The overall goal of the studies proposed here is to characterize the molecular mechanisms involved in the interactions of Salmonella with peanut plants and E. coli O157:H7 with lettuce and spinach plants. <P>This goal is built upon the hypothesis that the expression of specific genes in Salmonella and E. coli O157:H7 cells are essential for the colonization, and perhaps internalization of crop plants. Moreover, we hypothesize that whole genome transcriptome analyses will lead to the identification of these essential genes, and that some of these genes may prove to be useful in the development of strategies that reduce disease incidence.<P> The specific objectives of this proposal are to: 1) Identify changes in gene expression profiles among bacterial cells associated with roots, leaves and nuts of fresh produce. The following bacterial/plant associations will be examined: E. coli O157:H7 with lettuce and spinach, and Salmonella with peanuts. 2) Determine the physiological and functional role of bacterial genes in survival, attachment, biofilm formation, growth and internalization in these plant species. 3) Develop mitigation strategies to reduce food-borne contamination based on the identified genes and proteins required for plant colonization and proliferation.
Non-Technical Summary: Recent major gastroenteritis outbreaks due to contamination of food crops with Salmonella and enterohemorrhagic Escherichia coli have exposed our lack of understanding of the relationship of these pathogens with fresh leafy greens. Previous studies have suggested that these bacteria may possess unique characteristics that allow them to interact with plants, but very little is known about the molecular mechanisms involved in regulating these responses. The elucidation of the genetic principles that govern the ability of these food-borne pathogens is critical for the development of effective prevention strategies. Our laboratory has developed a model system, using hydroponically-grown lettuce plants inoculated with non-pathogenic E. coli, to study the interactions of pathogens with food plants. In this proposal, we plan to use a similar experimental system in combination with advanced genetic technologies to study the interactions of E. coli O157:H7 with lettuce and spinach, and Salmonella with peanuts. The identification of the bacterial genes involved in attaching or in growing on plant leaves and roots will be accomplished. Genes with important role in these interactions will be identified and grouped according to function. Those genes will be selected for further study using microbiological, microscopic and molecular techniques. Depending on identified genes and their functions, appropriate control strategies will be designed to inhibit colonization and prevent contamination. This project will advance the understanding of the fundamental phenomena that allow these pathogenic bacteria to interact and survive on produce and eventually cause disease. <P> Approach: In these studies we will obtain a comprehensive understanding of bacterial genetic mechanisms involved in the growth and survival of pathogens on plant roots, leaves, and nuts using genome-wide transcriptional analyses. This objective will concentrate on the interaction of E. coli O157:H7 with in spinach and lettuce and Salmonella with peanuts. The interactions or mechanisms to be studied will include attachment, biofilm development, survival and growth in the plant environment. Due to the dynamic nature of microbial population residing on plant tissue, our studies will involve time course experiments. In the studies proposed here, triplicate E. coli O157 microarrays (OciChip, Ocimum Biosolutions Inc., Indianapolis, IN) will be used for each tested condition and time point. These microarrays consist of 6,176 50-mer oligonucleotides specific for E. coli strain K12 (MG1655; a non-pathogenic commensal control strain), E. coli strain O157:H7 (EDL 933, a prototype pathogenic strain) and E. coli strain O157:H7 (RIMD0509952/VT2-Sakai); a strain isolated from a large outbreak associated with radish sprouts. The arrays also contain 93 control DNA spots. In the case of Salmonella we will use Salmonella enterica serovar Typhimurium as our model organism. Based on our preliminary results obtained with lettuce, we expect to identify a large number of bacterial genes that are up and down regulated in response to the plant environment. As was previously done in preliminary studies, these genes will be screened for phenotypic characteristics and further investigated in objective 2. Extensive databases of genes relevant for plant colonization and the confirmation of functional role will be generated. The microarray data generated in objective 1 will yield a large number of genes that are up and down-regulated as a response to the corresponding plant environment. A number of techniques will be employed to determine the physiological and functional roles of these genes and their importance in contributing to the fitness of these bacteria in the phyllosphere and rhizosphere of lettuce, spinach, and peanut. The first ideal outcome from this part of the study will be a set of colonization results revealing of mutants lacking the genes needed to successfully attach, colonize or create biofilms on plant tissues. All experimental measurements will be done in duplicate and each experiment will be repeated three times. The information gained from Objectives 1 and 2 will help us to develop strategies to control microbial contamination on food crops. Preliminary data already points to some areas that may be exploited in this objective. The expression of attachment fibrils that appear to be important for initial adherence of bacteria to leaf tissue may be affected by plant genotype and growing conditions. Objective 3 will be the most dynamic of the three objectives as it will be driven largely by the result obtained in the other two objectives. We hope to research and establish methods for at least two good intervention strategies or a combination of strategies to enhance the safety of vegetable crops.