industry stakeholders will be informed at appropriate meetings. I have already contacted key representative from each of the agencies and industry groups and have met with them all on different occasions. Please refer to the Impact Plan for a fuller description.

Publications

10 25 50
 
Description The aim of this work was to determine how a food-borne bacterial pathogen, Escherichia coli O157:H7, attach to plant hosts. Food-borne outbreaks can arise from plants, such as salad vegetables, but there is little known about the bacteria-plant interactions. Attachment is the first step of interaction between a bacterium and its host, and our longer term ambition is to determine whether there is any way we can prevent this step from occurring. In summary we identified several different E. coli O157:H7 surface factors that mediate attachment to plants. The work looked at factors with a known role in attachment (Objective 1), and identification of novel factors (Obj. 2), from where we characterised their roles in more detail (Obj. 3). Finally, we determined how the plant host perceive these adherence factors (Obj. 4). The project was carried out concurrently with a complementary project to identify any receptors in plant cells that were targeted by bacterial surface factors, which allowed us to build a detailed picture of the initial stages of bacterial-plant interactions progress. We identified at least four different cell- surface expressed factors in E. coli O157:H7 that mediated attachment to plants. These were the H7 flagella (flagella is normally only involved in chemotaxis and motility, but there is evidence to show a role in binding to animal cells); two different fimbrial types, E. coli common pilus (ECP) and F9; and a secretion apparatus, the Type 2 Secretion System (T2SS). The latter was identified from screening isolated regions of the E. coli O157:H7 genome to identify genes that provided a biological advantage in adherence. Together the complementary projects identified two different mechanisms of interaction with plant cells. On one hand, flagella interacted with lipids in plant cell membranes in a charged-based manner, whilst ECP and F9 targeted specific receptors in the plant cell wall. We have yet to identify how the T2SS interacts with plant cells, but to our knowledge, this is a novel role for a system that is largely uncharacterised in E. coli O157:H7. While ECP is almost ubiquitous in E. coli, F9 is restricted to only a few pathotypes, including serotype O157:H7, suggesting specificity arising from the bacteria. We found that ECP and F9 fimbriae are subject to temperature and catabolite control. Three of the adherence factors (H7, ECP and F9) induced a defensive response in plant hosts, tested in the model plant Arabidopsis thaliana. Intriguingly, although there were some commonalities in the pathways induced, there were specific differences between the surface factors. This work has led us to propose a model for the initial stages of interaction, where flagellate bacteria first encounter a plant cell and are able to bind in a generic manner. Since flagella are a significant elicitor of the plant defence, they are down-regulated and binding is then mediated by specific interactions between bacterial fimbriae and plant cell wall molecules. Differences in how fimbriae are perceived by the host may then facilitate subsequent establishment and colony formation.
Exploitation Route This work has elucidated the molecular mechanism for attachment of E. coli O157:H7 to plant cells. An important aspect is that E. coli encodes a large repertoire of potential adherence factors, many of which are completely uncharacterised. Therefore, this work is of interest to those working of E. coli attachment to biotic (and abiotic) surfaces. The role of flagella in attachment is of particular interest, given its target of charged membrane lipids, which are shared between biological kingdoms. There is very little information on how plant-associated bacteria attach to their plant hosts and much of the information here could be translated. For example, the T2SS in E. coli O157:H7 bears functional similarity to the T2SS in plant-associated Enterobacteriaceae, where it is expressed in planta to deliver cell wall degrading enzymes. A spin-out studentship has found that a T2SS-secreted protein from E. coli O157:H7 is secreted in plant tissue. Finally, information on the plant defence response to E. coli adherence factors will not only be informative on how the interaction progresses, but also for parallels with the animal host response to E. coli, and the plant response to other plant-associated bacteria. The work has mainly academic interest, but is also of interest to public health agencies and primary producers, showing an active interaction between E. coli and plant hosts.
Sectors Agriculture

