Progress 11/01/06 to 10/31/10
Outputs
OUTPUTS: The full genome sequence of an avirulent serotype 4a Listeria monocytogenes strain, HCC23, was released into GenBank and is publicly available (http://www.ncbi.nlm.nih.gov/sites/entrezdb=genomeprj&cmd=Retrieve&d opt=Overview&list_uids=29409). This is the first completed genome from genetic lineage III of Listeria monocytogenes. The genome sequence was used to add lineage III-specific gene probes to the Listeria monocytogenes microarray available at the Pathogen Functional Genomics Resource Center. Protein expression data was deposited in the Protein Identifications (PRIDE) database for public accessibility, and the original microarray data will also be made publicly accessible in a database following publication. PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine (MSU-CVM) is the project director. Dr. Shane Burgess (MSU-CVM) and Dr. Susan Bridges (MSU Department of Computer Sciences and Engineering) are the project co-directors. Dr. Janet Donaldson conducted phagocytosis assays and protein isolations as a postdoctoral scientist and collaborated on the project as an assistant professor in the MSU Department of Biological Sciences. Dr. James Watt conducted phagocytosis assays, RNA isolations, and microarray hybridizations. Chelsea Steele (MSU-CVM) completed the genome sequence of a serotype 4a strain. Mass spectrometry was conducted by Dr. Tibor Pechan at the MSU Life Sciences and Biotechnology Institute. Dr. Bindu Nanduri (MSU-CVM) conducted analysis of protein expression data. Comparative genomic analysis was conducted by undergraduate and graduate students in Dr. Bridges's computational biology class. TARGET AUDIENCES: This research will improve our basic understanding of the mechanisms of intracellular survival for Listeria monocytogenes, which will benefit both the human health and food safety research communities. However, the ultimate target audiences for this research are food producers, food processors, and consumers. L. monocytogenes is a significant foodborne pathogen that causes a number of food product recalls in the U.S. each year. This research could lead to the identification of markers to distinguish virulent and avirulent L. monocytogenes, which could reduce the number of food safety recalls in the U.S. due to L. monocytogenes, reduce the number of food-borne illnesses in the U.S., and provide an improved basis for regulation of food safety issues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
By comparing the genome sequence of serotype 4a isolate HCC23 with the genome sequences of other Listeria isolates, we gained important new knowledge about L. monocytogenes virulence genes. The genome sequence comparison allowed identification of 58 "virulence-specific" genes that are present in only pathogenic isolates and 66 "avirulence-specific" genes that are present in HCC23 and L. innocua. We also gained important information about lineage III strains: HCC23 contains 253 genes encoding proteins that are unique to this strain when compared to the completed genomes of strains from lineage I, lineage II, and L. innocua. A large number of these are hypothetical/conserved hypothetical along with phage proteins and some surface proteins. We also gained information about the genus: comparison of the protein sequences between genomes indicated that L. innocua is more similar to HCC23 than the other L. monocytogenes isolates. In addition, serotype 4b and 1/2a strains are more similar to each other than they are to the serotype 4a strain and L. innocua. Overall, we found that L. monocytogenes and L. innocua share a "core genome" composed of >2300 genes. We compared the ability of three L. monocytogenes strains (serovar 1/2a strain EGD, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGD and F2365 proliferated within macrophages for at least 7 hr. These findings indicate that sustained listerial intracellular growth in macrophages is potentially a pathogenic determinant. To elucidate the mechanism for listerial survival in macrophages, we compared the protein expression profiles of these three strains inside macrophages at 0 hr, 3 hr, and 5 hr post-infection using proteomics technology. Our results indicated that similar metabolic and cell wall associated proteins are expressed by all three strains at 3 hr post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5 hr post-infection. These proteins were only significantly increased in the two pathogenic isolates at this time point. We also detected proteomic evidence of decreased HCC23 replication at 5 hr PI and a potential compensatory response involving cell wall associated proteins. Overall, our results suggest that the oxidative stress response and protein chaperons are potentially important components for long-term listerial survival in macrophages. Further studies are being conducted to determine the roles of these proteins in macrophage survival and virulence.
