EHEC Tir and EspF_U promote more efficient colonization of polarized epithelial cells than EPEC Tir.

<p>(A) Polarized Caco-2 monolayers were infected with KC12 and EPEC strains for 6 h, fixed, and stained to visualize bacteria, F-actin, and DNA. Scale circles, 100, 500, 1000 μm². (B) Macrocolony sizes >100 μm² from experiments described in (A) were measured and binned into size groups. Each bar represents the mean number of macrocolonies (+/- SE) from 3 experiments, spanning 225–275 fields of view (FOV) and 4658–8434 colonies. All p-value significance is in reference to the 100–250 μm² bins. *p<0.05, **p<0.01 (ANOVA, Tukey post-hoc tests). (C) Data collected in (B) were reorganized to compare the strains within each category. Bars represent the mean (+/- SD) for 3 experiments, of the % of colonies falling into each bin. Asterisks are in reference to KC12+EspF_U. *p<0.05, **p<0.01 (ANOVA, Tukey post-hoc tests). (D) Macrocolony sizes measured from part (B) were averaged. Each bar represents the mean (+/- SD) from 3 experiments. All p-values are in reference to KC12+EspF_U. (E) Experiments were performed as in (A), but for 7 h. Each bar represents the mean (+/- SE) of the % of monolayer area infected for 58–60 FOVs. (F) The number of infected cells per macrocolony was calculated. Each bar represents the mean (+/- SE) calculated from 238–497 macrocolonies, taken from 17–19 representative fields of view from the images quantified in (E). *p<0.05, **p<0.01 (ANOVA, Tukey post-hoc tests).</p

EHEC and EPEC pedestals can be compared directly using engineered EPEC strains.

<p>(A) EPEC Y474*, EPEC Y474F, KC12+EspF_U, and KC12+vector are all EPEC strains engineered to express HA-tagged versions of Tir and/or myc-tagged EspF_U to reflect the WT EPEC or WT EHEC pathways of pedestal assembly. Green and purple boxes represent EPEC and EHEC proteins, respectively. The asterisk indicates phosphotyrosine residue 474. (B) HeLa cells were infected for 3 h with the indicated strains, fixed, and stained to visualize LPS, HA-Tir, and F-actin. Scale bar, 10 μm.</p

KC12+EspF_U and EPEC Y474* form macrocolonies that grow over time on polarized epithelial cells.

<p>(A) Polarized Caco-2 monolayers were infected with KC12+EspF_U for 6 h, fixed, and stained for LPS, DNA, and F-actin. Scale bar, 25 μm; inset 2.5 μm. (B) Cells infected in (A) with KC12+EspF_U or EPEC Y474* were imaged at a lower magnification. Scale circles have areas of 100, 500, and 1000 μm². (C) Polarized Caco-2 monolayers were mock infected (top panels) or infected for 6 h with KC12+EspF_U (bottom panels), and visualized by scanning electron microscopy. The inset highlights a portion of a macrocolony. Scale bars, 10 μm; inset,1 μm. (D) Cells infected in parallel with those in (C) were visualized by transmission electron microscopy. The inset shows a cross-section of a pedestal. Scale bars, 2 μm. (E) Polarized Caco-2 monolayers were infected for 3, 5, or 7 h, fixed, and stained to visualize bacteria, DNA, and F-actin. Scale circles, 100, 500, 1000 μm². (F) Macrocolony sizes were quantified from cells infected as in (E). Each bar represents the mean (+/- SE) of macrocolony sizes calculated from 3–6 coverslips (85–2309 colonies). Macrocolonies over 25 μm² were included in quantification. *p<0.05, ***p<0.001 (unpaired <i>t</i> tests).</p

EspF_U can enhance macrocolony size using either the EHEC or EPEC version of Tir.

<p>(A) Polarized Caco-2 monolayers were infected for 6 h with the indicated KC12 and KC12Δ<i>tir</i> strains, fixed, and stained to visualize bacteria, DNA, and F-actin. (B) Experiments described in (A) were quantified. Each bar represents the mean macrocolony size (+/- SE) calculated from 6 coverslips (2025–3179 colonies). (C) The experiments in (B) were also used to quantify the % of monolayer area infected. Each bar represents the mean (+/- SE) from 59–60 FOVs. (D-E) Polarized Caco-2 monolayers were infected with EHEC strains for 8 h, fixed, and stained as in (A). Bars represent the mean macrocolony size (+/- SE) calculated from 315–617 macrocolonies. (F-G) Polarized Caco-2 monolayers were infected with WT EPEC strains with or without EspF_U. Each bar represents the mean macrocolony size (+/- SE) calculated from 1163–2722 macrocolonies. KC12+EspF_U is shown in purple. For all panels, scale circles, 100, 500, 1000 μm². ** p <0.01, *** p <0.001 (ANOVA, Tukey post-hoc tests). To allow for a sufficient number of Δ<i>tir</i> colonies that could be analyzed, colonies larger than 50 μm² were included in quantification, unlike <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g004" target="_blank">Fig 4</a> where 100 μm² was the lower limit.</p

EspF_U-dependent actin pedestals allow for an efficient pathway of cell-to-cell transmission.

