The objective of our research program is to investigate the role of the gastrointestinal (GI) microflora, particularly LAB, in human and animal health, nutrition, and food safety. The proposed research objectives are: <OL> <LI> To identify mechanisms of adherence and colonization of probiotic bacteria in the GI tract using in vitro and avian animal models.<LI> Identify and understand host-microbe interactions at mucosal surfaces in the GI-tract, particularly those of probiotic (beneficial) bacteria. <LI> Identify applications and mechanisms of LAB in food safety. <LI> Develop recombinant lactic acid bacteria for the expression gene products important in bioprocessing/fermentations, improved human/animal nutrition and vaccine delivery.
NON-TECHNICAL SUMMARY: Lactic Acid Bacteria (LAB) are gram-positive, acid tolerant, non-sporulating bacteria that include the species Lactobacillus, Lactococcus, Pediococcus, Leuconostoc, and Streptococcus. LAB are naturally found in a wide range of environments including dairy products, meats, and plant materials and are associated with animal and human mucosal surfaces. LAB are important in the production and preservation of foods serving as starter cultures for fermented dairy products, meat, and vegetables and are important for the production of coffee, chocolate and wine. In addition to their role in food production, LAB are important in the production of renewable, low-carbon foot print energy and materials. l-lactic acid is an important building block for the synthesis of biodegradable polymers and is the substrate for the production of other compounds such as acrylic acid, acetic acid, propylene glycol, and ethanol LAB have long been thought to be beneficial to animal and human health. While LAB, particularly probiotic lactobacilli, have been shown to confer a number of beneficial health effects, the physiological and genetic basis of these is largely unknown. Recently, the genomes of several species of LAB have been sequenced including L. acidophilus (1), L. gasseri, L. johnsonii and others. The availability of these genome sequences will provide much insight as to the physiology, metabolism and biosynthetic capabilities of these organisms and will contributed to an improved understanding of the molecular mechanisms underlying the probiotic functionality of LAB. Because of their record of safety, importance in human health, genetic tractability and wide range of potential applications, LAB sit at the cutting edge of microbial biotechnology. Utilizing functional genomic and molecular genetic techniques we will investigate the physiology and genetics of these organisms which will be important in their development of microbially based biotech solutions for bioprocessing, human heath, and animal agriculture with particular focus on the use of lactic acid bacteria.
APPROACH: Objective 1. Identification of mechanisms of adherence and colonization of probiotic bacteria in the GI tract. In order to understand specific microbial factors involved in adherence and colonization (1) we will adapt the LMH cell culture model as an assay for the determination of microbial adherence to chicken epithelial cells, (2) determine the adherence of a number of difference probiotic LAB, (3) use functional genomic and molecular genetic techniques to identify specific microbial gene/gene products involved in adherence, and (4) apply and correlate knowledge gained from in vitro studies back into the live animal system. Objective 2. Indentify and understand host-microbe interactions at mucosal surfaces in the GI-tract. In these studies, we will create and employ a transgenic commensal Lactobacillus strain expressing model antigens. We will use these recombinant bacteria to colonize conventional and germ-free mice to minimize competition from other intestinal bacteria and track antigen-specific adaptive immune responses to this bacterium using common immunologic tools. Objective 3. Identify applications and mechanisms of LAB in food safety. One potential major area of application is the use of probiotic LAB, and similar strategies, as countermeasures to reduce the threat of foodborne infection. One strategy with strong potential is the reduction of the carriage of foodborne pathogens, including Campylobacter and Salmonella, in chickens using probiotics. Particular focus would on be four potential mechanisms: competitive exclusion through binding and occupation of attachment sights preventing colonization, in situ production of antagonistic molecules including bacteriocins and organic acids, modulation of host immunity using probiotics, and vaccine delivery via probiotic LAB. Objective 4. Develop recombinant lactic acid bacteria for the expression gene products important in bioprocessing/fermentations, improved human/animal nutrition and vaccine delivery Recombinant expression of proteins will be important in the future applications of LAB in bioprocessing/fermentation and human and animal health. We have previously developed a number of vectors for the recombinant expression of gene products by LAB. We will identify potential areas of application of LAB and use these molecular genetics tools to apply recombinant expression of proteins to a number of different areas including biotherapeutic and vaccine delivery, immune modulation, and improved digestion and nutrition.