Publications
- Donaldson, J. R., B. Nanduri, S. C. Burgess, and M. L. Lawrence. 2009. Comparative proteomic analysis of Listeria monocytogenes strains F2365 and EGD. Appl. Environ. Microbiol. 75:366-373. Steele, C. L., J. R. Donaldson, S. M. Bridges, S. Salkeld, R. Kumar, M. M. Banes, T. Arick, and M. L. Lawrence. 2009. Genome sequence of a lineage III serotype 4a Listeria monocytogenes strain. 109th General Meeting of the American Society for Microbiology, Philadelphia, Pennsylvania.
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Progress 11/01/08 to 10/31/09
Outputs
OUTPUTS: The full genome sequence of an avirulent serotype 4a Listeria monocytogenes strain, HCC23, has been released into GenBank and is publicly available (http://www.ncbi.nlm.nih.gov/sites/entrezdb=genomeprj&cmd=Retrieve&d opt=Overview&list_uids=29409). This is the first completed genome from genetic lineage III of Listeria monocytogenes. The genome sequence was used to add lineage III-specific gene probes to the Listeria monocytogenes microarray available at the Pathogen Functional Genomics Resource Center. Protein expression data will be deposited in the Protein Identifications (PRIDE) database for public accessibility, and the original microarray data will also be made publicly accessible in a database following publication. Research results are being disseminated at scientific meetings and are being published in peer-reviewed journals. PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine (MSU-CVM) is the project director. Dr. Shane Burgess (MSU-CVM) and Dr. Susan Bridges (MSU Department of Computer Sciences and Engineering) are the project co-directors. Dr. Janet Donaldson conducted phagocytosis assays and protein isolations as a postdoctoral scientist and continues to collaborate on the project as an assistant professor in the MSU Department of Biological Sciences. Dr. James Watt is conducting phagocytosis assays, RNA isolations, and microarray hybridizations. Chelsea Steele (MSU-CVM) completed the genome sequence of a serotype 4a strain. Mass spectrometry was conducted by Dr. Tibor Pechan at the MSU Life Sciences and Biotechnology Institute. Dr. Bindu Nanduri (MSU-CVM) conducted analysis of protein expression data. Comparative genomic analysis is being conducted by undergraduate and graduate students in Dr. Bridges's computational biology class. TARGET AUDIENCES: This research will improve our basic understanding of the mechanisms of intracellular survival for Listeria monocytogenes, which will benefit both the human health and food safety research communities. However, the ultimate target audiences for this research are food producers, food processors, and consumers. L. monocytogenes is a significant foodborne pathogen that causes a number of food product recalls in the U.S. each year. This research could lead to the identification of markers to distinguish virulent and avirulent L. monocytogenes, which could reduce the number of food safety recalls in the U.S. due to L. monocytogenes, reduce the number of food-borne illnesses in the U.S., and provide an improved basis for regulation of food safety issues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
By comparing the genome sequence of serotype 4a isolate HCC23 with the genome sequences of other Listeria isolates, we gained important new knowledge about L. monocytogenes virulence genes. The genome sequence comparison allowed identification of 58 "virulence-specific" genes that are present in only pathogenic isolates and 66 "avirulence-specific" genes that are present in HCC23 and L. innocua. We also gained important information about lineage III strains: HCC23 contains 253 genes encoding proteins that are unique to this strain when compared to the completed genomes of strains from lineage I, lineage II, and L. innocua. A large number of these are hypothetical/conserved hypothetical along with phage proteins and some surface proteins. We also gained information about the genus: comparison of the protein sequences between genomes indicated that L. innocua is more similar to HCC23 than the other L. monocytogenes isolates. In addition, serotype 4b and 1/2a strains are more similar to each other than they are to the serotype 4a strain and L. innocua. Overall, we found that L. monocytogenes and L. innocua share a "core genome" composed of >2300 genes. We compared the ability of three L. monocytogenes strains (serovar 1/2a strain EGD, serovar 4b strain F2365, and serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1. We found that the avirulent strain HCC23 was able to initiate an infection but could not establish prolonged infection within the macrophages. By contrast, strains EGD and F2365 proliferated within macrophages for at least 7 hr. These findings indicate that sustained listerial intracellular growth in macrophages is potentially a pathogenic determinant. To elucidate the mechanism for listerial survival in macrophages, we compared the protein expression profiles of these three strains inside macrophages at 0 hr, 3 hr, and 5 hr post-infection using proteomics technology. Our results indicated that similar metabolic and cell wall associated proteins are expressed by all three strains at 3 hr post-infection. However, increased expression of stress response and DNA repair proteins was associated with the ability to proliferate in macrophages at 5 hr post-infection. These proteins were only significantly increased in the two pathogenic isolates at this time point. We also detected proteomic evidence of decreased HCC23 replication at 5 hr PI and a potential compensatory response involving cell wall associated proteins. Overall, our results suggest that the oxidative stress response and protein chaperons are potentially important components for long-term listerial survival in macrophages.
