ABSTRACT

1. TOTAL NUMBER OF SIGNATURES 41294 139 576 23 SIGNIFICANTSIGNATURES 26821 NON-SIGNIFICANTSIGNATURES 14473 0 540 LB negative control Wild type sipA sopABDE2 1501 RELIABLE SIGNIFICANT 24596 UNRELIABLE SIGNIFICANT 2225 RELIABLE NONSIGNIFICANT 5987 UNRELIABLE NONSIGNIFICANT 8486 Overview of MPSS methodology2 Expression Level of Individual Genes as Calculated by MPSS 100 HITGENOME 12690 NO HIT TO GENOME 11906 HITGENOME 338 NO HIT TO GENOME 1887 HITGENOME 2038 NO HIT TO GENOME 3949 HITGENOME 1941 NO HIT TO GENOME 6545 10 Differentially down regulated Signatures Fold change as compared to control Differentially up regulated Signatures 1 C-C-L8 IL-1Ra NOS Angiotensin Claudin Legumain R1 0.1 443 Wild Type Wild Type Mutant Mutant 1501 128 184 Expression Level of Individual Genes by Real-Time-PCR LB LB 100 WT WT 28 10 0 800 Fold Change as compared to control (normalized against GAPDH) 1 C-C-L8 IL-1Ra NOS Angiotensin Claudin Legumain R1 MT MT 511 0.1 Signatures passed via significant and reliable filter Overview Total RNA samples were collected from bovine ligated ileal loops inoculated with Luria-Bertani broth, Wild Type S. Typhimurium, or an isogenic sipA, sopABDE2 mutant at seven time points (15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 12 hours) from the same four animals that were used for the MPSS analysis. Bovine microarrays were hybridized with cDNA generated from this RNA. Analysis of the gene expression profile from these samples is currently ongoing. Perinatal Calf Ileal Loop Model Fluid Accumulation Bacteriology Electron Microscopy Histopathology RNA Extraction LB control 30 min WT 30 min MT 30 min * * Excision Time 0 Process Bovine Gene Expression in Acute Salmonella enerica Serovar Typhimurium Infection Analyzed by MPSS and Microarray.Sara D. Lawhon1, Sangeeta Khare1, Carlos A. Rossetti1, Cristi L. Galindo4, Robin E. Everts3 , Josely F. Figueiredo1, Jairo E. S. Nunes1, Tamara Gull1, Christen Haudenschild6, George S. Davidson5, Harold R. Garner4, Ken Drake7, Harris A. Lewin3, Andreas J. Bäumler2, and L. Garry Adams11 Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467; 2 Department of Medical Microbiology and Immunology, College of Medicine, University of California at Davis, CA 95616-8645; 3 Department of Animal Sciences, University of Illinois, Urbana, IL, 61801; 4 Eugene McDermott Center for Human Growth and Development, The University of Texas Southwestern Medical School, Dallas, TX 75390-8591; 5 Sandia National Laboratories, Computation, Computers and Mathematics Center, Albuquerque, NM 87123; 6 Solexa, Inc., Hayward, CA 94545; 7 Serologix, Austin, TX 78730 ABSTRACT Salmonella enterica Serovar Typhimurium (S. Typhimurium) is the leading cause of death in humans due to foodborne illness in the United States. Infection in humans and young calves is characterized by polymorphonuclear cell influx to the intestinal mucosa and diarrhea. The clinical similarities between human and bovine infection make the calf an ideal model for human salmonellosis. Previous studies have shown that fluid secretion caused by S. Typhimurium is caused by effector proteins secreted by the Type III Secretion System (T3SS), specifically SipA, SopA, SopB, SopD, and SopE2. Loss of the genes encoding these effectors reduces not only fluid secretion in bovine ligated ileal loops but also Interleukin 8 (IL-8) gene expression. The objective of this study was to create a profile of host gene expression during acute infection with wild type S. Typhimurium and also an isogenic sipA sopABDE2 mutant. This was accomplished using microarray analysis and Massively Parallel Signature Sequencing (MPSS). A microarray time-course study was performed by collecting and analyzing samples from bovine ligated ileal loops over seven time points, 15 minutes, 30 