Submitted to: Mammalian Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2005
Publication Date: 2/13/2006
Citation: Zhao, S., Kuhar, D.J., Lunney, J.K., Dawson, H.D., Guidry, C.A., Uthe, J., Bearson, S., Recknor, J., Nettleton, D., Tuggle, C.K. 2006. Gene expression profiling in salmonella choleraesuis infected porcine lung using a long oligonucleotide microarray. Mammalian Genomics. 17:777-789. Interpretive Summary: Our studies are aimed at preventing food safety related infections. To do we must understand how the host reacts to the infection and controls its spread within the body. Thus lung tissues were collected from control pigs and at 24 hr and 48 hr after Salmonella enterica serovar Choleraesuis infection and RNA prepared and analyzed using microarrays and real-time gene expression assays. To determine the breath of the systemic transcriptional response to Salmonella infection, the first-generation, long oligonucleotide Qiagen-NRSP8 swine array was used to analyze RNA samples from the porcine lung tissue. This microarray, representing 13,297 cDNAs and ESTs, was designed by Qiagen-Operon, Inc. in cooperation with NRSP8 swine genome scientists. Arrays were hybridized with fluorescently labeled RNA/cDNA “targets” produced from lung tissues from Salmonella Choleraesuis infected pigs. Results showed that 57 genes showed differential expression (p < 0.001), and 1,300 genes at p < 0.05 level, on the array. Real-time gene expression assays (Q-PCR) results confirmed statistically differential expression for 23 of 33 genes compared genes, and correlated changes in another 17. Specifically, these data demonstrated that protective, interferon-gamma dominated, T-helper 1 responses were activated in the Salmonella infected lung. These responses are required to control lung pathology as well as prevent further spread of the infection to other pigs. The data revealed substantial changes in cell death, or apoptotic genes, as well as inflammatory pathways in infected lung tissue. This data now gives researchers a broader array of targets for controlling Salmonella infections, and thus should lead to improved pork safety. Swine researchers have had limited comprehensive genomic tools to assess physiologic and disease responses. This manuscript is among the first to describe the use of the Qiagen-NRSP8 long oligonucleotide array for porcine transcriptional profiling during an infectious disease response. Probing the transcriptional response to pathogenic bacterial infection within food animals is of fundamental interest and should result in improved methods to detect diseased animals, control spread of the infection, and lead to improved food safety.
Technical Abstract: Understanding the transcriptional response to pathogenic bacterial infection within food animals is of fundamental interest and should result in improved methods to detect diseased animals and improve food safety. To determine the systemic transcriptional response to Salmonella infection, the long oligonucleotide Qiagen-NRSP-8 swine array was used to analyze RNA samples from control, 24 hour (hr), and 48 hr Salmonella enterica serovar Choleraesuis infected porcine lung tissue. In total, 1,300 genes showed differential expression at p < 0.05 level (false discovery rate, FDR = 27 %), while 57 genes showed differential expression at p < 0.001 level (FDR = 12 %). Quantitative real-time PCR (Q-PCR) of 62 genes was used to confirm the microarray results and to identify pathways responding to infection. Of the 33 genes identified by microarray analysis as differentially expressed, statistical analysis of Q-PCR results confirmed 23 genes. A novel finding was that two protein-glutamine glutamyltransferase family genes (TGM1 and TGM3) showed dramatic increases in expression post-infection; combined with several other apoptotic genes, they indicated the induction of apoptotic pathways with S. Choleraesuis infection. A predominant T helper 1 type immune response occurred during infection, with IFNG significantly increased at 48 hpi. Genes known to be induced by IFNs, such as guanylate binding proteins (GBP1, GBP2), acute phase response genes (C1S, C1R), and antigen processing genes (MHC2TA, PSMB8, TAP1, TAP2) showed increased expression during porcine lung infection. This data is the first thorough investigation of gene regulation pathways that control an important porcine respiratory and foodborne bacterial infection.