Publications
- Steele, C. L., J. R. Donaldson, S. M. Bridges, S. Salkeld, R. Kumar, M. M. Banes, T. Arick, and M. L. Lawrence. 2009. Genome sequence of a lineage III serotype 4a Listeria monocytogenes strain. 109th General Meeting of the American Society for Microbiology, Philadelphia, Pennsylvania.
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Progress 11/01/07 to 10/31/08
Outputs
OUTPUTS: The full genome sequence of an avirulent serotype 4a Listeria monocytogenes strain, HCC23, has been released into GenBank and is publicly available (http://www.ncbi.nlm.nih.gov/sites/entrezdb=genomeprj&cmd=Retrieve&d opt=Overview&list_uids=29409). This is the first completed genome from genetic lineage III of Listeria monocytogenes. The genome sequence is being used to add lineage III-specific gene probes to the Listeria monocytogenes microarray available at the Pathogen Functional Genomics Resource Center. Protein expression data will be deposited in the Protein Identifications (PRIDE) database for public accessibility, and the original microarray data will also be made publicly accessible in a database following publication. Research results are being disseminated at scientific meetings and are being published in peer-reviewed journals. PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine (MSU-CVM) is the project director. Dr. Shane Burgess (MSU-CVM) and Dr. Susan Bridges (MSU Department of Computer Sciences and Engineering) are the project co-directors. Dr. Janet Donaldson conducted phagocytosis assays and protein isolations as a postdoctoral scientist and continues to collaborate on the project as an assistant professor in the MSU Department of Biological Sciences. Dr. James Watt is conducting phagocytosis assays, RNA isolations, and microarray hybridizations. Chelsea Steele (MSU-CVM) completed the genome sequence of a serotype 4a strain. Mass spectrometry was conducted by Dr. Tibor Pechan at the MSU Life Sciences and Biotechnology Institute. Dr. Bindu Nanduri (MSU-CVM) conducted analysis of protein expression data. Comparative genomic analysis is being conducted by undergraduate and graduate students in Dr. Bridges's computational biology class. TARGET AUDIENCES: This research will improve our basic understanding of the mechanisms of intracellular survival for Listeria monocytogenes, which will benefit both the human health and food safety research communities. However, the ultimate target audiences for this research are food producers, food processors, and consumers. L. monocytogenes is a significant foodborne pathogen that causes a number of food product recalls in the U.S. each year. This research could lead to the identification of markers to distinguish virulent and avirulent L. monocytogenes, which could reduce the number of food safety recalls in the U.S. due to L. monocytogenes, reduce the number of food-borne illnesses in the U.S., and provide an improved basis for regulation of food safety issues. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Case fatality rates from listeriosis outbreaks have approached 30%-40%, which is much higher than the case mortality rates for other common foodborne pathogens such as Salmonella enteritidis, Campylobacter jejuni, E. coli O157:H7, and Vibrio. Despite this potential for human virulence, not all L. monocytogenes strains pose a food safety risk to humans. However, the underlying mechanisms for the variation in pathogenic potential are not well understood. Identification of genes and proteins that are unique to different strains may allow the identification of biomarkers that can be used to better identify L. monocytogenes serotypes and/or predict pathogenic potential. 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 macrophages is a distinguishing feature between virulent and avirulent L. monocytogenes. This research will further our understanding of the basis for variation in listerial virulence by using functional genomics (proteomics and transcriptomics) to identify genes uniquely expressed by virulent L. monocytogenes in response to phagocytosis. The identified genes could provide the basis for reliable identification of pathogenic L. monocytogenes, which would result in improved L. monocytogenes risk assessment. The proposed research will also increase our understanding of how virulent strains are able to avoid being cleared by professional phagocytes. In addition, it will result in application and further development of state-of-the-art proteomics methodologies (2-D LC ESI MS2) to bacterial pathogenesis.