minutes, 1 hour, 2, 4, 8, and 12 hours. MPSS analysis measures signatures from individual mRNA sequences. MPSS was performed on pooled samples from 1 hour, 2 hours, and 4 hours and identified 5,460 unique signatures. Signatures included those for genes previously identified in host response to Salmonella such as chemokine (C-C motif and C-X-C motif) ligand, tumor necrosis factor receptor superfamily (member A1 and 5), prostaglandin E synthase, lymphotoxin- and fibronectin 1 as well as signatures for genes with previously unrecognized roles in host response to Salmonella infection including dipeptidylpeptidase IV (adenosine deaminase complexing protein-2), angiotensin receptor 1, angiotensin II receptor-associated protein, thyroid receptor interacting protein, and adhesion molecule syndecan-4. MPSS also revealed evidence of extensive antisense transcript expression. Signatures identified only in the wild type inoculated loops may serve as potential biomarkers specific for early infection. AIM OF THE STUDY To identify host responses to Salmonella typhimurium wild type at the transcriptome level and to compare the responses of the host in the absence of key virulence genes, sipAsopABDE2 Antisense transcripts: regulation of gene expression due to antisense nature INTRODUCTION Invasion of the intestinal epithelium by Salmonella requires a type III secretion system (T3SS) encoded in Salmonella Pathogenicity Island 1 (SPI-1) to translocate effector proteins into host epithelial cells. Effector proteins, SipB and SipC, together form a pore in the host cell membrane through which additional effector proteins are translocated into the host cell cytoplasm. The functions of some of these proteins are as follow: Our lab has previously shown1 these five effectors (SipA, SopA, SopB, SopD, and SopE/E2), act in concert to induce inflammatory changes and fluid secretion in the bovine ligated ileal loop model of S. typhimurium infection. We employed gene expression profiling by massively parallel signature sequencing (MPSS) to evaluate global alterations in in vivo gene expression of the bovine ileal Peyer’s patch caused by infection with wild type S. typhimurium strain IR715 (WT) and compared these changes to those elicited by a sipAsopABDE2 mutant (ZA21) and also by LB broth. MPSS captures and identifies transcript sequences and analyzes the level of expression of virtually all genes expressed in a sample by counting the number of individual mRNA molecules derived from a geneto the level of only a few transcripts present per one million transcripts reported as transcripts per million (TPM). Transcripts present in S.typhimurium infected group only (POTENTIAL BIOMARKERS) Classification of signatures Bovine Microarray Analysis Signatures present in one experimental group CONCLUSIONS *Several unique transcripts were identified in the absence of virulence factorsthus predicting novel functions of the five SPI-1 T3SS translocated proteins. *Several biomarkers were discovered that were solely present in only one experimental group. *MPSS-generated data also provided valuable insight of natural antisense genes during the early phase of infection that had not been identified by other genomics tools. • REFERENCES: • Zhang, S., Santos, R. L., Tsolis, R. M., Stender, S., Hardt, W. D., Bäumler, A. J. & Adams, L. G. (2002) Infect Immun 70, 3843-55. • Brenner, S., Johnson, M., Bridgham, J., Golda, G., Lloyd, D. H., Johnson, D., Luo, S., McCurdy, S., Foy, M., Ewan, M., Roth, R., George, D., Eletr, S., Albrecht, G., Vermaas, E., Williams, S. R., Moon, K., Burcham, T., Pallas, M., DuBridge, R. B., Kirchner, J., Fearon, K., Mao, J. & Corcoran, K. (2000) Nat Biotechnol 18, 630-4. Acknowledgements: This work was supported by a Texas A&M University, Life Sciences Task Force grant.