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Title:   Author  item
                                            He, Louisitem
                                            Genovese, Kenneth

                                                         - *Ken*item
                                            Swaggerty, Christina

                                                         - *Christi*item
                                            Nisbet, Daviditem
                                            Kogut, Michael

                                                         - *Mike*
  Submitted to: PLOS ONE
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 2/7/2013
                                    Publication  Date: 3/18/2013
                                    Citation: 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.Interpretive Summary: Salmonella enterica serovar Enteritidis is one of the most common Salmonella species in poultry and is often associated with human salmonellosis.  In light of the emergence of increased resistance to commonly used antibiotics in Salmonella, development of new antibacterial agents is urgently needed.   Macrophage cells are one of the white blood cells that can produce chemicals to kill bacteria.  However, S. Enteritidis is able to inhibit the production of antibacterial substances, such as nitric oxide (NO), and survives within the chicken macrophage cells.  In this study, we have developed a screening tool using NO as a biomarker to identify chemicals with antibacterial activity against Salmonella inside the chicken macrophage cells.  Using this tool, we have tested a group of pharmaceutical compounds and found that one of the compounds, H-89, was able to kill S. Enteritidis within the macrophage cells and reversed the inhibition on NO production.  Additionally, H-89 was found to inhibit the bacterial growth in the culture medium.  This information is important to the pharmaceutical and poultry industries in the United States because it offers a potential new tool to control Salmonella in poultry.
                                Technical Abstract: Salmonella enterica serovar Enteritidis is one of the most prevalent Salmonella serovars in poultry and is often associated with human salmonellosis.  S. Enteritidis is known to suppress nitric oxide (NO) production in infected chicken macrophage HD11 cells, while dead S. Enteritidis stimulate a high level of NO production, suggesting a bacterial inhibitory effect on NO production.  Based on this observation, the present study was conducted to evaluate whether the NO production in S. Enteritidis-infected HD11 cells can be used as a biomarker to identify molecules that kill intracellular Salmonella.  Since Salmonella are known to manipulate the host cell kinase network to facilitate intracellular survival, we screened a group of pharmaceutical inhibitors of various kinases to test our hypothesis.  Among them, H-89, a protein kinase A inhibitor, was found to reverse the suppression of NO production in S. Enteritidis-infected HD11 cells.  Production of NO in S. Enteritidis-infected HD11 cells increased significantly when the cells were treated with H-89 at or above 20 μM.  Inversely, the numbers of viable intracellular Salmonella decreased significantly in the cells treated with H-89 at or above 30 μM.  Furthermore, the growth rate of S. Enteritidis in culture was significantly inhibited by H-89 at concentrations from 20 to 100 μM.  Our results demonstrate that NO-based screening using S. Enteritidis-infected HD11 cells is a viable tool to identify chemicals with anti-intracellular Salmonella activity.  Using this method, we have identified that H-89 has a bacteriostatic property against Salmonella which was not previously known.


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