Publications
- Donaldson, J. R., B. Nanduri, S. C. Burgess, and M. L. Lawrence. 2009. Comparative proteomic analysis of Listeria monocytogenes strains F2365 and EGD. Appl. Environ. Microbiol. 75:366-373.
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Progress 11/01/06 to 10/31/07
Outputs
OUTPUTS: We are using 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 used a published method for phagocytosis of L. monocytogenes strains with murine macrophage cell line J774A.1. RNA and proteins were isolated from bacteria released from lysed macrophages and from bacteria cultivated in bacterial growth medium. For analysis of protein expression, we used 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. 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. PARTICIPANTS: Dr. Mark L. Lawrence at the Mississippi State University College of Veterinary Medicine (MSU-CVM) is the project director. Dr. Shane Burgess (MSU-CVM) and Dr. Susan Bridges (MSU Department of Computer Sciences and Engineering) are the project co-directors. Dr. Janet Donaldson conducted phagocytosis assays, protein isolations, and RNA isolations as a postdoctoral scientist and continues to collaborate on the project as an assistant professor in the MSU Department of Biological Sciences. Chelsea Steele (MSU-CVM) is completing the genome sequence of a serotype 4a strain. Mass spectrometry was conducted by Dr. Tibor Pechan at the MSU Life Sciences and Biotechnology Institute. Dr. Bindu Nanduri (MSU-CVM) conducted analysis of protein expression data. Comparative genomic analysis is being conducted by undergraduate and graduate students in Dr. Bridges's computational biology class. TARGET AUDIENCES: This research will improve our basic understanding of the mechanisms of intracellular survival for Listeria monocytogenes, which will benefit both the human health and food safety research communities. However, the ultimate target audiences for this research are food producers, food processors, and consumers. L. monocytogenes is a significant foodborne pathogen that causes a number of food product recalls in the U.S. each year. This research could lead to the identification of markers to distinguish virulent and avirulent L. monocytogenes, which could reduce the number of food safety recalls in the U.S. due to L. monocytogenes, reduce the number of food-borne illnesses in the U.S., and provide an improved basis for regulation of food safety issues. PROJECT MODIFICATIONS: There are no major changes to report.
Impacts
Our data show that conducting comparisons between bacterial strains at the protein expression level can complement information that is gained from genome sequence comparisons. In particular, protein expression comparisons can be valuable in helping to fully understand the virulence capabilities of L. monocytogenes strains. Our next step in this project is to conduct protein expression comparisons between these strains in the context of phagocytosis by murine macrophages, which will allow a better delineation of how protein expression relates to virulence. Identification of proteins that are unique to different strains may also allow the identification of biomarkers that can be used to better identify L. monocytogenes serotypes and/or predict pathogenic potential.
Publications
- Karsi, A. and M. L. Lawrence. 2007. Broad host range fluorescence and bioluminescence expression vectors for gram negative bacteria. Plasmid 57:286-95.
- Karsi, A., S. Menanteau-Ledouble, and M. L. Lawrence. 2006. Development of bioluminescent Edwardsiella ictaluri for non-invasive disease monitoring. FEMS Microbiol. Lett. 260:216-223.
- Karsi, A. and M. L. Lawrence. 2006. Development of fluorescent Edwardsiella ictaluri and visualization of the pathogen in channel catfish neutrophils. 106th General Meeting of the American Society for Microbiology, Orlando, Florida.
- Karsi, A., S. Menanteau, and M. L. Lawrence. 2005. Real-time monitoring of bioluminescent Edwardsiella ictaluri infections in living catfish. 105th General Meeting of the American Society for Microbiology, Atlanta, Georgia.
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