2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Conduct research on the differential host-pathogen interactions of Salmonella in human, chicken, and swine intestinal key host immune cells using emerging genomic technologies.
Objective 2: Analyze and characterize both host and Salmonella proteins that are modulated in expression during infection using quantitative proteomics.
Objective 3: Research on the molecular and cellular details of the host-microbe interactions will be used to identify virulence-associated microbial genes and host defense strategies. Identify potential intervention targets (e.g., host kinases) for Salmonella infections in food animals.
Objective 4: Develop strategies for the reduction of foodborne pathogens by targeting the host innate immune system (by identifying the use of immunomodulatory antimicrobial or host-defense peptides) and targeting and identifying virulence factors.
Sub-objective 4A: Molecular characterization of anti-infectives that target the host innate immune system to facilitate pathogen-specific immune responses.
Sub-objective 4B: Develop a high-throughput assay to screen a series of commercial libraries of small molecules for their ability to inhibit virulence factors produced by S. typhimurium.
1b.Approach (from AD-416):
Objective 1: Utilize deep sequencing and mutagenesis technologies to dissect the differential host-pathogen interactions of Salmonella in human, chicken, and swine intestinal epithelial cells and macrophages. Specifically, we will determine the differential transcriptome of S. Typhimurium in mammalian versus chicken epithelial cells and develop gene-deletion mutants of S. Typhimurium to elucidate the differential mechanisms of intestinal pathogenicity of S. Typhimurium in humans compared to chickens and swine. Objective 2: A newly described technique of purifying live Salmonella expressing green fluorescent protein from either infected tissues or cell cultures using flow cytometry will be used to analyze and characterize both host and Salmonella proteins that are modulated in expression during infection. Quantitative proteomics including conventional two-dimensional electrophoresis, difference gel electrophoresis (DIGE), and mass spectrometry will be used to facilitate accomplishment of this objective. Objective 3: Using a matched comparative model (Salmonella characterized by contrasting degrees of pathogenicity and/or gene-deletion mutants of S. Typhimurium), newly developed peptide arrays will be used for studying the kinome of chicken and swine intestinal epithelial cells and macrophages. Cell lysates will be analyzed on a kinomics array containing 1,024 peptides derived from known phosphorylation sites annotated with reported upstream kinases. In addition, reverse chemical genetics will be used to identify host kinases that are essential in controlling intracellular Salmonella infections. This procedure will enable us to identify a class of kinases using selective chemical inhibitors of kinases with relevant biological activities to control in vitro and in vivo infections. Objective 4: We will develop a high-throughput assay to search for inhibitors of the Type 3 secretion systems. We will screen a series of commercial libraries of small molecules for their ability to inhibit type 3 secretion by S. Typhimurium. A systems approach will be employed to understand and characterize the host-pathogen interactions that are manipulated in food animals using novel therapeutic approaches with BT peptides and CpG oligonucleotides without engendering antimicrobial resistance. Microarray analyses of avian and porcine peripheral blood granulocytes and monocytes following treatment with BT peptides or CpG oligonucleotides will be performed. Using InnateDB, bioinformatic interrogation of gene ontology, signaling pathways and transcription factor binding sites will be undertaken; confirmation will be achieved experimentally by qRT-PCR and inhibitor studies of in vitro functional biological assays, and followed up by direct biochemical confirmation. Collectively, these will lead to substantial advances in understanding the complexity of signaling pathways and transcription factors involved in the responses to BT peptides and CpG modulation.
