Submitted to: Methods in Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/18/2009
Publication Date: 6/17/2011
Publication URL: http://DOI: 10.1007/978-1-61779-102-4_6
Citation: Quinones, B., Swimley, M.S. 2011. A Vero Cell Based Fluorescence Assay to Assess Relative Toxicities of Shiga Toxin 2 Subtypes from Escherichia coli. Methods in Molecular Biology. 739(1): 61-71. Interpretive Summary: The enteric pathogen Shiga toxin-producing Escherichia coli (STEC) is known to cause human gastrointestinal illnesses ranging from bloody diarrhea and hemorrhagic colitis to the life-threatening hemolytic-uremic syndrome (HUS). The most commonly reported STEC serotype associated with large outbreaks and the development of HUS in North America is serotype O157:H7. Recently, findings from molecular typing studies have demonstrated that there is a prevalence of disease caused by other non-O157 serotypes, such as O26:H11, O103:H2, O111:H-, O121:H19, O145:H- in Europe and parts of Latin America, demonstrating that certain non-O157 STEC strains are potentially as virulent as O157:H7 strains. One of the best characterized virulence factors in STEC pathogenicity and a determinant thought to be responsible for causing HUS are Shiga toxins (Stxs). In contrast to Stx1, the Stx2 group is composed of diverse and heterogeneous group of subtypes. In addition to Stx2, different subtypes have been identified in STEC strains implicated in causing human illnesses; these subtypes are Stx2c (Stx2v-a), Stx2d (Stxd-OX3a and Stx2d-Ount), mucus-activatable Stx2d (Stx2dactivatable) (Stx2vh-a and Stx2vh-b), Stx2e, and Stx2f (10-14). Sequence analyses have shown that these Stx2 subtypes have a high sequence similarity to Stx2, and it is thought that these sequence variations may affect the ability of a particular strain to cause disease. Molecular typing studies have demonstrated that there is a strong correlation between STEC strains harboring certain stx2 subtypes and severe disease outcomes such as bloody diarrhea and HUS. Recent evidence has shown that the Stx2 subtypes appear to have different toxicities in cultured mammalian cells. One factor that may determine the cytotoxic response of the Stx2 subtype is the receptor binding specificity on the target cells. The glycolipid globotriosylceramide (Gb3) is the functional receptor for Stx2 and Stx2 subtypes. Although globotetraosylceramide (Gb4) was shown to be the preferred receptor for Stx2e, Gb3 can substitute for Gb4 in mediating the cytotoxic response of Stx2e. In addition to receptor binding specificities, the amounts of Stx2 produced may define the severity of disease caused by STEC strains. The differential expression and induction of the Stx2 subtypes appears to contribute to the relative virulence of the STEC strain. To examine the relative toxicities of Stx2 and Stx2 subtypes, the Vero-d2EGFP fluorescence assay was employed. The assay uses the Vero-d2EGFP cell line, generated from Vero cells to constitutively express a destabilized variant of the enhanced green fluorescent protein (EGFP). The short, in vivo half-life of this EGFP variant makes it a sensitive marker for measuring the inhibition of protein synthesis by Stx. Vero cells are a suitable and sensitive system to examine Stx cytotoxicity since these cells contain large amounts of both Gb3 and Gb4 glycolipid, receptors used by Stx2 and Stx2 subtypes. Thus, the Vero-d2EGFP fluorescence assay is a simple, highly sensitive, and quantitative method to examine the relative cytotoxicities of STEC strains expressing Stx2 and Stx2 subtypes.
Technical Abstract: Shiga toxin-producing Escherichia coli is a leading cause worldwide of human gastroenteritis from food and waterborne sources. Shiga toxins 1 and 2 are important virulence factors linked to severe human illness. In particular, Shiga toxin 2 is composed of a diverse and heterogeneous group of subtypes with differential receptor binding interactions and cytotoxicities in mammalian cells. In this report, we describe the use of the Vero-d2EGFP fluorescence assay to examine the relative toxicities of Stx2 subtypes expressed by strains of Shiga toxin-producing Escherichia coli.