Source: AGRICULTURAL RESEARCH SERVICE submitted to
A SYSTEMS BIOLOGY APPROACH TO UNDERSTANDING THE SALMONELLA-HOST INTERACTOME IN POULTRY AND SWINE
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0420971
Grant No.
(N/A)
Project No.
3091-32000-031-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 3, 2011
Project End Date
Jan 2, 2016
Grant Year
(N/A)
Project Director
KOGUT M H
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
COLLEGE STATION,TX 77845
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3083210109035%
7123220109043%
3083230109010%
7123520109012%
Goals / Objectives
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.
Project Methods
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.

Progress 01/03/11 to 01/02/16

Outputs
Progress Report 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. 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 project expired in FY 2016 and was replaced by 3091-32000-034-00D which is expanding upon the work of this project. Work during the life of this project developed new insights into individual regulatory control mechanisms of Salmonella and Campylobacter persistence in poultry, and its ability to evade the innate host defenses by way of using genomic (quantitative real time-PCR, microarray analysis), and proteomic (kinome peptide arrays) technologies (Objectives 1,4). Investigations of mechanistically unique components of the leukocyte-mediated innate immunity and genetic control of the functional innate response provided convincing evidence for the potential use of innate immunity in anti- infective therapy through the boosting of innate responses (Objectives 3, 4). Furthermore, project scientists found that recognition of Salmonella by a variety of host immune receptors induced various extracellular activation cascades and intracellular signaling pathways, leading to the inflammatory response, recruitment of immune cells for clearance of the pathogens, and mobilization of professional antigen-presenting cells (Objectives 1,3). The research identified these signaling pathways, their component proteins, and their resultant cytokine profiles in avian intestine following infections with Salmonella. Project research found that the induction of cytokine mRNA transcripts were regulated by transcription factors, and showed that they are required steps in intracellular signaling that result in changes in gene expression (Objectives 2,4). The signal transduction events that lead to the activation of transcription factors and the induction of specific, pathogen-directed immune responses are controlled not at the transcriptional or translational level, but post-translationally via a reversible series of phosphorylation of proteins. Lastly, the role of some bacterial virulence factors and their intestinal colonization and persistence were evaluated (Objectives 3,4). Specifically, it was discovered that the AvrA gene functions similarly in poultry as it does in mammals, but the molecular difference lies in the host response to the release of this protein, where it functions to suppress the local mucosal immune response.

Impacts
(N/A)

Publications

  • Arsenault, R.J., Kogut, M.H. 2015. Immunometabolism and the kinome peptide array: A new perspective and tool for the study of gut health. Frontiers in Veterinary Infectious Diseases. 2:1-5.
  • Kogut, M.H., Arsenault, R.J. 2015. A role for the non-canonical Wnt-�- Catenin and TGF-� signaling pathways in the induction of tolerance during the establishment of a Salmonella enterica serovar Enteritidis persistent cecal infection in chickens. Frontiers in Veterinary Infectious Diseases. 2:1-11.
  • Swaggerty, C.L., McReynolds, J.L., Byrd, J.A., Pevzner, I.Y., Duke, S.E., Genovese, K.J., He, H., Kogut, M.H. 2016. Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens- induced necrotic enteritis. Poultry Science. 95:370-374.
  • Arsenault, R.J., Genovese, K.J., He, H., Wu, H., Neish, A.S., Kogut, M.H. 2016. Wild-type and mutant AvrA- Salmonella induce broadly similar immune pathways in the chicken ceca with key differences in signaling intermediates and inflammation. Poultry Science. 95:354-363.
  • Oakley, B.B., Kogut, M.H. 2016. Spatial and temporal changes in the broiler chicken cecal and fecal microbiomes and correlations of bacterial taxa with cytokine gene expression. Frontiers in Veterinary Infectious Diseases. doi: 10.3389/fvets.2016.00011.


