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The objectives of these studies are to characterize the mechanisms of Stx-triggered programmed cell death and to elucidate Stx-2 regulation transcriptionally and translationally within the pathogen. Specifically, it is proposed to define the nature of cell death during different points in the host cell cycle, to ascertain the binding affinities of intracellular Stx with the pro-apoptotic and anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1, Bax and Bak) and create a model pathway for the molecular mechanism of Stx-triggered apoptosis. <P>In addition, we will determine the mechanism by which Stx suppresses normal apoptosis in neutrophils and thus facilitates the delivery of Stx-2 to the kidneys and other organs by prolonged carriage of the toxin in the blood stream within neutrophils. The rates of stx2 gene transcription (Real Time RT-PCR) and Stx-2 production (ELISA) will be compared in order to define discrepancies between gene expression and the quantity of protein produced during organism stress induced by sub-inhibitory concentrations (SIC) of antibiotics, different carbohydrate energy sources and the presence of organic acids.
Non-Technical Summary: Escherichia coli O157:H7 is a potential contaminant of the human food supply. Some strains of the pathogen produce Shiga-like toxins which is a virulence factor responsible for severe, life threatening disease, especially in children. The purpose of this study is to define the mechanisms of cell death induced by the Shiga-like toxin produced by E. coli O157:H7. Gene regulation will also be investigated at the level of transcription and translation. Characterizing these will give a better understanding of disease induction by this pathogen.<P> Approach: Stx positive and Stx negative E. coli O157:H7 strains will be used in these studies (ATCC 43888, Shiga negative; NLEP 95-2157, Stx-1 positive; NLEP 90-2380, Stx-2 positive and ATCC 43895, Stx-1 and Stx-2 positive). Using stx1 and stx2 primers, which we previously published, together with PCR the presence of Stx genes in these strains will be defined. Toxin will be purified using size exclusion filtration techniques and Centricon concentration. The toxin will be further purified by elution on an immunoaffinity column using Stx-2 antibody bound to protein G beads. Native- and SDS-PAGE will be performed to ensure the identity of the toxin and this will be confirmed by Western blot. Further purification of the Stx-2 A1 fragment will be done by binding the toxin to Gb3 followed by trypsin or furin (the natural substrate) to cleave the A subunit which will be confirmed for purity by SDS-PAGE. Three host cell types, Vero, Caco-2 and human neutrophils will be used in these investigations. Vero cells remain the cells of choice for studies on Shiga-like toxins (also known as Verotoxins) and these will be used to define the activity of Stx throughout the experimental procedures. Caco-2 cells will serve as the source of the Bcl-2 proteins and will be used in protein interaction and affinity binding assays using far Western blot analysis. Human neutrophils will be exposed to wild-type and mutated Stx2 toxin to investigate the rate of apoptosis/anti-apoptosis and to define the role these play in disease development and outcome. To achieve this, wild-type toxin will be mutated from the amino acid sequence NWGRI to NWARI resulting in a non-dimerizing toxin. Cell cycle studies will be conducted to determine if the stage in cell development (G and S-phases) at which exposure to Stx occurs affects the outcome, i.e. apoptosis versus necrosis, and these will be measured using a cell death detection Elisa kit. Cell cycle determinations will be made by FACS coupled with Modfit software. Induction of Stx will be investigated by using the pathogen in both exponential and stationary phases of growth and in the presence of (i) low doses of antibiotics, (ii) glucose and glycerol as sole carbon sources and (iii) acetic acid or lactic acid. The use of these criteria will define the impact of (i) stress on the organism as well as (ii) the lysogeny or lysis of the toxin-carrying phage and (iii) the result of probiotics on toxin synthesis. The data will be quantified using the MTT assay to define cytotoxic effects on host cell viability, real time RT-PCR to determine the rates of stx2 gene transcription and by ELISA to quantify Stx-2 production translationally.