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

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.

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.

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.