Location: Food and Feed Safety Research2012 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.
3. Progress Report:
In FY 2012, significant focus was on the design, development, and optimization of a novel chicken species-specific immune function assay (known as kinome peptide array) for experiments involving changes in host cell kinase activity following infection. Kinases are enzymes involved in immune responses. The process of array design involved finding known human kinases that take part in immune-related signaling. Over 500 human kinase target sites were chosen. The equivalent target sites were then searched for within the chicken (Gallus gallus) proteome. Using these search results, enzyme "equivalency" (known as homology) was determined first by ensuring that the human and chicken proteins were in fact equivalent. Then, the target sequence was confirmed to have minimum amino acid conservation, usually 8 out of 15 amino acids matching, and the correct phosphorylated residue was confirmed as present at the correct position within the sequence. When all these criteria were met, the chicken phosphorylation target sequence was selected for incorporation onto the peptide array. Of the 500 sites queried, 358 were selected for incorporation onto the array. In other work, a chicken metabolism peptide array was used in an animal trial involving the infection of untreated chicks with Salmonella typhimurium. A total of 4 time points and 24 birds were sampled. The goal was to determine if any metabolic changes occurred in the host during infection and to identify those changes. Preliminary results following infection include: 1) an increase in phosphoglycerate acid mutase (PGAM) phosphorylation; PGAM is an enzyme involved in glycolysis (glucose metabolism), 2) an increase in calponin phosphorylation, suggesting calponin may negatively affect bone growth, and 3) an increase in AMPKG2 phosphorylation, known to be involved in fatty acid biosynthesis. Project work in FY 2012 developed foundational information on how the chicken immune system functions to protect the birds against microbial infections. This work has direct implications for microbial food safety of poultry meat products.
1. New insights on how Salmonella interacts 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 produce poultry that are not colonized by these harmful bacteria, given that absence of the pathogens in living birds will largely translate into pathogen-free meat products for human consumption. ARS researchers at College Station, Texas, found that after exposure to S. typhimurium, a type of chicken immune cell known as a heterophil responded by up-regulating (turning on) genes associated with regulation of cell differentiation, protein transport, macromolecule localization, and heterocycle metabolic processes. The work also established that bacteria attacked by the heterophils responded by increasing fatty acid biosynthesis, flagellar assembly, glutathione metabolism, and the Type III secretion system. This work is important because for the first time, it has been shown how Salmonella and the bird host interact and how these interactions ultimately result in protection versus illness/death. This work has identified specific targets to design new immune modulatory and/or antimicrobial compounds that can be utilized by the poultry industry to produce microbiologically safer poultry meat products for the consumer.
2. Genetic analysis of Salmonella-resistant and -susceptible chickens. 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 produce poultry that are not colonized by these harmful bacteria, given that absence of the pathogens in living birds will largely translate into pathogen-free meat products for human consumption. ARS researchers at College Station, Texas, showed that certain genes in chickens that convey immune protection against Salmonella are more active in the Salmonella-resistant chicken line A than in the susceptible line B. These findings are important because they identify new targets for genetic selection of chickens to increase resistance to bacterial infections. Such resistant birds will be much less likely to harbor microorganisms that can contaminate poultry meat products and cause food poisoning in humans.
He, L.H., Genovese, K.J., Swaggerty, C.L., MacKinnon, K.M., Kogut, M.H. 2011. Co-stimulation with TLR3 and TLR21 ligands synergistically up-regulates Th1-cytokine IFN-gamma and regulatory cytokine IL-10 expression in chicken monocytes. Developmental and Comparative Immunology. 36:756-760.