In FY 2013, significant focus was on describing an integrated picture of how dietary changes influence bird gut health, intestinal structure and function, gut mucosal immunity, and the intestinal microbiota. We monitored the effect of changing the diet during a broiler grow-out on gut microbiota composition, mucosal host defenses, and susceptibility to colonization of the intestine by opportunistic bacteria. Using multiple molecular approaches (pyrosequencing, quantitative real-time PCR, peptide microarrays), we have preliminary findings that the three dietary periods (starter, grower, finisher) that commercial broilers undergo during a normal grow-out dramatically alter the gut microbiota composition. This microbial perturbation leads to a severe dysregulation of the physiological and immunological intestinal homeostasis (immune responses, mucus production, intestinal barrier functions) and provides a "window of opportunity" for increasing the susceptibility to intestinal colonization by Salmonella. Migratory birds play an important role in the ecology, circulation, and dissemination of pathogenic organisms. We established that the innate immune defenses were significantly more efficient in two migratory parasitic (lays eggs in other bird species' nests) cowbird species than in the non-parasitic red-winged blackbird. Additionally, immune defenses were more efficient in the brown-headed cowbird, an extreme host-generalist brood parasite, than in the bronzed cowbird, a moderate host-specialist with a smaller migratory range and lower exposure to other bird species and their parasites (e.g., mites, insects, etc.). Thus, the relative effectiveness of these two innate immune responses corresponds to the diversity of parasites in the niche of each species and to their relative resistance as carriers of food safety pathogens such as Salmonella, and other human pathogens such as West Nile virus. This work has direct implications for microbial food safety of poultry meat products.
Persistent intestinal Salmonella colonization affects chicken muscle metabolism. Food-poisoning bacteria, such as Salmonella, colonize the cecum of chickens and are then persistently shed in the chicken excreta into the surrounding environment. New approaches are needed to identify poultry that shed these harmful bacteria in order to reduce Salmonella persistence in the intestine and increase pathogen-free meat products for human consumption. ARS scientists at College Station, Texas, found profound changes in fatty acid and glucose metabolism in the skeletal muscle of chickens that were persistently infected with Salmonella. The infections resulted in the deposition of fat instead of protein in the muscle. This work identified previously unknown effects of infection on host metabolism and sheds light on mechanisms used by Salmonella to cause disease and by the host to counter infection. This work identifies new targets for the design of antimicrobial compounds directed towards the host metabolism and that will greatly reduce infection/colonization of poultry by harmful microorganism.
Salmonella interactions with chicken immune cells. Food-poisoning bacteria, such as Salmonella, are significant causes of human disease; these pathogens can often be found as contaminants in poultry meat products. New approaches are needed to reduce bacterial colonization of poultry, given that absence of the pathogens in living birds will largely translate into pathogen-free meat products for human consumption. ARS scientists at College Station, Texas, found that a certain chicken immune system receptor interacts with another cellular component (double-stranded RNA) to increase the expression of disease-fighting cells and the production of nitric oxide, the hallmark immune responses that promote protective immunity against Salmonella in chickens. This work is important because it provides a more detailed understanding of how Salmonella and the host interact, and how these interactions eventually result in protection. The work has identified specific targets to design new immune modulatory and/or antimicrobial compounds to enhance the microbial safety of poultry products reaching the consumer.
He, L.H., Genovese, K.J., Swaggerty, C.L., Nisbet, D.J., Kogut, M.H. 2012. A comparative study on invasion, survival, modulation of oxidative burst, and nitric oxide responses of macrophages (HD11), and systemic infection in chickens by prevalent poultry Salmonella serovars. Foodborne Pathogens and Disease. 9:1104-1110.
Hahn, D.C., Summers, S.G., Genovese, K.J., He, L.H., Kogut, M.H. 2012. Enhanced innate immune responses in a brood parasitic cowbird species: Degranulation and oxidative burst. Avian Diseases. 57:285-289.
Kogut, M.H., Chiang, H., Swaggerty, C.L., Pevzner, I.Y., Zhou, H. 2012. Gene expression analysis of toll-like receptor pathways in heterophils from genetic chicken lines that differ in their susceptibility to Salmonella enteritidis. Frontiers in Genetics. 3:1-10.
He, L.H., Genovese, K.J., Swaggerty, C.L., Nisbet, D.J., Kogut, M.H. 2013. Nitric oxide as a biomarker of intracellular Salmonella viability and identification of the bacteriostatic activity of protein kinase A inhibitor H-89. PLoS One. 8:1-7.
Genovese, K.J., He, L.H., Swaggerty, C.L., Kogut, M.H. 2013. The avian heterophil. Developmental and Comparative Immunology. 41(3):334-340. doi: 10.1016/j.dci.2013.03.021.
Arsenault, R.J., Napper, S., Kogut, M.H. 2013. Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling. Veterinary Research. 44:35-50.
Arsenault, R.J., Kogut, M.H. 2013. Chicken-specific peptide arrays for kinome analysis: Flight for the flightless. Current Topics in Biotechnology. 7:79-89.