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The ability to determine the pathogenic potential of L. monocytogenes isolated from food products will enable better prediction of the health risk to consumers. An important event in the pathogenesis of listeriosis is the bacterial encounter with host phagocytes; there is evidence that the ability to resist killing by macrophages is a distinguishing feature between virulent and avirulent L. monocytogenes. Therefore, the objective of this grant is to determine how virulent and avirulent isolates of L. monocytogenes respond to phagocytosis by macrophages. To accomplish this objective, we will conduct a genome-wide comparison of protein and RNA expression profiles from virulent and avirulent L. monocytogenes strains in response to phagocytosis by the murine macrophage cell line J774A.1. We will conduct our comparisons at the protein level because proteins are the ultimate effectors of bacterial virulence. Subsequent comparisons at the transcriptome level will elucidate whether differences in the proteome are due to transcriptional or post-transcriptional effects. We expect that this project will result in identification of genes and proteins expressed by virulent L. monocytogenes that are critical in defining the outcome of survival in macrophages and in determining the outcome of infection. This research will also result in an expressed protein database for L. monocytogenes, which will contribute significantly to functional annotation of the L. monocytogenes genome. These findings will contribute to our basic understanding of the mechanisms of intracellular survival for this important pathogen and will allow the identification of markers for identification of virulent L. monocytogenes.
NON-TECHNICAL SUMMARY: Listeria monocytogenes is a significant bacterial foodborne pathogen that causes a number of food product recalls in the U.S. each year. However, while many L. monocytogenes strains can cause disease and/or mortality, others have low virulence or are avirulent. The ability to determine the pathogenic potential of L. monocytogenes isolated from food products will enable better prediction of the health risk to consumers. There is evidence that the ability to resist killing by host immune cells (macrophages) is a distinguishing feature between virulent and avirulent L. monocytogenes. We hypothesize that virulent and avirulent L. monocytogenes strains have different protein expression responses to the stressful intracellular environment of host macrophages; this difference is likely one of the contributing factors to the outcome of infection. The immediate objective of this proposal is to conduct a comparison of protein and RNA expression profiles from virulent and avirulent L. monocytogenes strains in response to phagocytosis by mouse macrophages. We expect that this project will result in identification of genes and proteins expressed by virulent L. monocytogenes that are critical in defining the outcome of survival in macrophages and in determining the outcome of infection. These findings will contribute to our basic understanding of the mechanisms of intracellular survival for this important pathogen and will allow the identification of markers for identification of virulent L. monocytogenes.
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APPROACH: We will use one L. monocytogenes isolate from each of the major genetic lineages: serovar 1/2a, serovar 4b, and serovar 4a. The full genome sequence is publicly available for each of the 1/2a and 4b strains, and we will also use the genome sequence of an avirulent serotype 4a strain. We will use a published method for phagocytosis of L. monocytogenes strains with murine macrophage cell line J774A.1. RNA and proteins will be isolated from bacteria released from lysed macrophages and from bacteria cultivated in bacterial growth medium. For analysis of protein expression, we will use multidimensional protein identification technology (MudPIT), which utilizes two dimensional liquid chromatography with electrospray ionization tandem mass spectrometry (2-D LC ESI MS2) to quantitatively compare protein expression profiles. Non-electrophoretic proteomics eliminates confusion from host proteins because we conduct Sequest searches on our own species-specific protein and nucleotide databases. In this way, we can remove ambiguous mass spectral identifications and can clearly differentiate Listeria monocytogenes peptides from host peptides. Comparisons of RNA expression profiles under phagocytic and non-phagocytic conditions will be conducted by microarray analysis using an L. monocytogenes array constructed from PCR products from 2819 genes from serovar 1/2a and 96 genes unique to serovar 4b (2915 total genes). In addition, more genes unique to 4b, 4c, and 4a strains are being added. Microarray data will allow determination of whether differential protein expression is due to transcriptional regulation or posttranscriptional events. RNA expression for a subset of the genes will be confirmed by real-time RT-PCR. Protein and RNA expression data will be statistically analyzed to identify genes and proteins that are differentially expressed under phagocytic and non-phagocytic conditions. This expression data will allow us to then compare the differential response of the three L. monocytogenes strains to phagocytosis.