Submitted to: Iowa State University, Ames, Thesis
Publication Type: Other
Publication Acceptance Date: 4/22/2005
Publication Date: 5/7/2005
Citation: Winter, K. 2005. Identification of Shiga toxin binding sites in porcine tissues and to porcine leukocytes [M.S. Thesis]. Ames, IA: Iowa State University. 94 p.
Interpretive Summary: Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 cause severe diarrhea and sometimes kidney failure and death in humans. Pigs are susceptible to both natural and experimental infection with STEC, and are useful models for studying how STEC cause disease. Exposure to Stxs is toxic for cultured cells and causes damage to blood vessels, paralysis, and death in experimental animals. However, the mechanisms of Stx-mediated damage are not understood. The two studies presented in this thesis focused on the development and use of in vitro methods to identify where Stx binds to isolated porcine tissues and white blood cells. In the first study, an immunohistochemical Stx overlay assay was used to identify Stx binding sites in porcine tissues obtained from small and large intestines, liver, kidney, and brains of neonatal pigs and white blood cells obtained from 3- to 6-week-old pigs. Both Stx1 and Stx2 bound to all tissues tested and to white cells isolated from the lungs (macrophages) or peripheral blood (granulocytes and mononuclear cells). Globotriaosylceramide (Gb3), a receptor for Stx, was identified by immunoassay in all of the tissues and cells where Stx bound. In the second study, a flow cytometric assay was used to show that Stx binds to porcine PMN isolated from peripheral blood. Similar Stx binding to human PMN from peripheral blood has led to the hypothesis that human PMN (which lack Gb3) are involved in transport of Stx to target tissues (which contain Gb3). In contrast to human PMN, porcine PMN contain Gb3. We showed that crude Stx preparations (which can be readily prepared from broth cultures of STEC strains without time-consuming or expensive purification procedures) are suitable alternatives to purified Stx for Stx binding studies. The use of porcine peripheral blood PMN and crude Stx preparations in the flow cytometric Stx binding assays will facilitate studies to determine the role of PMN in the pathogenesis of STEC disease in pigs and in humans.
Technical Abstract: Shiga toxin (Stx)-producing Escherichia coli (STEC) cause disease in humans and pigs. The pathogenesis of STEC disease depends on the production of a number of different virulence factors by the bacteria, including Stxs. All STEC strains produce one or more Stx types (Stx1, Stx2), important virulence factors that mediate systemic STEC disease. Stxs are cytotoxic for vascular endothelial and renal epithelial cells and cause lesions in the kidney, colon, and central nervous system. Human monocytes and polymorphonuclear leukocytes (PMN) also bind Stxs, but there is limited information regarding the roles these leukocytes may play in Stx-mediated damage. The mechanisms of Stx-mediated damage remain to be fully elucidated. The identification of tissues and cell types to which Stxs bind will facilitate elucidation of the mechanisms of Stx-mediated tissue damage. In this thesis, we used tissues isolated from pigs, a natural host for systemic STEC disease, to identify Stx binding sites. The objectives of the first study in this thesis were to (i) identify Stx1 and Stx2 binding sites in multiple porcine tissues, and (ii) determine if Stxs bind to isolated porcine leukocytes. An immunohistochemical Stx overlay assay that was previously used for the identification of Stx binding sites in bovine tissues was modified for use with porcine tissues and leukocytes. Stx binding sites were identified in all tissue types tested (i.e., kidney, brain, intestine, and liver obtained from neonatal pigs), and we demonstrated Stx binding to fixed porcine alveolar macrophages and peripheral blood monocytes and PMN. In the second study in this thesis, a flow cytometric assay to monitor Stx binding to isolated porcine peripheral blood PMN was developed. Stx2 was used as a representative Stx type. The resulting assay is amenable for use in functional studies with isolated porcine PMN.