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:
In order to gain knowledge of genetic and molecular activities of Salmonella sp. a transcriptome analysis approach was taken. High quality RNA isolation was found to be critical for sequencing entire messenger RNA populations at any given time point. Methods were designed to generate reliable sequencing libraries avoiding artifacts which could be generated by high-throughput RNA analysis. Substantial progress has been achieved in obtaining 10-20-fold coverage of Salmonella genome in RNA-Seq reactions. Pathogen strains exhibiting enhanced virulence and infectivity are frequently isolated in food-borne diarrheal outbreaks. Citrobacter rodentium, a mouse pathogen that mimics many aspects of enterohemorrhagic E. coli O157:H7 infection of humans, can serve as a useful model for studying virulence mechanisms. C. rodentium developed a hyperinfectious state once it was passed through the mouse gastrointestinal tract. Hyperinfectious C. rodentium cells were extremely acid sensitive compared to cells grown in laboratory media. Growth under anaerobic environment or on fecal components did not induce this hyperinfectious state. Developing tools to identify traits of hyper-virulent outbreak strains will provide a better measure of causality and food safety risk. Cell surface appendages facilitate bacterial attachment to biotic and abiotic surfaces. Procedures for proteomic analyses of bacterial cell surface appendages are being developed, including trypsin digestion(shaving) and selective labeling and analysis using LC-MC. E. coli and Salmonella mutants for cell surface proteins have been generated. These mutants will be tested for interactions with fresh produce and abiologic surfaces.
1. Determination of detergent tolerance of food-borne pathogens. Osmoregulated periplasmic glucans (OPGs) are synthesized by members of the family Enterobacteriaceae when grown under low osmotic conditions. Enteropathogens such as Shigella flexneri spend considerable time outside the host intestinal environment, such as in irrigation waters where low nutrient low osmolarity conditions normally exist. We demonstrated that OPGs of S. flexneri are required for optimal growth under low osmolarity low nutrient conditions. OPGs of S. flexneri are anionic. Based on homology of the OPG biosynthesis genes to those of Escherichia coli, the most likely function of opgC and opgB genes is to add succinate and phosphoglycerol residues respectively onto OPGs. We constructed opgB, opgC and opgBC mutants of S. flexneri. The mutant strain defective in opgC and opgB genes synthesized neutral OPGs which, although were beneficial for the organism’s growth in hypoosmotic media, were ineffective in combating stress caused by anionic detergents. Cloned wild type genes opgB, opgC, and opgBC upon mobilization to respective opg mutants, simultaneously restored anionic charges to OPGs and tolerance to detergents to wild type levels. It appears that anionic charges on the OPGs contribute towards overcoming the stress by anionic detergents such as sodium dodecyl sulfate (SDS) and sodium deoxycholate.
2. Native microflora influences biofilm formation by food-borne pathogens. Biofilm formation is an important mechanism for bacterial survival in stressful environments. Many foodborne bacterial pathogens are poor biofilm formers. However they are capable of interacting with other bacteria with strong biofilm forming potential. Ralstonia insidisa, a bacterial species that was frequently isolated from fresh produce processing plants was shown to be a strong biofilm producer and strongly enhanced the incorporation of E. coli O157:H7 cells in the dual-species biofilms. This suggests that native microflora-based biofilms may play an important role in the survival of bacterial pathogens in produce processing environments.
Bhagwat, A.A., Yi Ning, L., Liu, L., Mahesh, D., Porteen, K. 2012. Role of anionic charges of periplasmic glucans of Shigella flexneri in overcoming detergent stress. Foodborne Pathogens and Disease. 9(7):632-637.