Analysis of T3S and LEE1 expression in <i>E. coli</i> with and without integrated Stx prophages.

<p>(A) Western blot analysis of paired <i>E. coli</i> O157 and O26 strains. EspD was detected from bacterial supernatants and EscJ and RecA from whole cell samples prepared as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s4" target="_blank">Materials and Methods</a>. EDL is the sequenced EDL933 strain and this was originally compared with a published strain TUV93-0 (lane 4) that has lost both Stx1- and Stx2-encoding prophages. This strain demonstrates higher secretion levels and EscJ expression. The Stx2-encoding prophage and the Stx1-encoding prophage were deleted from EDL933 (lanes 2 and 3 respectively) leading to increased T3S. A marked Stx2 phage <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Dahan1" target="_blank">[67]</a> was conjugated into TUV93-0 expressing a cloned <i>cI</i> from Sakai Sp5 (lane 5) and this resulted in a reduction in T3S and EscJ expression. The final two lanes contain samples from a published isogenic pair of EHEC O26 strains <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Mellmann1" target="_blank">[47]</a> from which one has lost the Stx2 phage leading to a marked increase in T3S. (B–C) To quantify differences in T3S expression, the same strain sets as in part A, were transformed with a LEE1-GFP construct and fluorescence measured throughout the growth curve. This was repeated three times with one experiment shown in (B). A minimum of three values were determined from the expression curves for OD₆₀₀ = 0.9 and plotted in (C) as mean and 95% confidence intervals. *** : <i>p</i><0.001 for the TUV strains and <i>p</i> = 0.001 for the EDL strains compared. (D) The <i>E. coli</i> O26 pair of strains blotted in (A) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat-1002672-t001" target="_blank">table 1</a>) were transformed with the LEE1-GFP expression construct or a control plasmid and population fluorescence levels measured through the growth curve. Taken together the data demonstrates that lysogeny with an Stx2-encoding phage represses T3S expression.</p

Regulatory scheme for prophage control of T3S and impact on cattle colonisation.

<p>(A) Schematic diagram showing that the integrated Shiga toxin prophage represses type III secretion (T3S) by restricting Ler-mediated LEE promoter activation. Under the conditions shown there is no expression of the PchA/B regulators associated with other integrated prophages that express effector proteins secreted by the T3S system. (B) Repression is overridden by the activity of activators such as PchA/B that are induced following sensing of niche specific signals in the animal host. A number of environmental signals are known to control T3S <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Tree1" target="_blank">[13]</a> including quorum sensing <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Sperandio1" target="_blank">[63]</a> which is proposed to contribute to the tropism of EHEC O157 for the terminal rectum of cattle <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Naylor1" target="_blank">[43]</a>. However, much less is known about whether these can act through Pch activation. PchA/B stimulate LEE1 and Ler expression leading to production of the T3S apparatus and secretion of LEE-encoded regulators, indicated as blue circles marked ‘LEE’ in the figure. (C) Psr regulators on effector-encoding prophages increase <i>gadE</i> expression leading to repression of LEE-encoded effector protein secretion. It is proposed that this prophage regulation allows non-LEE encoded effectors (Nle) to compete for export through the T3S system <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Tree2" target="_blank">[14]</a>. (D) A model for EHEC interaction with the epithelium. SOS stress responses result in prophage induction and Stx release in a subset of the population. Potentially certain stresses associated with the interaction with epithelial cells may induce this response. The released toxin induces the expression and redistribution of receptors to the epithelial cell surface <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Robinson1" target="_blank">[32]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Liu1" target="_blank">[62]</a>. T3S is repressed but can be induced by Pch regulators <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Abe1" target="_blank">[12]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Tree1" target="_blank">[13]</a>, RgdR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Flockhart1" target="_blank">[15]</a> and further controlled by PsrA/B <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Tree2" target="_blank">[14]</a> present on cryptic prophages to ensure co-ordinate T3S apparatus expression and effector protein secretion (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat-1002672-g005" target="_blank">figure 5A–C</a>). The induction of T3S includes intimin expression on the outer membrane of the bacteria allowing binding to Stx-induced receptors, including nucleolin <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Robinson1" target="_blank">[32]</a>. This leads to intimate attachment and lesion formation. Secreted effector proteins can repress inflammation as can Stx <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Gobert1" target="_blank">[33]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#ppat.1002672-Mhlen1" target="_blank">[39]</a>. It is proposed that the degree and nature of this modulation will be different between strains impacting on bacterial replication and therefore the extent of excretion from the animal.</p

Bacterial strains associated with the study.

