Comparison of Shiga-Like Toxin H Expression Genetically Diverse Lineages of Escherichia

The existence of two separate lineages of Escherichia coli 0157:H7 has previously been reported, and research indicates that one of these lineages (lineage 1) might be more pathogenic toward human hosts. We postulated that the lineage more pathogenic expresses higher levels of Shiga toxin 2 (Stx2) than do the nonpathogenic lineage II. A comprehensive set of methodologies were used to investigate the difference in Stx2 protein and mRNA expression between the two lineages. An initial Stx2-specific enzyme-linked immunosorbent assay was conducted, and lineage I overall demonstrated significantly more toxin proteins expressed (P < 0.01). Gene expression analyses all showed significantly higher stx2 gene expression in lineage I (P = 0.02). PCR mapping revealed a possible explanation for decreased amounts of six2 transcripts in the potentially nonpathogenic lineage II isolates, suggesting that genomic changes have modified the toxin-encoding region of the phage. This study provides additional data to support the existence of two diverse lineages of E. coli 0157:H7, one of which may have lower pathogenic potential in relation to human hosts. The PCR described also provides a possible screening tool for E. coli 0157 populations to differentiate these lineages. This study provides useful information on the ecology of E. coli 0157, with broad implications within the clinical, scientific, and livestock industries. Escherichia co/i 0157:1-17 isolates are associated with diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (21) and are noted for their ability to produce Shiga toxin 2 (Stx2). Although Stx2 plays an important role in the virulence of enterohemorrhagic E. co/i (20) and is the focus of this study, it should be noted that there are also other virulence factors including those involved in intimate adherence (8, 9, 15, 17). Stx2 is composed of a single Stx2a subunit and five smaller Stx2b subunits (4, 5, 11, 18, 25). The genes encoding Stx2 are thought to be located solely within lambdoid-like prophages, and these phages are all thought to share similar means of genome organization and regulation (3, 10, 12, 13, 19, 24). Transcription of six,genes are thought to be a result of phage induction through activation of the bacterial SOS response. In short, phage induction results in Q modification, which initiates at PR' and transcends rR' (23), resulting in stx2 expression during late stages of phage production. Statistical analyses has correlated disease occurrence with E. co/i isolates that express the stx2 gene (2, 6). Previously, octamer-based genome scanning of enterohemorrhagic E. co/i serotype 0157:1-17 isolates revealed the potential existence of two genetically distinct lineages, which grouped primarily bovine isolates into lineage II, while human clinical isolates fell into lineage I (14). Subsequent research has also suggested that one lineage may be less pathogenic or have less potential for transmission * Author for correspondence. Tel: 806-746F\t I??. 1:s\, 806-74O28: E-naII : sdowd9 hkars.usda.gO\ to humans (7). Forty 0157:H7 isolates were studied, which included 20 lineage I and 20 lineage II isolates. The purpose of this study was to compare both stx 2 transcription and protein expression levels between these representatives of the two lineages. We hypothesized that lineage I, containing the majority of human 0157:1-17 clinical isolates would produce significantly more stx2 transcript and toxin when compared with lineage II, which contained primarily isolates derived from bovine sources. MATERIALS AND METHODS Bacterial isolates. Twenty lineage I isolates and 20 lineage II isolates were kindly provided by Dr. Andrew Benson (University of Nebraska). Of these 40 isolates, three of the lineage II isolates did not possess the six, gene based on initial screening using reverse transcriptase PCR (RT-PCR). These 3 isolates were excluded from enzyme-linked immunosorbent assay (ELISA) calculations and the 10 isolates chosen from lineage II. One of the isolates had low amplification efficiency, using the primers in this study. This isolate was also excluded. Stx2-specific ELISA. Twenty lineage I isolates and 20 lineage 11 isolates were cultured to log phase in LuriaBertani broth, their optical density normalized (0D 600 0.35), and then centrifuged at 3,000 X g for 10 mm. One milliliter of supernatant was transferred to a fresh 1.5-ml microcentfifuge tube. Triton X-lOO was added to a final concentration of 0.1% and vortexed briefly to disrupt Stx2containing membrane vesicles (27). A custom Stx2-specific ELISA (SafePath Laboratories. Carlsbad, Calif.) was conducted, following the protocol recommended by the manufacturer. A hichromatic reading was taken with the measuremen t and cl ci ciscc ldtci ct at ittid ( ni p . repecticlY All aa