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Applications of FLHD/FLHC Medicated Gene Regulation in Escherichia Coli


The goal is to provide a long-term perspective for the reduction of infectious diseases that are caused by Escherichia coli O157:H7. Specifically, we will develop a spray that can be used as a post-harvest treatment on beef carcasses to protect consumers from infection and contribute to the development of materials that do not promote biofilm formation.<P>
During the project period, we will pursue three objectives: <OL> <LI> We will investigate gene regulation by E. coli grown on the surface of beef, confirm hypothesized phenotypes and attempt to affect these phenotypes by the addition of metabolic intermediates that the bacteria will consider as 'food'. This will make the new spray difficult to become resistant towards. <LI> We will determine environmental and genetic factors that underlie biofilm formation. Biofilms contribute to 80% of all infectious diseases and have been researched extensively. Still, a compilation of all environmental and genetic factors that contribute to their formation is novel. <LI> We will determine temporal and spatial expression of specific genes within biofilms. </ol> This addresses the concern that biofilms really contain many micro-environments, yet have been researched as an average over the multicellular colony in the past. Objectives 2 and 3 will yield a vast amount of information that will have the development of materials as a translational aspect, which will be done by the Center for Nanoscale Science and Engineering. Algorithms to analyze the high-throughput data sets will be developed in collaboration with the Department of Computer Sciences.

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NON-TECHNICAL SUMMARY: Enterohemorrhagic E. coli (EHEC) is a highly pathogenic virotype of E. coli that is found in the intestines of warm-blooded animals, such as cattle. E. coli O157:H7 is of particular interest, as it was first recognized as a human pathogen during two outbreaks in 1982. While E. coli O157:H7 is not a pathogen for adult cattle, cattle do serve as a reservoir during the transmission of the bacteria to humans. In humans, the bacteria may cause severe cases of diarrhea, hemolytic uremic syndrome, and a variety of other diseases. To ensure the safety of cattle products, a variety of pre- and post- harvest treatments are available. Among the post-harvest treatments, spraying the carcasses with organic acids is commonly used, but poses problems due to the unusually high acid resistance of E. coli O157:H7. Non-acid sprays or combinations of different chemicals address this concern. Despite all efforts, E. coli O157:H7 remains the predominant cause of E. coli associated food-borne disease in the United States. The long-term goal of Objective 1 is to develop antibacterial sprays that can be used as post-harvest treatment with the goal to protect consumers from infection. The sprays will be novel because they constitute metabolic intermediates that feed into the central metabolic pathways of the bacteria. Bacteria will consider these food and, consequently, will find it more difficult to develop resistance against. We will use Objectives 2 and 3 to expand previous biofilm research. According to the NIH, microbial biofilms account for more than 80% of human infections. The three-dimensional aggregates of microbes are highly resistant to antibiotic treatment and host immune defenses. The colonies give rise to chronic, difficult-to-treat infections that can cause disease in nearly any host system and are particularly problematic on medical devices, such as prosthetic joints and catheters. The goal of Objective 2 is to identify specific nutrients that inhibit biofilm formation. Objective 3 will determine targets for the effective prevention and treatment of biofilm-associated infection by identifying proteins that will be expressed early during biofilm development (prevention) or at the surface of the biofilm (treatment).


APPROACH: Objective 1 will involve microarray analysis and quantitative PCR to determine the expression levels of genes that are differentially expressed between a mutant in the flagellar master regulator FlhC and its isogenic parent strain. Preliminary data indicate that these genes are involved in cell division, biofilm formation, and virulence. We will then perform phenotype experiments to determine whether cell division, biofilm formation, and virulence are indeed affected by FlhC. This will be done with plate count technology, 96 well biofilm assays and crystal violet quantifaction, and the chicken embryo lethality assay. All these assays have been established in the lab and published in multiple peer-reviewed articles. Objective 2 constitutes a high-throughput experiment where the effect of a large number of chemicals will be tested on a selection of E. coli mutants. In collaboration with Dr. Denton from the Computer Science Department, algorithms will be developed to analyze the data. This is a continuation of a long-term collaboration that has resulted in four peer-reviewed Journal articles in the past. The goal is to identify specific chemical properties (e.g. an amino group in a certain position) that inhibits or promotes biofilm formation. Information of this kind may aid CNSE in their development of novel biofilm preventing materials. Objective 3 will involve the construction of promoter::fluorescence protein fusions. Fluorescence microscopy will be used to determine the temporal pattern of expression from the fused promoters. Confocal microscopy will be used to determine spatial expression of the same genes. Genes that are expressed early in biofilm development will be proposed as biofilm prevention targets, while genes that are expressed late, but at the outermost edge of the colony will be proposed as treatment targets.

Pruess, Birgit
North Dakota State University
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