<p>Bacterial strains associated with the study.</p

Homologous <i>cI</i> expression represses prophage control of T3S.

<p>Western blot of Secreted EspD and whole cell pellet RecA levels (preparation control) for the indicated strains: (A) Conjugation of a marked Sp5 Stx2-prophage from <i>E. coli</i> O157 Sakai was carried out into TUV93-0 containing an induced clone of Sp5 <i>cI</i> to prevent zygotic induction. This pBAD-CI clone was then displaced with a variant of pBR322. The <i>cI</i> clone limited the repressive impact of the Sp5 prophage on T3S. (B) <i>E. coli</i> O157 Sakai was transformed with both pBAD18 and pBAD-CI. The presence of pBAD-CI with or without arabinose induction increased the level of EspD secretion. (C) An <i>E. coli</i> O157 PT21/28 isolate (15602, table S1) was transformed with both pBAD18 and pBAD-CI. The presence of the pBAD-CI clone led to detectable EspD secretion that was increased on induction of <i>cI</i>. There was no increase in T3S when the same plasmid was induced in <i>E. coli</i> O157 EDL933 (data not shown), indicating that the induction may be specific to strains containing a cognate <i>cI</i> on the Stx2-encoding bacteriophage.</p

Comparison of EHEC O157 PT 21/28 and PT32 strains.

<p>(A) Comparative genome hybridisation of six PT21/28 and six PT32 strains. From left to right the PT21/28 strains are: 09807, 13425, 09064, 11204, 16438 & 17482; the PT32 strains are: 11805, 16117, 177706, 17478, 17489 & 08997 (table S1). The heat map shown indicates relative hybridisation levels of the defined strains to the Stx2 phage genome and flanking gene sequences from the O157:H7 Sakai strain which does not contain a Stx2c prophage. Red through to orange indicates a positive hybridization signal, with yellow a weakening signal through to blue colouration indicative of poor hybridization. All the PT21/28 strains show good relative hybridization to the sequences especially the <i>gam</i>-<i>cII</i> immunity region that is indicated. This is not the case for the PT32 strains with only 1 showing positive hybridisation over this same region, indicating that the remaining 5 strains are unlikely to contain the Stx2 phage. This was confirmed for the strains using a PCR to detect Stx2 and Stx2c phage control regions (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s4" target="_blank">Materials and Methods</a>), the results of which are shown under the heat map lanes. The Stx phage distribution was then determined by PCR for 60 strains (table S1), please see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s2" target="_blank">Results</a> section. (B) Western blot analysis of a subset of PT21/28 and PT32 strains as defined (table S1). EspD was detected from bacterial supernatants and EscJ and RecA from whole cell samples prepared as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002672#s4" target="_blank">Materials and Methods</a>. (C) Relative fluorescence levels from a LEE1-GFP reporter construct transformed into 14 PT21/28 and 16 PT32 strains with levels measured at OD₆₀₀ = 1. The strains with mean values are defined in table S1. The median level of fluorescence from the PT32 strains is significantly higher than for the PT21/28 (p<0.001), the variability in expression levels correlates with the Western blotting data in part B. Also included is the overall median level (horizontal dashed line) (D) LEE1-GFP expression levels compared between strains containing either one or both Stx2 or Stx2c prophages. Strains containing both Stx2 phages have a significantly reduced (P = 0.016) median level of LEE1 expression compared with strains containing only one type of Stx2 prophage as determined at OD₆₀₀ = 1 cultured in MEM-HEPES medium.</p