Performing Department
Biological & Environmental Sciences
Non Technical Summary
The proposed research project is designed to address the NIFA priority area of Food Safety and the Reduction of food-borne illness.The center for disease control estimates that nearly 1 in 6 (46 million) Americans per year report food-borne illness. Of that amount, 250,000 require hospitalization and 3,000 deaths occur each year (Scallan 2011). Equally alarming is the economic burden of food-borne illnesses as it is estimated that food-borne illnesses cost the economy more than $15.6 Billion annually (USDA). Taken together, these data support the need for research programs in Food Safety and the reduction of food borne illnesses as a major priority in the Unites States It is reported that 39% of food-borne illness is caused by bacteria, making bacterial infections a major public health threat. Pathogens such as Escherichia coli (E.coli) that contaminate our foods and cause food-borne illness utilize various pathways that facilitate their ability to adapt to stress and survive in dynamic environments. Food safety efforts are dependent on an understanding of these pathways well enough to prevent them (Behravesh, Casey B).E.coli O157:H7 has been identified as one of the top five pathogens that cause food-borne illnesses that result in hospitalization (Scallan, E. 2011). This emerging food-borne pathogen is the most common cause of outbreaks of E.coli infection, making it a public health priority. (Gould, L. Hannah). E.coli O157:H7 releases one of the most potent toxins known to man, the Shiga-like toxin. These toxins are very dangerous as they have a really small infectious dose (ref). The resulting infection can lead to severe, life-threatening complications and even death (ref).E.coli O157 is constantly evolving, mutating and acquiring new characteristics that creates new challenges in food safety and public health. This ability to exploit different mechanisms of survival necessitates additional research to further understand its pathogenicity. Current data provides an alarming depiction of how antibiotic resistance in E.coli has increased over time(solomakos, N 2009). (Tadesse DA, 2012). Studies show that small regulatory RNA molecules influence bacterial antibiotic resistance (Lalaouna, David 2014).E.coli is one of the most extensively studied pathogens and although great progress has been made to reduce the burden of food-borne illness overall, little progress has been made to reduce the incidence of E.coli O157 infections (ref). If we are to make food safer, we must focus on reducing levels of microbial contamination.In order to broaden knowledge and ultimately reduce the burden of food-borne illness, a new look at solutions for preventing contamination of food is necessary.The envelope stress response is an important pathway that is critical in bacterial adaptation ability. An improved understanding of how this pathogen behaves under various environmental conditions or in response to stressors will improve our understanding of bacterial cell biology and opportunities for development or improvement of effective prevention and control strategies in order to have a safer food supply (King, T 2016) (Newell DG 2010). (Aboutaleb, N 2014).The findings of this research project have the potential to elucidate pathways that E.coli O157 depend on for a survival advantages and thus aid in their ability to use food as a vehicle of transmission. The findings of this research project can also reveal possible opportunities for further dialogue and interdisciplinary collaboration to address this very important public health concern. (Meng J, 1997).Basic molecular biology techniques will be uitlized to conduct a laboratory investigation of mechanisms that influence that ability of E.colirespond and adapt to changing environmental conditions. Such ability may be a contributing factor to its fitness to survive and contaminate the food supply.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The USDA reports that bacteria are responsible for 39% of reported food-borne illness. E.coli O157:H7 is one of the top five causes of bacterial-related food borne illnesses. The resulting infection can range from mild to severe and huge public health concern.The presence of receptors and surface proteins play a major role in pathogenic E.coli O157 ability to cause infection. The bacterial envelope stress response is trigged when there is perturbation of outer membrane protein. Thepossible post-transcriptional regulation of the RseAP3 transcript by small regulatory RNAs has not been fully investigated.This gap in the current knowledge of RseA regulation provides the basis for this investigation. The primary goal of this project is to investigate the possible post-transcriptional regulation of RseA by small regulatory RNAs (sRNAs). The rationale driving the project goal is the discovery of a relatively long (228 nucleotides) 5' un-translated region of a novel RseA transcript and the fact that the envelope stress is so tightly regulated.Objective 1: Determine if the small RNA RyhB directly regulates RseA translation. This will be done by identifying nucleotide point mutaitons in the RyhB small RNA that affect its regulatory activity on PBAD-rseA27-lacZ.Next, an identification of compensatory nucleotide point mutations in the RseA 5'UTR portiion ofPBAD-rseA27-lacZtranslational fusion that restore the regulatory ability of RyhB point mutants.Objective 2: Determine if RyhB regulation of RseA affects σE Dependendent promoter activity in vivo.Objective 3: Determine if there is a link between iron metabolism and envelope stress.
Project Methods
This investigation will primariliy be conducted in a research laboratory setting and utilize a variety of molecular biology techinques to carry out the experimental investigation of the regulatory effect of small RNA molecules onRseA, a major regulatory point in the bacterial envelope stress response.Generally, the efforts will involve bacterial strain cultivation and construction. The construction of bacterial strains will require transformation and recombineering, a technology to create gene fusions and marked mutations.Other methods to be utilized includeDNA manipulation (site-specific mutagenesis) and plasmid isolation, Nucleic acid quantification.A library screen was conducted to identify small regulatory RNAs that can stimulate a lacZ translational fusion. Two small RNAs returned postivie results, the small RNA RyhB and FnrS.The construction of bacterial strains into a rseA-lacZtranslational fusionwill give the ability to examine the effect of small regulatory RNAs on RseA translation. The translational fusion will be transformed with plasmids containing with the wild-type and mutated RhyB small RNA. Four strains containing mutated RyhB will be created for this investigation.Qualitative and quantitative analyses will be conducted to determine the effect of the small RNA RyhB on RseA translation.The beta-galactosidase assay will be used for a quantitativeanalysis. Beta-galactosidase expression will be measured using a spectrophotometer.It is expected that there would be a significant increase in translational fusion activity in the presence of the wild-type small RNA RyhB and decrease in the presence of the mutant small RNA RyhB.A complementary mutatant bacterial strian wil also be constructed. This strain will contain point mutations in the regions of RseA that is expected to base-pair with the RyhB small RNA. This compensatory mutant strain will be assayed after being transformed with plasmids carrying the mutant RyhB small RNA.The findings of this part of the investigation would confirm or reject that the small RNA RyhB regulates RseA translation through direct interaction via complementary base-pairing. We hypothesize that the complementary mutations in the 5' UTR of the RseA translational fusion would restore the ability of the respective episomal RyhB point mutants to stimulate the activity of the mutant rseA-lacZ translational fusion.Next, we will utilize the beta-galactosidase assay to determine how the expression of RyhB wil affectσE-dependent promoter activity. A lacZ transcriptional fusion to the small RNA RybB will be used as the assay tool. A 2-plasmid system will be created.Finally, the small RNA RyhB is expresed during low iron levels in the cell. We hypothesize that low iron conditions results in decreased envelope stress response through RyhB. We will investigate the possible relationship between iron metabolism and envelope stress using the rybB-lacZ transcriptional fusion. The goal is to determine if iron depletion affectsσE-dependent promoters in vivo. An iron-depleted environment will be created using iron chelators.The beta-galactosidase assay will be used to quantify the effects of iron depletion onσE?-dependent promoters.