One of the most important causative agents of foodborne illness in the USA is the pathogenic bacterium, Listeria monocytogenes. This bacterium is responsible for about 2,500 cases of the severe disease known as listeriosis and 500 deaths each year. Research has shown that growth of. L. monocytogenes in foods can be controlled by antimicrobial peptides known as class IIa bacteriocins. These peptides are produced by a number of beneficial lactic acid bacterial species that are used in food production. However, as with all antimicrobial agents, L. monocytogenes can become resistant to class IIa bacteriocins. High-level resistance is caused by mutations that knock out the Ell-t(Man) glucose transport protein encoded by the mpt operon. This protein serves as the docking site for class lla bacteriocins in the cytoplasmic membrane. The long-term goals of the laboratory are to characterize the regulatory networks required for Ell-t(Man) expression and bacteriocin resistance, and apply this knowledge in food preservation technology. It is important to study the mechanism of Ell-t(Man) regulation as this protein is the primary cellular target recognized by class II peptides. It further is important from a clinical standpoint to determine if bacteriocin resistance is influenced by virulence gene expression, and if virulence and susceptibility to antimicrobial agents are altered in resistant strains. To address the long-term goals, genes associated with Ell-t(Man) -based resistance to the class lla bacteriocin known as pediocin AcH were isolated in the model, non-pathogenic species, L innocua. It now is proposed as the objective of this study to use forward and reverse genetics to obtain the corresponding mutations in L. monocytogenes and determine their mechanism of action and influence on virulence. Mutants will be characterized by real-time RT-PCR to quantify mpt regulator and virulence gene transcript levels, and two-dimensional gel electrophoresis and mass spectrometry to study alterations in total protein expression. The susceptibility of strains to antibiotics and antimicrobial peptides of the innate immune system also will be measured. Finally, the mechanism of action of a newly identified mpt regulatory protein, Lmo0095 will be studied. The research will generate novel information about genes involved in antimicrobial peptide resistance mechanisms in L. monocytogenes and establish their influence on virulence. ? ? Foodborne illnesses are a major public health concern in the USA. It is estimated that about 75 million cases of foodborne disease resulting in 325,000 hospitalizations, 5,000 deaths, and $5 to 6 billion in costs occur each year. The research ultimately will lead to better methods for prevention of food contamination and disease caused by the deadly pathogen, Listeria monocytogenes. ? ? ?

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-DDR-N (01))
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Mills, Melody
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University of Wyoming
Schools of Earth Sciences/Natur
United States
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