Progress 10/01/14 to 09/30/15

Outputs
Progress Report 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. 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 2015, using genomic and proteomic technologies, this project developed new insights into individual regulatory control mechanisms of Salmonella persistence in poultry and its ability to evade the innate host defenses (Objective 4). Investigations of mechanistically unique components of the leukocyte-mediated innate immunity and genetic control of the functional innate response provided convincing evidence for the potential use of innate immunity in anti-infective therapy through the boosting of innate responses. Furthermore, we found that recognition of Salmonella by a variety of host immune receptors induce various extracellular activation cascades and intracellular signaling pathways, leading to the inflammatory response, recruitment of immune cells for clearance of the pathogens, and mobilization of professional antigen- presenting cells. We identified these signaling pathways, their component proteins, and their resultant cytokine profiles in avian intestine following infections with Salmonella (Objective 1). We found that the induction of cytokine mRNA transcripts are regulated by transcription factors and showed that they are required step(s) in intracellular signaling that result in changes in gene expression. The signal transduction events that lead to the activation of transcription factors and the induction of specific, pathogen-directed immune responses are controlled, not at the transcriptional or translational level, but post-translationally via a reversible series of phosphorylation of proteins (Objective 2). Protein phosphorylation and dephosphorylation are regulated by a balanced activity of protein kinases and protein phosphatases. Thus, phosphorylation acts as a molecular switch, controlling the activities of signaling molecules and downstream target proteins. We evaluated the role of some bacterial virulence factors and their intestinal colonization and persistence. Specifically, we found that the AvrA gene functions similarly in poultry as it does in mammals, but the molecular difference lies in the host response to the release of this protein where it functions specifically to suppress the local mucosal immune response. Lastly, we identified in FY 2015 a diet-based immunotherapy that selectively stimulates protective immune responses as a treatment option for Salmonella. A group of small peptides isolated from a soil bacterium, when provided to neonatal chicks as a feed additive for four days after hatch, significantly decreased intestinal and extra-intestinal colonization by Salmonella Enteritidis (Objective 4). More importantly, feeding the peptide-supplemented diet primed cecal tissue for immune-mediated antibacterial activity without being directly antimicrobial, thus avoiding the development of bacterial resistance. The significance of this work is that the orally delivered cationic peptides stimulate the innate response during the first week after hatch, normally a time of immunologic inefficiency and increased susceptibility to bacterial infections. Significant focus was on the interface between the host immune system and Salmonella, and studies on the interactions between the two. Accomplishments 01 High and low loads of cecal colonization by Salmonella Enteritidis in chickens trigger distinct immune kinome profiles. Salmonella-related infections originating from poultry and/or poultry products are a major cause of human foodborne illness, and S. Enteritidis is the leading cause worldwide. Despite the importance of Salmonella to human health and chickens being a reservoir, little is known of the response to infection within the chicken gastrointestinal tract. ARS researchers at College Station, Texas, used chicken-specific kinome immune peptide arrays to conduct a detailed kinomic analysis of the chicken gut immune response in birds with low and high levels of Salmonella colonization. Birds with lower loads of S. Enteritidis had increased activity in key immunological pathways associated with chemokine signaling, ErbB signaling, Jak-Stat signaling, and MAPK signaling compared to birds that maintained higher loads of S. Enteritidis colonization. Further analysis identified BLNK, Raf1, and AKT3 as specific proteins that may be associated with increased resistance against S. Enteritidis, and could provide poultry breeders with additional biomarkers to identify birds naturally more resistant to this important foodborne pathogen, potentially reducing the need for antibiotics and creating a safer food supply. 02 Novel technology to identify species-specific protein changes during Salmonella infections. Due to a multitude of post-transcriptional regulatory events that occur during and after an infection, there are concerns that descriptions of patterns of gene expression, no matter how comprehensive, do not accurately describe or predict cellular phenotypes. Specifically, a major criticism of genetic approaches is their inability to consider post-transcription regulatory events, such as activation and deactivation of proteins by any number of post- translational modifications. ARS researchers at College Station, Texas, designed, developed, and optimized novel chicken, turkey, and bovine species-specific immune-metabolism kinome peptide arrays which were then used to delineate the physiological changes that occur in host tissues during and following infection with Salmonella. The development of this new technology resulted in the creation of a CRADA with an industry partner for use in identifying new phenotype biomarkers for avian and bovine infectious and metabolic diseases. The implications of this work are that monitoring global responses at the level of cellular kinase activity (the kinome) is an effective approach to understand complex biology, as well as to identify therapeutic targets and biomarkers. This accomplishment advances work to minimize or prevent colonization of poultry by harmful microorganisms.

