Abstract
Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway.
Highlights
Despite extensive efforts to understand and prevent Salmonella infection, the number of people infected with Salmonellae each year has not changed appreciably in the United States over the last two decades [1]
Microarray Results Validation Ten selected bovine genes with differential expression by microarray results were analyzed by quantitative RT-PCR
Prior to analyzing gene expression, bacterial invasion and uptake by M cells and phagocytes were assessed by bacterial counts measuring tissue-associated bacteria and analyzing morphologic changes in the tissue (Figure 1 and data not shown)
Summary
Despite extensive efforts to understand and prevent Salmonella infection, the number of people infected with Salmonellae each year has not changed appreciably in the United States over the last two decades [1]. 1.4 million people in the United States are infected each year with Salmonellae. This infection is devastating to children and immunocompromised adults. A recent study estimates that there are annually 93.8 million cases of Salmonella gastroenteritis worldwide, resulting in 155,000 deaths [3]. In immunocompromised individuals, Salmonellae penetrate the intestinal mucosa and enter the bloodstream leading to systemic infection. Understanding the acute phase of intestinal infection is important to designing intervention strategies for children and immunocompromised adults where CD4+ T cell help, which is required to prevent disseminated infection, may be diminished or absent
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