Food and Drink

Environment

Healthcare

 
Description The information has contributed to knowledge of how the bacteria interact with crop plants, which in turn has been presented to & discussed with growers and producers. For example, N. Holden was involved in a series of grower workshops run by the Agri. and Hort. Dev. Board, 'Keeping it clean' (Jan 2016), designed to inform growers on the state of knowledge for microbial contamination of fresh produce. The work has been used to inform risk assessment in a model for microbial contamination of fresh produce currently under development in a separate project (joint with Aberdeen University), and have contributed to a EU networking project on human pathogens in fresh produce (COST CA16110). The findings have also been used in an undergraduate teaching programme (Dundee university, 2017, 2018, 2019, 2020). The findings have been extended to investigate secretion of an E. coli O157:H7 protein in plant tissue (Glasgow university, 2019-2020).
First Year Of Impact 2015
Sector Agriculture, Food and Drink
Impact Types Policy & public services

 
Description Microbiome Working Group - AgriFood group (Microbiology Society)
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
URL http://www.microbiologysociety.org/policy/microbiome-policy-project.cfm
 
Description PATH-SAFE Science Advisory Group (SAG)
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
URL https://www.food.gov.uk/our-work/pathogen-surveillance-in-agriculture-food-and-environment-programme
 
Description Scottish Alliance for Food: Advisory Steering Board Member
Geographic Reach National 
Policy Influence Type Participation in a guidance/advisory committee
URL https://www.gla.ac.uk/research/az/scaf/
 
Description Food Safety Research Network (FSRN) Seeding Award
Amount £28,000 (GBP)
Funding ID 43266FSRN-2023S12 
Organisation Quadram Institute Bioscience 
Sector Academic/University
Country United Kingdom
Start 09/2023 
End 02/2024
 
Description Joint PhD studentship scheme: James Hutton Institute & Univ. Reading
Amount £60,000 (GBP)
Organisation James Hutton Institute 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2019 
End 09/2022
 
Description Secretion dynamics of a STEC Type 2 secreted protein, StcE
Amount £0 (GBP)
Organisation University of Glasgow 
Sector Academic/University
Country United Kingdom
Start 08/2019 
End 06/2020
 
Description Strategic call
Amount £350,444 (GBP)
Funding ID FS101056 
Organisation Food Standards Agency (FSA) 
Sector Public
Country United Kingdom
Start 02/2014 
End 06/2016
 
Title Extraction of bacterial mRNA from inoculated plants 
Description As part of this project we optimised and developed a method to extract bacterial mRNA from inoculated plants. The RNA is of sufficient quality and quantity to carry out gene expression analysis using quantitative reverse-transcriptase PCR. This method is a significant advance for the community because it is challenging to obtain sufficient quality of mRNA for gene expression studies from living plants. 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Too early to say, but the related publication (Holmes et al, 2014, Front Microbiol) has been viewed >1250 times (as of 03/11/2014) with>350 pdf downloads. The article has also generated interest through direct contact to me. 
URL http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00286/abstract
 
Title A model to identify Escherichia coli O157:H7 genes enriched in adherence to spinach root tissue 
Description E. coli O157:H7 gene loci enriched from adherence to spinach tissue (see associated microarray dataset) were quantified and measured using a Bayesian hierarchical model. This resulted in 115 CDS credible candidates across seven contiguous genomic regions. 
Type Of Material Data analysis technique 
Year Produced 2018 
Provided To Others? Yes  
Impact The dataset and approach have wider applications for those investigating plant hosts interactions for any bacterial species; the use of positive-selection approaches for gene enrichment under different scenarios; and adoption of hierarchical modelling to identify candidate targets. 
URL https://widdowquinn.github.io/SI_Holmes_etal_2019/
 
Title Dataset of Escherichia coli O157:H7 genes enriched in adherence to spinach root tissue 
Description A high-throughput positive-selection approach was taken to generate a dataset of Shigatoxigenic Escherichia coli (STEC) O157:H7 genes enriched in adherence to plant tissue. A BAC clone library derived from STEC isolate 'Sakai' was inoculated with spinach roots to generate a treatment library, in parallel to a non-plant control library. After four rounds of successive rounds selection and amplification, a total of 7.17 x 10^8 cfu/ml of bacteria were recovered from the plant treatment and 1.13 x 10^9 cfu/ml of bacteria from the no-plant control. Genomic DNA from both libraries was hybridised to an E. coli DNA microarray chip. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact This dataset was used to identify enriched genes that potentially play a role in adherence of E. coli O157:H7 to plants. A model was developed to identify the genes. Candidates were pursued leading to identification of the type 2 secretion system and associated secreted proteins involved in E. coli-plant interactions. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5923/
 
