Role for a Phage Promoter in Shiga Toxin 2 Expression from a Pathogenic Escherichia coli Strain

Abstract

Shiga toxins (Stxs), encoded by the stxA and stxB genes, are important contributors to the virulence of Escherichia coli O157:H7 and other Stx-producing E. coli (STEC) strains. The stxA and stxB genes in STEC strains are located on the genomes of resident prophages of the family immediately downstream of the phage late promoters (p R ). The phage-encoded Q proteins modify RNA polymerase initiating transcription at the cognate p R promoter which creates transcription complexes that transcend a transcription terminator immediately downstream of p R as well as terminator kilobases distal to p R . To test if this Q-directed processive transcription plays a role in stx 2 AB expression, we constructed a mutant prophage in an O157:H7 clinical isolate from which p R and part of Q were deleted but which has an intact pStx, the previously described stx 2 AB-associated promoter. We report that production of significant levels of Stx2 in this O157:H7 isolate depends on the p R promoter. Since transcription initiating at p R ultimately requires activation of the phage lytic cascade, expression of stx 2 AB in STEC depends primarily on prophage induction. By showing this central role for the prophage in stx 2 AB expression, our findings contradict the prevailing assumption that phages serve merely as agents for virulence gene transfer.Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) strains are responsible for outbreaks and sporadic cases of diarrhea. In some patients, exposure to STEC leads to hemorrhagic colitis and hemolytic uremic syndrome that may lead to death (20). Two major classes of Stxs, Stx1 and Stx2, encoded respectively by stx 1 AB and stx 2 AB, have been identified in STEC (2). The severe clinical consequences of STEC infections are thought to be caused by the activities of Stxs, although Stx2 appears to be more closely associated with these sequelae than does Stx1 (7,28,36). Shiga toxins are of the A-B type, with the glycolipid-binding B subunits being involved in the transport of the enzymatic A subunits into the eukaryotic cell where the A subunit, acting as a glycosylase, catalyzes a cleavage at a unique site in the 28S rRNA (2). The resulting inactivation of the ribosome leads to an inhibition of protein synthesis. More than 60 serotypes of STEC have been associated with human disease (1). The stx genes of many, if not all, STEC strains are in the genomes of prophages of the lambdoid family (19,23,27). This fact probably accounts for the wide dissemination of these genes in diverse E. coli serotypes.Comparison of lambdoid phage genomes (9) has revealed a common arrangement of functionally similar genes and a shared strategy governing gene expression (Fig. 1). In the lysogenic state, the repressor silences transcription of most phage genes (30). Removal of repression, which can occur when DNA damage activates the bacterial SOS response causing RecA-mediated cleavage of the repressor (21), leads to a cascade of regulatory events beginning with expression of the N transcription ant...

Shiga toxins and stx phages: highly diverse entities

“…Oxidative stress has been suggested as one of the conditions that may occur in the intestine of an infected human and could influence the induction of stx phages (Łoś et al, 2010). This idea is supported by in vitro experiments that show that hydrogen peroxide and neutrophils increase Stx2 production (Wagner et al, 2001), and by the fact that stx phages are induced in cultures of STEC strains treated with hydrogen peroxide (Łoś et al, 2009, 2010). However, human microbiota and their secreted products can inhibit Stx production (Gamage et al, 2003(Gamage et al, , 2006de Sablet et al, 2009).…”

“…This switch from the lysogenic state to the lytic state is called induction. Thus, expression of stx in STEC depends primarily on prophage induction (Wagner et al, 2001;Tyler et al, 2013), although stx 1 transcription can be also driven by its own promoter under low iron conditions (Calderwood & Mekalanos, 1987;Aertsen et al, 2005b).…”

“…Such factors include hydrogen peroxide (Wagner et al, 2001;Łoś et al, 2009, 2010, high temperature in combination with UV irradiation (Yue et al, 2012), EDTA (Imamovic & Muniesa, 2012), sodium citrate (Imamovic & Muniesa, 2012;Nejman-Faleń czyk et al, 2012), amino acid starvation (Nejman-Faleń czyk et al, 2012), phenethyl isothiocyanate (Nowicki et al, 2014), DNase colicins (Toshima et al, 2007), high hydrostatic pressure (Aertsen et al, 2005a), sodium chloride (Łoś et al, 2009;Harris et al, 2012), nitric oxide (Vareille et al, 2007), 60 Co irradiation (Yamamoto et al, 2003) Łoś et al, 2009;Nassar et al, 2013), as well as those antibacterials used as growth promoters in animal production (Köhler et al, 2000).…”

The players in a mutualistic symbiosis: Insects, bacteria, viruses, and virulence genes

“…In Shiga toxin-producing E. coli, the amplification of stx copy number, the regulation of stx transcription, and the delivery of Shiga toxin to host tissues are all achieved through phage-mediated processes, which culminate in the lysis of a subset of the bacterial cells (49)(50)(51). It has been proposed that the phage persist as mutualists of their bacterial hosts, as lysis of some bacteria improves the environment for the surviving bacterial cells, which retain lysogenic phage (50).…”

Cumulative effect of prophage burden on Shiga toxin production in Escherichia coli

“…The direct impact of Stx has been established, and its presence undoubtedly increases severe disease outcomes (Ashkenazi et al, 1990). Bacteriophage-encoded Stxs are usually co-transcribed with the phage late genes, which include many of the components required for assembly, packaging and release of infective phage particles from the host cell (Brüssow et al, 2004;Herold et al, 2005;Neely & Friedman, 1998;pR9 and the phage-mediated lytic cascade (Wagner et al, 2001(Wagner et al, , 2002. In a lysogen culture, Stx can be found at detectable levels in the medium, but this is primarily due to the relatively high level of background spontaneous lytic pathway induction (Shimizu et al, 2009;Wagner et al, 2002).…”