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Listeria monocytogenes is a bacterial pathogen that is responsible for the disease listeriosis, that has a high fatality rate. Contamination of food, particularly ready-to-eat meats, with L. monocytogenes leads to expensive product recalls. A key aspect of the role of L. monocytogenes as a foodborne pathogen is its unusual ability to grow at refrigeration temperatures. There is a need for new and improved methods for the control of the organism. The cytoplasmic membrane of the organism is a lipid-protein bilayer structure that provides a variety of essential function for the organism. In order for the membrane to function properly it must have an appropriate fluidity. The fatty acids of membrane lipids are the major determinants of membrane fluidity and fatty acid anteiso C15:0 imparts an essential fluidity to the membrane of Listeria, allowing the bacterium to grow at refrigeration temperatures. The first objective is to investigate the underlying mechanisms that result in the characteristically high content of anteiso C15:0 in L. monocytogenes. In specific aim 2 attempts will be made to modulate the fatty acid composition of the organism to produce one that will not support growth at low temperatures. In the final objective gene expression patterns of L. monocytogenes growing in an actual food matrix will be determined. It is expected that achievement of these objectives will lead to improvements in the control of the growth of L. monocytogenes in food.
NON-TECHNICAL SUMMARY: The foodborne bacterial pathogen Listeria monocytogenes is the cause of listeriosis, a serious disease with a fatality rate of about 25%. The organism has a unique ability to grow in food at refrigeration temperatures, and this leads to expensive product recalls. This research addresses the mechanisms operating that enable Listeria to grow at low temperatures with particular reference to ready-to-eat meats. We are searching for novel mechanisms for control of growth at refrigeration temperatures. In its cytoplasmic membrane lipids L. monocytogenes produces high amounts of the branched-chain fatty acid anteiso C15:0. We believe that this is a major determinant of the ability of Listeria to grow at low temperatures through imparting an essential fluidity to the membrane. We will investigate the mechanisms operating to ensure the high levels of anteiso C15:0 through cloning, over expressing, purifying and determining the substrate preferences of key enzymes in the biosynthesis of branched-chain fatty acids. We will investigate whether the fatty acid composition of Listeria can be modified to one that does not support growth at low temperatures by feeding selected fatty acid precursors to the bacterium. Finally, we will use DNA microarray technology to study gene expression of the organism in an actual food matrix. It is our expectation that these studies will lead to improved methods for control of the growth of L. monocytogenes
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APPROACH: The first objective is to investigate the mechanism operative that ensures the characteristically high content of fatty acid anteiso C15:0 in L. monocytogenes. Key enzymes of the branched-chain fatty acid biosynthesis pathway, Bkd, FabH and BabF, will be cloned, over expressed and purified. The substrate preferences of the enzymes will be determined at different temperatures to investigate the hypothesis that the intrinsic activities of the enzymes are responsible for producing high levels of fatty acid anteiso C15:0. The second objective is to investigate whether the fatty acid composition of L. monocytogenes can be manipulated to lower the production of anteiso C15:0 and thereby produce a fatty acid composition that produces a rigid membrane that does not allow the organism to grow at refrigeration temperatures. Various fatty acid precursors will be added to bacteriological medium and turkey slurries and the growth and fatty composition of of the organism will be determined at low temperatures. In the third objective gene expression by L. monocytogenes growing on an actual food matrix will be compared to cells growing on bacteriological media. Genome-wide transcriptional profiling using DNA microarrays will be employed.
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PROGRESS: 2007/09 TO 2008/08 <BR>
OUTPUTS: Outputs: Further experiments have been undertaken on analyzing the kinetics of FabH, modeling the activity of the enzyme in the presence of multiple substrates. FabH is a critical enzyme in the biosynthesis of branched-chain fatty acids, particularly anteiso C15:0, which are critical in the growth of the organism at refrigeration temperatures. The fatty acid composition of a branched-chain fatty acid deficient mutant grown in the presence of different precursors has been determined to validate in vivo inferences from studies of FabH kinetics in vitro. Further studies on the influence of various fatty acid precursors, including food preservatives, on the fatty acid composition and growth of Listeria monocytogenes have been carried out. Manuscripts describing these studies are in preparation for publication. An extensive metabolomics study of the small molecular weight compounds present in L. monocytogenes grown at 37 and 8 degrees C have been carried out. I attended the USDA CSREES Principal Investigator\'s meeting in Washington, DC in November and reported on our FabH and fatty acid studies. Studies have been initiated on the growth of L. monocytogenes on turkey slices with a view to studies of gene expression in this food environment. <BR>
PARTICIPANTS: Dr Brian J. Wilkinson, Professor of Microbiology, Dr Atul Singh, Postdoctoral Fellow, Mudcharee Julotok, PhD student, all at Illinois State University, Normal, IL. Collaborator: Dr Charles Rock, Saint Jude Research Hospital, Memphis, TN. <BR>
TARGET AUDIENCES: Target audiences are food scientists interested in food safety in academia, government, and industry. Two publications have appeared this year and a presentation has been made to CSREES principal investigators. <BR>
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IMPACT: 2007/09 TO 2008/08<BR>
The affinity (Km) of FabH for 2 methylybutyryl CoA increased at low temperatures thus ensuring the high content of fatty acid anteiso C15:0, which is important for growth of the organism at low temperatures. Other CoA substrates effectively competed with 2 methylbutyryl CoA at 30 degrees C, but were much less effective at 10 degrees C. Consistent with the kinetic properties of FabH, only a limited number of fatty acid precursors, related to 2 methylybutyrate, were able to modulate the fatty acid composition and inhibit the growth of Listeria at low temperatures. Combinations of precursors may be an effective means of controlling the growth of Listeria at low temperatures. Major changes were noted in the metabolome of L. monocytogenes grown at 8 compared to 37 degrees C. This helps us understand the mechanisms involved in the psychrotolerance of this foodborne pathogen. Two papers were published on the alkali tolerance response of L. monocytogenes. Alkali stress induced extensive changes in gene and protein expression. The alternative sigma factor, SigB, played a role in the alkali stress response. Alkali stress induced cross protection against osmotic and ethanol challenges. This has implications for food safety and preservation given the role of these conditions in achieving them.