Description PhD project - Risk assessment of microbial contamination of fresh produce 
Organisation University of Aberdeen
Department College of Physical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution The findings from the BBSRC grant have provided data that can directly be used in the risk assessment model. The PhD project involves generation of the model framework and additional primary data. This will all be used to generate the risk assessment itself.
Collaborator Contribution The partner (Norval Strachan) brings expertise in risk modelling and risk assessment. He has helped in generation of the framework and will oversee the risk assessment calculations.
Impact Merget, B., U. Dobrindt, K. J. Forbes, N. J. C. Strachan, F. Brennan and N. J. Holden (2020). "Variability in growth responses of non-O157 EHEC isolates in leafy vegetables, sprouted seeds and soil extracts occurs at the isolate level." FEMS Microbiol Lett 367(1). https://doi.org/10.1093/femsle/fnaa030 Merget, B., K. J. Forbes, F. Brennan, S. McAteer, T. Shepherd, N. J. C. Strachan and N. J. Holden (2019). "Influence of plant species, tissue type, and temperature on the capacity of Shiga-Toxigenic Escherichia coli to colonize, grow, and be internalized by plants." Appl Environ Microbiol 85(11). DOI: 10.1128/AEM.00123-19 Wright, K. M., L. Crozier, J. Marshall, B. Merget, A. Holmes and N. J. Holden (2017). "Differences in internalization and growth of Escherichia coli O157:H7 within the apoplast of edible plants, spinach and lettuce, compared with the model species Nicotiana benthamiana." Microbial Biotechnology 10(3): 555-569. https://doi.org/10.1111/1751-7915.12596
Start Year 2013
 
Description Agricultural Show exhibit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interactive exhibit on 'Microbes & Your Food' for the Royal Highland Show (22-25 June 2017), an interactive exhibit for the education centre (RHET), covering microbial interactions in food production, digestion and pathogen detection, and a SEFARI joint exhibit that engaged with primary producers (livestock & arable), public health workers (e.g. clinicians, nurses) and policy-makers (SG, food safety agency). Related directly to findings from this project and in the wider context of how microbes interact with foodstuffs (positively and negatively).
Year(s) Of Engagement Activity 2017
URL https://www.royalhighlandshow.org/
 
Description COST action HuPlantControl workshop, IS-MPMI satellite meeting (June 2019) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was a joint organiser for a workshop on the molecular mechanisms that allow human pathogenic bacteria to colonise plants. It was arranged as a satellite session for a large plant pathology conference and so sparked discussions about parallels & differences between plant and human pathogens, and enabled us to reach an audience that we don't normally access. The outcome was partnerships for cross-disciplinary work.
Year(s) Of Engagement Activity 2019
URL https://ismpmi.confex.com/ismpmi/2019/meetingapp.cgi/Session/1061
 
Description Microbiology conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Kath Wright presented a poster "E. coli O157:H7 - Watch out for the salad, as well as the undercooked beefburger!" to the Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis, University of Dundee, Scotland, 29-30th June 2016
Year(s) Of Engagement Activity 2016
URL http://www.lifesci.dundee.ac.uk/other/yms/
 
Description Scientific exhibit (Dundee Food & Flower festival) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Scientific exhibit "Putting microbes on the table" aimed at school children and the general public. Interactive activities to show how beneficial and pathogenic microbes interact with and adhere to food to either help or hinder in the food making processes.

Nothing formal, but good feedback from teachers, school children and the general public about activities
Year(s) Of Engagement Activity 2014
URL http://www.dundeeflowerandfoodfestival.com/
 
Description Talk on the endophytic lifestyle of E. coli O157 presented at Microscience Microscopy Congress 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact A number of questions were asked leading to exchange of information from others involved in imaging.

Information was gained to aid future work.
Year(s) Of Engagement Activity 2014
 
Description VTEC 2015 Conference - poster presentation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Poster presentation stimulated discussion during the presentation session. This was both with those that are familiar with the area, but probably more importantly also with those completely unfamiliar who did not appreciate that VTEC could interact with plants and use them as hosts.

I managed to make some new contacts and to increase my professional network. This has started discussions on where common ground lies for new grant applications.
Year(s) Of Engagement Activity 2015
URL http://www.vtec2015.org/