1a. Objectives (from AD-416)
Objective 1: Characterize pathogenic E. coli and Salmonella cell surface structures (fimbriae, pili, flagella) and elucidate their functions in interacting with abiotic environmental matrices and plant surfaces. Sub-objective 1.1. Develop methods for profiling and characterizing bacterial cell surface structures. Sub-objective 1.2. Determine the effects of environmental factors on the expression of various surface components of E. coli and Salmonella. Sub-objective 1.3. Determine the role of pathogenic E. coli and Salmonella surface structures in attachment to plant surfaces and to abiotic surfaces, and in biofilm formation and persistence. Objective 2: Elucidate survival strategies of E. coli and Salmonella strains under produce production, processing, and storage conditions. Sub-objective 2.1. Determine if produce sanitation and fresh-cut preparation environments promote rpoS related adaptive mutations in enteric foodborne pathogens. Sub-objective 2.2. Determine the role of periplasmic components of pathogenic E. coli and Salmonella in cell survival in low nutrient and low osmolarity environments.
1b. Approach (from AD-416)
Objective 1: A proteomic approach will be applied for developing the surface profiling technologies. Various cell surface proteins will be harvested using sheering or enzymatic shaving techniques or membrane- impermeable biotin mediated affinity purification. Proteins and pipetides will be identified using MALDI-TOF mass spectrometry and various liquid chromatography (LC) coupled MS detection technology. Besides the proteomic approach, antibody and micelle glycoprotein libraries will be tested in collaboration with CRADA partners. Similar approaches wil be used to determine the effects of environmental factors on the expression of surface proteins. Selected genes for targeted cell surface proteins will be mutated using site directed allelic change procedures and the effect of mutation on cell interacting with plant and environments will be studied using genetic and proteomic tools. Objective 2: Short-term and long-term nutrient starvation studies using Salmonella and E. coli O157:H7 under varying physiological conditions will be applied to determine the role of rpoS mediated adaptive mutations. In vitro growth conditions such as nutrient limited chemostat cultures, or vegetable wash waters in batch cultures will be utilized. Induction of acid tolerance by EHEC during different packaging conditions on various acidic and non-acidic produce during storage will be characterized. In collaboration with Dr. Sadowsky (U. Minisoda), natural Salmonella and E. coli O157:H7 isolates undergone minimal subculturing (>3) in the laboratory media will be used to determine rpoS heterogeneity. Genes encoding for osmoregulated cytoplasmic glucans (OPGs) will be cloned and characterized using site directed mutagenesis. Functions of OPGs in cell surface and cytoplasmic protein expression, cell motility, biofilm formation and survival in adverse environments will be studied using genetics and proteomic approaches.
3. Progress Report
Considerable progress has been made towards meeting the sub-objective 1.1 i.e. to develop methods for profiling and characterizing bacterial cell surface structures. Initial trials have indicated that we can harvest cell surface protein without having any adverse effects on cell viability. This is an important step in order to prevent contamination of cytoplasmic proteins into surface protein fraction. Further tests are being conducted to identify surface proteins. The research team has also made progress toward meeting goals of sub-objective 1.3 in constructing mutants with altered cell surface properties.