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The broad objective of the research is to develop methods for control of the foodborne pathogen, Listeria monocytogenes, in food products. While not as prevalent as food poisoning resulting from Salmonella or Staphylococcus contamination, the disease listeriosis which is caused by this microorganism is responsible for a greater number of deaths and a much higher rate of mortality in the USA than any other disease of foodborne origin. the major objects are to isolate Listeria mutants that are resistant to pediocin AcH, identify genes responsible for resistance, and determine the roles played by these genes in resistance to pediocin AcH and other peptides. We expect to identify genes encoding molecules that mediate cell wall and membrane binding of pediocin AcH, proteases thatn can degrade the peptide, and sensor proteins and other signaling molecules involved in stress response to peptide agents. Knowledge of the pathway of uptake and resistance mechanisms will allow us to design altered molecules that by virtue of enhanced protease resistance, for example, could circumvent resistance mechanisms. In addition the identification of gene products that mediate resistance will permit design of new therapeutic agents against these potential drug targets.
NON-TECHNICAL SUMMARY: The disease listeriosis, which is caused by Listeria monocytogenes, is responsible for a greater number of deaths and a much higher rate of mortality in the USA than any other disease of foodborne origin. The long-term goal is to develop improved methods for eradicating L. monocytogenes from food sources.
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APPROACH: Several new methodologies important for the successful completion of the project are that we, in summary: 1) have obtained a suitable plasmid vector for transposon mutagenesis and introduced it into L. Innocua, 2) created transposon knockout libraries, and 3) currently are developing methods for screening mutants for resistance to podiocin AcH. We plan to generate additional libraries and continue screening until enough mutants are available for cloning and sequencing studies. Investigation and experiments include isolation of mutants from transposon libraries and determination of the fold increase, evaluation of the stability of mutant phenotypes, cloning and sequencing of Listeria genome segments containing transposon insertions, identification of transposon insertion sites by genome database searching, identification of genes responsible for pediocin AcH resistance, and roles of FL monocytogenes genes in pediocin AcH resistance, analysis of antimicrobial peptide cross-resistance in Listeria mutants.
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PROGRESS: 2000/12 TO 2004/09<BR>
The research was aimed at identifying genes that encode proteins and other cellular components that are necessary for interaction of the antimicrobial peptide pediocin AcH with Listeria. Previously, we reported on the isolation of a pediocin AcH-resistant mutant of strain L. innocua Lin11 (mutant G7) by transposon Tn917 mutagenesis. The basic characterization of this mutant now has been completed. The G7 mutant is ~1,000-fold more resistant to pediocin AcH than the wild-type strain. In G7, the transposon appears to disrupt the promoter of the lin0142 gene. Real-time reverse transcriptase (RT)-PCR analysis of mRNA levels confirms that lin0142 transcripts are undetectable in G7. Importantly, the loss of lin0142 expression abolishes transcription of the mpt operon (encodes the mannose-specific phosphotransferase system transporter) which is the docking protein for pediocin AcH in cells. The introduction of the intact lin0142 gene into the G7 mutant on a complementation vector restores transcription of mpt and sensitivity to pediocin AcH. In conclusion, a novel activator required for mpt expression and pediocin AcH resistance has been identified through the research.
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IMPACT: 2000/12 TO 2004/09<BR>
Listeria monocytogenes is a foodborne pathogen responsible for hundreds of deaths in the United States annually and losses of millions of dollars to food producers due to product recalls. The long-term goal of this research is to apply the antimicrobial peptide, pediocin AcH, which is produced by the food-grade bacterium, Pediococcus acidilactici, to help control growth of Listeria in foods. In the short term, this research project has helped determine how pediocin AcH interacts with Listeria cells and how the bacterium potentially could become resistant to its action. Knowledge of these points will enable strategies to be developed to circumvent resistance, should it arise.