Impacts
(N/A)

Publications

  • Arsenault, R.J., Trost, B., Kogut, M.H. 2014. A comparison of the chicken and turkey proteomes and phosphoproteomes in the development of poultry- specific immuno-metabolism kinome peptide arrays. Frontiers in Veterinary Infectious Diseases. 1:22. doi: 10.3389/fvets.2014.00022.
  • Daigle, J., Van Wyk, B., Trost, B., Scruten, E., Arsenault, R.J., Kusalik, A., Griebel, P., Napper, S. 2014. Peptide arrays for kinome analysis of livestock species. Frontiers in Veterinary Infectious Diseases. 1:4. doi:0. 3389/fvets.2014.00004.
  • Cohen, N., Bourquin, J., Bordin, A., Kuskie, K., Brake, C., Weaver, K., Liu, M., Felippe, M., Kogut, M.H. 2014. Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals. PLoS One. 9:1-10. doi: 10.137/journal.pone. 0109865.
  • Tellez, G., Latorre, J., Kuttappan, V., Kogut, M.H., Wolfenden, A., Hernandez-Velaso, X., Hargis, B., Bottje, W.G., Bielke, L.R., Faulkner, O. B. 2014. Utilization of rye as energy source affects bacterial translocation, intestinal viscosity, microbiota composition, and bone mineralization in broiler chickens. Frontiers in Nitrigenomics. 5:1-7.
  • Kogut, M.H., Swaggerty, C.L., Chiang, H., Genovese, K.J., He, L.H., Zhou, H., Arsenault, R.J. 2014. Critical role of glycogen synthase kinase-3� in regulating the avian heterophil response to Salmonella enterica serovar Enteritidis. Frontiers in Veterinary Infectious Diseases. 1:Article 10.
  • Kogut, M.H. 2014. Perspectives and research challenges in veterinary infectious diseases. Frontiers in Veterinary Infectious Diseases. 1:Article 21.
  • Latorre, J.D., Hernandez, X., Kogut, M.H., Vicente, J., Wolfenden, R., Wolfenden, A., Hargis, B., Kuttappan, V., Tellez, G. 2014. Role of Bacillus subtilis direct-fed microbial on digesta viscosity, bacterial translocation, and bone mineralization in turkey poults fed with a rye- based diet. Frontiers in Veterinary Infectious Diseases. 1:Article 26.
  • Kapczynski, D.R., Jiang, H., Kogut, M.H. 2014. Characterization of cytokine expression induced by avian influenza virus infection with real- time RT-PCR. In: Spackman, E., editor. Animal Influenza Virus. 2nd edition. New York, NY: Springer. p. 217-233.
  • Swaggerty, C.L., Pevzner, I., Kogut, M.H. 2015. Selection for pro- inflammatory mediators produces chickens more resistant to Eimeria tenella. Poultry Science. 94:37�42.
  • Shanmugasundaram, R., Kogut, M.H., Arsenault, R.J., Swaggerty, C.L., Cole, K., Reddish, M.J., Selvaraj, R.K. 2015. Effect of Salmonella infection on cecal tonsil regulatory T cell properties in chickens. Poultry Science. 94:1828-1835. doi: 10.3382/ps/pev161.
  • Oakley, B.B., Lillehoj, H.S., Kogut, M.H., Kim, W.K., Maurer, J.J., Pedroso, A., Lee, M.D., Collett, S.R., Johnson, T.J., Cox Jr, N.A. 2014. Mini-review of the chicken gastrointestinal microbiome. FEMS Microbiology Letters. 360(2):100-122. doi: 10.1111/1574-6968.12608.


