2011 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.
This is a new project that replaced 6202-32000-021-00D and which is continuing and expanding upon the work of the precursor project. Work under this project in FY 2011 had significant focus on the design, development, and optimization of new high-throughput assays to be used in a systems biology approach to evaluate the genomic, proteomic, transcriptomic, and metabolomic mechanisms involved in the host-pathogen interactions between Salmonella and Campylobacter and their poultry and swine hosts. New information was developed in work to identify innate immune mechanisms that regulate protection against food-poisoning bacteria. We established that a group of small peptides (named BT peptides) isolated from a soil bacterium possess antimicrobial activities against food-poisoning microorganisms in swine. Mechanistic studies with these peptides indicate that they may be an effective immunomodulator with subsequent effects on the carriage of Salmonella in both the swine gut and tissues. Project work in FY 2011 also established that certain avian cells (heterophils) produce lipid mediators of inflammation, specifically leukotrienes and prostaglandins, in response to various bacterial components that activate certain cell receptors.
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, TX, showed that day-old birds of an immunologically efficient line developed by the project's work were much more resistant to colonization by Salmonella enteritidis than were birds of a less immunologically efficient line. The immunologically efficient birds were also significantly more resistant to three species of Eimeria (parasitic protozoa that cause coccidiosis). This is clear evidence that ongoing efforts to breed pathogen-resistant new commercial poultry lines will likely succeed, resulting in healthier birds during growout and in meat products for consumption that are much safer from a microbiological contamination standpoint.
Protein kinases regulate resistance/susceptibility to food-borne pathogens in poultry. Salmonella and Campylobacter are the two leading causes of bacteria-derived human food-borne illness in the U.S. It is known that of two genetically distinct lines of chickens (lines A and B), the line A chickens are more resistant to both of these bacterial pathogens than are the line B chickens. ARS researchers at College Station, TX, hypothesized that the differences in resistance were due at least partly to differences in intracellular signaling pathways, and conducted experiments which clearly showed that certain enzymes in the birds (protein kinases) and the relevant signaling pathways did in fact contribute to the pathogen resistance patterns exhibited in the different bird lines. The work is important because it has identified key regulatory points that can be targeted in genetic selection for pathogen-resistant and possibly even pathogen-immune commercial poultry.
Stimulation of innate immunity genes in poultry. 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, TX, showed that feeding certain small peptides to chickens as part of the normal diet induces an increase in innate immune response genes in the intestine of Salmonella-infected birds. The work is important because it has identified a new target in birds that can be manipulated to increase resistance to pathogen infection, and thus result in safer poultry meat products reaching the consumer.