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 and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Expression of NleH-GFP constructs in <i>E. coli</i> O157:H7 grown in defined media.

<p>Constructs consisting of 120 bp (pAHE18), 283 bp (pAHE19) or 531 bp (pAHE8) of the NleH1 5′ UTR and 113 bp (pAHE20), 291 bp (pAHE21) or 655 bp (pAHE22) of the NleH2 5′ UTR cloned upstream of <i>gfp</i> were transformed into ZAP193, grown in MEM-HEPES (A) or DMEM (B) and GFP fluorescence measured during growth. All values were corrected for background from a promoter-less GFP (pAJR70) control measured at the same optical density. Graphs represent the average of three experimental repeats.</p

NF-κB activity in the presence of NleH variants.

<p>HEK293T cells were co-transfected with a luciferase reporter plasmid under the control of consensus κB sites, a β-galactosidase plasmid and a control (pCMV), NleH or OspG vector. After 40 hours, cells were stimulated by the addition of TNF-α (25 ng/ml; 24 hours). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033408#s2" target="_blank">Results</a> represent three biological replicates, where variants were tested in triplicate and assayed in duplicate. Statistical analysis with one-way ANOVA shows no significant difference compared with the pCMV control. Error bars represent the standard error of the mean.</p

Expression of NleH-GFP upon <i>E. coli</i> O157:H7 ZAP193 contact with EBL cells.

<p>ZAP193 transformed with plasmids expressing GFP constitutively (pAJR145; <i>rpsm</i>::<i>gfp</i>) or translational fusions of <i>nleH</i> or <i>tir</i> to <i>gfp</i> under the control of their native promoter (pAHE8; NleH1-GFP, pAHE22; NleH2-GFP, pAJR75; Tir-GFP) were added to EBL cells and incubated for 0, 5, 30, 60 or 180 minutes at 37°C, 5% CO₂ before the removal of supernatant and fixation of cells with 4% paraformaldehyde. The panel of images is representative of all time points tested, apart from Tir-GFP, that showed strong early expression during cell contact but was markedly reduced at 180 minutes.</p

Quantitative PCR of NleH transcripts in LEE regulator knockouts.

<p>RNA was collected from ZAP193 strains WT, Δler and ΔgrlA grown to OD₆₀₀ = 1.2 in MEM and cDNA prepared. NleH1, NleH2, GapA, Tir and 16S RNA transcript was then quantified by q-PCR, NleH values normalised to that of 16S RNA, and the fold change calculated comparing mutant to wild-type. Bars represent the average of three biological samples. Error bars represent the standard error of the mean.</p

Fluorescence microscopy of NleH-GFP.

<p>pAHE8 (NleH1-GFP) and pAHE22 (NleH2-GFP) were transformed in ZAP193, ZAP193Δler and ZAP193ΔgrlA and at OD₆₀₀ = 0.8, dried onto a microscope slide in 4% PFA and stained for EspA filaments. Volocity quantification software was used to determine the average GFP fluorescence per voxel of 100 individual bacteria for NleH1 (A) and NleH2 (B). Each point represents the average GFP fluorescence from a composite from 16 z-slice images thus reducing planar effects. Error bars represent the standard deviation.</p

Expression of NleH-GFP and Tir-GFP in <i>E. coli</i> O157:H7 defined LEE regulator mutants.

<p><i>E. coli</i> O157:H7 ZAP193, ZAP193Δ<i>ler</i> and ZAP193Δ<i>grlA</i> were transformed with constructs expressing NleH1-GFP (pAHE8; A), NleH2-GFP (pAHE22; B) and Tir-GFP (pAJR132; C). GFP expression was monitored during growth of the transformants in MEM media, with a promoterless GFP construct (pAJR70) as a background control. Fluorescence values were corrected for background and lines represent the average of three biological repeats.</p