Progress 10/01/13 to 09/30/14

Outputs
Progress Report 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. 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 2014, significant focus was on the interface between the host immune system and Salmonella, and studies on the interactions between the two. We have shown that by combining two unique immune stimulants, which individually induce some protection against Salmonella, a synergistic immune response resulted. We characterized this synergy and found that when combined together, these immune materials activate a totally new signaling pathway that resulted in the induction of a protective immune response (known as Th1 based against Salmonella). Finding such a unique, protective, non-pathogenic signaling pathway may be an important consideration for the induction of pathogen specific immune responses without the use of live vaccines. Work in FY 2014 further established the genetic basis for bird phenotypic and genotypic biomarkers associated with increased resistance to intestinal pathogens without any detrimental effects on performance. Development of such �green� commercial lines of birds will have significant effects in reducing the incidence of human food-poisoning in the U.S. and abroad, and will improve public perception of naturally grown poultry and eggs. Other work in FY 2014 was in response to the gradual loss of effective classical antibiotics for control of bacterial pathogens. Project work identified an adjuvant immunotherapy that selectively stimulates protective immune responses as a treatment option for Salmonella and Campylobacter. These immune peptides, when provided to neonatal chicks as a feed additive for four days after hatch, significantly decreased colonization by Salmonella. More importantly, feeding the peptide-supplemented diet primed cecal tissue for immune-mediated antibacterial activity without the peptides themselves being directly antimicrobial. The work is important because the orally delivered cationic peptides stimulate the innate response during the first week after hatch, normally a time of immunologic inefficiency and increased susceptibility to bacterial infections. Accomplishments 01 Mechanism of persistent Salmonella infection in chickens. Non- typhoidal Salmonella enterica induces an early pro-inflammatory response in chickens. However, the response is short-lived, asymptomatic of clinical disease, results in a persistent colonization of the gastrointestinal (GI) tract, and can transmit infections to na�ve hosts via fecal shedding of bacteria. ARS researchers at College Station, Texas, found that persistent colonization of the ceca of chickens by Salmonella is accompanied by increased expression of anti- immune factors that reduce the local immune response. The tolerogenic response is mediated by an alteration of host signaling pathways that maintain the anti-inflammatory immune responses. Taken together, these results established that a state of immunological tolerance is developed within four days post-infection and is maintained during persistent Salmonella colonization in the gut of chickens. This work provides new and unique insights into the cross-talk between Salmonella and its avian host. It also provides a target time and site for the development of immune control measures to eliminate the pathogen from living birds. 02 Novel technology to identify species-specific protein changes during Salmonella infections. A major criticism of genetic approaches is their inability to consider post-transcription regulatory events such as activation and deactivation of proteins by any number of post- translational modifications. ARS researchers at College Station, Texas, designed, developed, and optimized a novel chicken species-specific immune-metabolism kinome peptide array, to delineate the physiological changes that occur in host tissues during and following infection with Salmonella. Development of this new technology has catalyzed establishment of a CRADA with an industry partner to identify new biomarkers for avian infectious and metabolic diseases and to develop porcine and bovine species-specific kinome arrays. The implications of this work are that monitoring global responses at the level of cellular kinase activity (the kinome) is an effective approach to understand complex biology as well as to identify therapeutic targets and biomarkers.

Impacts
(N/A)

Publications

  • Swaggerty, C.L., Pevzner, I.Y., Kogut, M.H. 2014. Selection for pro- inflammatory mediators yields chickens with increased resistance against Salmonella enterica serovar Enteritidis. Poultry Science. 93:535-544.
  • Kogut, M.H. 2013. The gut microbiota and host innate immunity: Regulators of host metabolism and metablic diseases in poultry? Journal of Applied Poultry Research. 22:637-646.
  • Arsenault, R.J., Kogut, M.H., He, L.H. 2013. Combined CpG and poly I:C stimulation of monocytes results in unique signaling activation not observed with the individual ligands. Cellular Signaling. 25:2246-2254.
  • Kogut, M.H., Genovese, K.J., He, L.H., Swaggerty, C.L., Jiang, Y. 2013. Modulation of chicken intestinal immune gene expression by small cationic peptides as feed additives during the first week posthatch. Clinical and Vaccine Immunology. 20:1440-1448.
  • Lowenthal, J.W., Bean, A.G.D., Kogut, M.H. 2013. What's so special about chicken immunology? Developmental and Comparative Immunology. 41:307-309. doi: 10.1016/j.dci.2013.07.012.
  • Kallapura, G., Kogut, M.H., Morgan, M.J., Pumford, N.R., Bielke, L.R., Wolfenden, A.D., Faulkner, O.B., Latorre, J.D., Merconi, A., Hernandez- Velasco, X., Kuttappam, V.A., Hargis, B.M., Tellez, G. 2014. Fate of Salmonella Senftenberg in broiler chickens evaluated by challenge experiments. Avian Pathology. 17:1-5.


