Identification of epitopes recognised by mucosal CD4+ T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7

Additional file 5. Sequence alignment of Intimin epitopes against Intimin sequences from non-O157 EHEC serotypes. Alignment of Intimin CD4+ T-cell epitope sequences with representative Intimin sequences from EHEC serotypes O145, O127, O26, O103, O121, O45 and O111. Percentage values indicate % similarity to the EHEC O157:H7 reference sequence

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins

Bacterial Flagella: Twist and Stick, or Dodge across the Kingdoms

The flagellum organelle is an intricate multiprotein assembly best known for its rotational propulsion of bacteria. However, recent studies have expanded our knowledge of other functions in pathogenic contexts, particularly adherence and immune modulation, e.g., for Salmonella enterica, Campylobacter jejuni, Pseudomonas aeruginosa, and Escherichia coli. Flagella-mediated adherence is important in host colonisation for several plant and animal pathogens, but the specific interactions that promote flagella binding to such diverse host tissues has remained elusive. Recent work has shown that the organelles act like probes that find favourable surface topologies to initiate binding. An emerging theme is that more general properties, such as ionic charge of repetitive binding epitopes and rotational force, allow interactions with plasma membrane components. At the same time, flagellin monomers are important inducers of plant and animal innate immunity: variation in their recognition impacts the course and outcome of infections in hosts from both kingdoms. Bacteria have evolved different strategies to evade or even promote this specific recognition, with some important differences shown for phytopathogens. These studies have provided a wider appreciation of the functions of bacterial flagella in the context of both plant and animal reservoirs

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

Bacterial strains associated with the study.

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

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