Characterisation of Escherichia coli O157:H7 Metabolism During Colonization of Plants

Overview:
Fresh produce is a recognised source of foodborne illness from bacterial, parasitic and viral infections. Enteric bacteria, including enterohamorrhagic E. coli (EHEC) can use plants as secondary hosts, colonising the plant surface and penetrating internal tissues. Large scale outbreaks, as seen in Germany and northern Europe in 2012, with > 4000 cases and 50 fatalities, have raised particular concerns about colonisation of sprouted seeds. Sprouting seeds and young plants release nutrients in the form of sugars, amino acids and polysaccharides that combined with relatively high temperature and humidity, provide permissive conditions for bacterial colonisation and growth. As such, inadequately washed sprouts and young plants pose a significant risk to the consumer. Identification of the nutrients utilised by EHEC and their availability in a variety of plant-species, will inform on which species present a higher risk of EHEC colonisation and aid in defining a control strategy.
Background leading up to the project:
We have shown that EHEC (isolate Sakai) are able to proliferate on fresh produce plants and increase on micro-leaf species by several orders of magnitude and interaction with plant tissue induces metabolic pathways(1). The bacteria then can penetrate the internal tissue(2), where, in certain species, they form large biofilm-like colonies(3). This leads to the potential use of carbohydrates by the bacteria, made available during apoplastic loading. Sucrose is one of the dominant sugars in plant tissue and although EHEC has genes for sucrose metabolism its transport into bacterial cells may be limiting(4), yet, our data shows 10-fold induction of the fructokinase gene (cscK, ECs3242) in root exudates, under conditions of translational stalling. Metabolic biosensors for plant-associated bacteria have previously been used to calculate the concentration of sucrose on a bean leaf (~20 µM)(5) and similar sensors can be used for EHEC due to the genetic relatedness of the bacteria. Our global transcriptomic analysis has identified induction of various metabolic pathways of EHEC (Sakai) on exposure to plant extracts including arabinose, likely to be relevant since it is a component of seedling exudates(6) and of plant cell wall polymers. Therefore, the over-arching aim of the project is to better understand the metabolic pathways that EHEC (Sakai) uses on colonisation of edible plants classed as ‘microleaf’, using a combination of cell biology, biochemical and microbiological approaches.
Objectives:
1. Identify the sources of nutrition for EHEC bacteria colonising the surface and internal spaces of the phyllosphere and rhizosphere of model and crop plants.
2. Determine the concentrations of carbon sources in the plant apoplast
3. Assess the role of the ‘native’ microbiome in EHEC colonisation and nutrient sensing
Methodology:
Metabolic biosensors: Genetic promoter regions from E. coli O157:H7 (Sakai) for carbon utilisation, including sucrose, glucose, fructose and arabinose, will be fused to a transcriptional fluorescent reporter (GFP) on a plasmid, and transformed in E. coli O157:H7 (Sakai), carrying a second, constitutive fluorescent marker (mKate) to aid identification (Year 1). Quantitative PCR(7) will be used to validate expression (and as a contingency if necessary). The possibility of showing sequential utilisation of different sources following depletion of the primary source will be investigated using combinations of reporters (e.g. different fluorophores). The student will be encouraged to identify potential metabolic pathways from our microarray datasets(1).
Cell biology: Transformed EHEC (Sakai) will be inoculated into the apoplast of the model plant species Nicotiana benthamiana and examined by confocal microscopy to (i) determine if the biosensors are effective and (ii) assess the availability and concentration of each carbon source (Year 1). Plants classed as micro-herb & micro-leaf will also be inoculated to identify the available carbon sources on the leaf surface, within the rhizosphere and/or phylloshpere and where internalised, within the apolastic space (Year 2+).
Biochemical analysis: Apoplastic fluid and other plant extracts from N. benthamiana and micro herb species will be analysed by HPLC to identify to quantify available primary carbohydrates (Year 1-2).
Microbiology: Bacteria will be enumerated from infected tissue to quantify the rate of colonisation and identify differences between the plant species (Year 1+). Micro herb species will be co-infected with EHEC and other environmental plant epiphytic bacteria (e.g. Pectobacterium atrosepticum, Pseudomonas fluorescens), and on axenic plants to identify potential competition for available nutrients (Year 3). The student will be encouraged to explore the most suitable species & isolates with ecological relevance.