Progress 10/01/12 to 09/30/13

Outputs
Progress Report 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. 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. Accomplishments 01 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. 02 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.

Impacts
(N/A)

Publications

  • 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.


Progress 10/01/11 to 09/30/12

Outputs
Progress Report 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. 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 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. Accomplishments 01 New insights on how Salmonella interacts with chicken immune cells. Foo 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 no 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 kno as a heterophil responded by up-regulating (turning on) genes associated with regulation of cell differentiation, protein transport, macromolecul 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 bir host interact and how these interactions ultimately result in protection versus illness/death. This work has identified specific targets to desi new immune modulatory and/or antimicrobial compounds that can be utilize by the poultry industry to produce microbiologically safer poultry meat products for the consumer. 02 Genetic analysis of Salmonella-resistant and -susceptible chickens. Foo 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 no 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, showe that certain genes in chickens that convey immune protection against Salmonella are more active in the Salmonella-resistant chicken line A th 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.

Impacts
(N/A)

Publications

  • 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.
  • Kogut, M.H., He, L.H., Genovese, K.J. 2011. Bacterial toll-like receptor agonists induce sequential NF-kB-mediated leukotriene B4 and prostaglandin E2 production in chicken heterophils. Veterinary Immunology and Immunopathology. 145:159-170.
  • Kogut, M.H., McReynolds, J., He, L.H., Genovese, K.J., Jesudhasan, P.R., Davidson, M.A., Cepeda, M.A., Pillai, S.D. 2012. Electron-beam irradiation inactivation of Salmonella: Effects on innate immunity and induction of protection against Salmonella enterica serovar Typhimurium challenge of chickens. Procedia in Vaccinology. 6:47-63.
  • Swaggerty, C.L., He, L.H., Genovese, K.J., Duke, S.E., Kogut, M.H. 2012. Loxoribine pretreatment reduces Salmonella enteritidis organ invasion in 1- day-old chickens. Poultry Science. 91:1038-1042.
  • Kogut, M.H., Genovese, K.J., Nerren, J., He, L.H. 2012. Effects of avian triggering receptor expressed on myeloid cells (TREM-A1) activation on heterophil functional activites. Developmental and Comparative Immunology. 36:157-165.
  • Patterson, R., Nerren, J., Kogut, M.H., Court, P., Villareal-Ramos, B., Seyfert, H., Dalby, P., Werling, D. 2011. Yeast-surface expressed BVDV E2 protein induces a Th1/Th2 response in na�ve T cells. Developmental and Comparative Immunology. 37:107-114.
  • Kogut, M.H., Genovese, K.J., He, L.H., Swaggerty, C.L., Jiang, Y. 2011. BT cationic peptides: Small peptides that modulate innate immune responses of chicken heterophils and monocytes. Veterinary Immunology and Immunopathology. 145:151-158.
  • Kogut, M.H., Swaggerty, C.L. 2011. The effects of pre- and probiotics on the host immune response. In: Callaway, T.R., Ricke, S.C., editors. Direct Fed Microbials/Prebiotics for Animals: Science and Mechanisms of Action. New York, NY: Springer Verlag Publishing. p. 61-72.


Progress 10/01/10 to 09/30/11

Outputs
Progress Report 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. 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. Accomplishments 01 Stimulation of innate immunity genes in poultry. Food-poisoning bacteri 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 feedin 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 pathog infection, and thus result in safer poultry meat products reaching the consumer. 02 Genetic analysis of Salmonella-resistant and -susceptible chickens. Foo 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 no 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. T immunologically efficient birds were also significantly more resistant t 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. 03 Protein kinases regulate resistance/susceptibility to food-borne pathoge 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 two genetically distinct lines of chickens (lines A and B), the line A chickens are more resistant to both of these bacterial pathogens than ar the line B chickens. ARS researchers at College Station, TX, hypothesiz that the differences in resistance were due at least partly to differenc in intracellular signaling pathways, and conducted experiments which clearly showed that certain enzymes in the birds (protein kinases) and t 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.

Impacts
(N/A)

Publications