The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively

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

A variety of mechanisms employed to “dodge” the flagellin innate immune response.

<p>(A) Flagella filaments degrade, releasing monomeric flagellin, the residues of which are recognised by receptor TLR5, NLRC4, or FLS2, resulting in cytokine release or PTI. The example residues and receptor (right) shown are involved in TLR5 recognition. (B) Flagellin recognition by TLR5, NLRC4, or FLS2 is evaded by variation in key residues involved in flagellin detection, which can necessitate compensatory mutations. (C) Bacteria secrete enzymes that specifically target and degrade monomeric flagellin, preventing its recognition by TLR5, NLRC4, or FLS2. (D) Post-translational glyosylation of flagellin is thought to enhance flagella stability; reduced release of flagellin from flagella filaments will result in reduced recognition by TLR5 or FLS2. (E) Bacteria secrete effector proteins that interfere with TLR5, NLRC4, or FLS2 recognition either by direct inhibition of receptor expression or binding, or by inhibition of downstream signalling pathways. (F) Bacteria down-regulate or switch off flagella expression when motility and/or binding are no longer required.</p

Cross-kingdom immune recognition of flagellin structures.

<p>Top: backbone of the key residues of flagellin recognized by plant (left) and animal (right) innate immune receptors are highlighted in red. FliC from <i>S. enterica</i> is presented as a “model” flagellin, with reports for recognition by both TLR5 and FLS2 receptors. These residues are superimposed on the solved flagellin structure (PDB# 1UCU) in UCSF Chimera [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004483#ppat.1004483.ref113" target="_blank">113</a>]. Surfaces and backbone are coloured according to previously assigned structural domains as indicated below each monomer [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004483#ppat.1004483.ref057" target="_blank">57</a>]. Bottom: recognition of flagella filaments by plant (left) and animal (right) innate immune receptors does not occur as key residues (surfaces highlighted in red) are hidden within the filament structure. However, immune recognition still occurs in animals via antibody recognition of the D3 domain.</p

The biophysical properties of flagella, to “twist and stick,” lend themselves towards nonspecific adhesion.

<p>Left, a summary of key characteristics of the flagella apparatus that are advantageous for adherence, and right, specific properties highlighted on the left. (A) Flagellum length results in a long reach towards colonization surfaces as an early-stage anchor—higher affinity binding can occur at closer proximity with specific adhesins and receptors. (B) Flagella rotation generates force that promotes membrane interactions during initial adherence. (C) Flagella are highly repetitive structures, non-specific low affinity binding can result in adhesion at high avidities.</p

Bacterial strains associated with the study.

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

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