<p>(A) JEG-3 and HeLa cells infected for 6 h and 5 h, respectively, were stained to show bacteria (blue), F-actin (red), and HA-Tir (green). Individual events were ordered into the following sequence based on live imaging in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g007" target="_blank">Fig 7</a>: bacteria (i) use pedestals to protrude and contact an uninfected neighboring cell, (ii) translocate effectors including Tir (arrowheads) into the second cell, (iii) polymerize a second pedestal (arrows), and (iv) use the secondary pedestal to dock bacteria at junctions as the bacteria divide. Scale bars, 3 μm. (B) The proposed model is based on data from experiments in Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g007" target="_blank">7</a> and 8, and is shown to incorporate every step of the infectious life cycle. Green circles represent translocated effectors, red lines indicate F-actin in pedestals, and green ovals represent Tir.</p

The actin pedestals assembled by KC12+EspF_U and EPEC Y474* promote motility and exploration of the host cell surface.

<p>(A) NIH3T3 cells stably expressing mCherry-actin were infected with EPEC expressing GFP for 3 h and imaged live for 45 min. Scale bar, 10 μm. (B) mCherry-actin expressing cells were infected with the indicated strains for 3 h and imaged live for 18–20 min. 15–20 bacteria per host cell for each strain were tracked, and data from representative cells were plotted such that points starting at t = 0 were centered at the origin. (C) Bacterial motility rates were quantified from cells infected as in (B). Each bar represents the mean speed (+/- SE) of bacteria on 6–20 host cells (95–293 total bacteria). ** p<0.01, *p<0.05 (ANOVA, Tukey post-hoc tests). (D) The fraction of pedestals that were considered moving was quantified, using the average speed of the pedestal deficient counterpart strain as a minimum cutoff to define movement. Each bar represents the mean (+/- SE) from 227–320 pedestals. p = 0.3 (Fisher’s exact test). (E) Directional persistence of pedestals was calculated by dividing the maximum displacement by the total path length for pedestals considered to be moving. Each bar represents the mean (+/- SE) for 205–297 pedestals on 19–20 cells. p = 0.1 (unpaired <i>t</i> test) (F) Cells were infected with EHEC strains with or without EspF_U and imaged live. Each bar represents the mean speed (+/- SE) of bacteria on 6 cells (60 bacteria). *p<0.05 (ANOVA, Tukey post-hoc test).</p

Experimental clustering of the EspF_U repeats bypasses the requirement for the Tir C-terminus during actin pedestal formation.

<p>(A) A fusion of membrane-targeted HN-TirFL to EspF_U-myc is shown. Treatment of transfected cells with Tir antibodies and <i>S. aureus</i> particles can promote clustering of membrane-localized Tir-EspF_U fusions. (B) Murine fibroblast-like cells (FLCs) transfected with plasmids encoding Tir-EspF_U fusion constructs comprising the N-terminal domain or truncations of the C-terminal repeats were treated with Tir antibodies and <i>S. aureus</i>, fixed, and stained with HA antibodies to identify both transfected cells and <i>S. aureus</i> (which binds the fluorescent antibodies) and with phalloidin to detect F-actin. (C) Pedestal formation indices were determined by calculating the percentage of transfected cells harboring five or more <i>S. aureus</i> particles generating actin pedestals. Data represent the mean+/−SD from three experiments. The Tir-EspF_U R1 construct triggered actin assembly significantly less efficiently than the other truncations (p<0.05).</p

EspF_U repeats synergistically activate actin assembly mediated by recombinant N-WASP/WIP complex in vitro.

<p>(A) N-terminally His6-Flag-tagged N-WASP and His6-Myc-tagged WIP were co-expressed in insect cells, purified as a stoichiometric complex, resolved by SDS-PAGE, and stained with Coomassie blue. (B) Actin (2 µM) was polymerized in the presence of Arp2/3 complex, N-WASP/WIP complex, and the indicated concentrations of EspF_U. F-actin fluorescence was measured in arbitrary units (AU). (C) Actin polymerization was examined in the presence of 20 nM Arp2/3 complex, 20 nM N-WASP/WIP complex, and the indicated concentrations of EspF_U derivatives. Polymerization rates at half-maximal F-actin concentrations were measured relative to the rate of polymerization in control Arp2/3+N-WASP/WIP samples lacking EspF_U. Curves were fit using Prism software. (D) Actin polymerization was measured as in (C), except that EspF_U concentrations have been scaled to the number of repeats in each protein.</p

The GTPase-binding domain (GBD) of N-WASP is a dominant negative inhibitor of EHEC pedestal formation and binds with high efficiency to a single EspF_U repeat in yeast two-hybrid assays.

<p>(A) The modular structure of N-WASP is depicted, featuring WASP homology-1 (WH1), GTPase-binding (GBD), proline-rich (PRD), and WH2/verprolin-connector-acidic (VCA) domains. Several N-WASP-binding partners are shown above their interacting domains. (B) HeLa cells transfected with plasmids encoding Flag-N-WASP constructs were infected with EHECΔ<i>dam</i> (a mutant that binds to mammalian cells and generates pedestals with considerably higher efficiency than wild type EHEC <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000191#ppat.1000191-Campellone4" target="_blank">[44]</a>), fixed, and treated with DAPI to identify bacteria, a Flag antibody to visualize tagged N-WASP (top panels), and phalloidin to detect F-actin (bottom panels). Flag-N-WASP recruitment was only evaluated in cells expressing low levels of these tagged proteins (top panels), while effects on actin pedestal formation were only assessed in cells expressing high levels of Flag-N-WASP (bottom panels). Pedestal formation indices were determined by calculating the percentage of mock-transfected or Flag-N-WASP overexpressing cells harboring five or more actin pedestals. Data represent the mean+/−SD of three experiments. (C) HeLa cells expressing GFP alone, a GFP-tagged GBD, or a GFP-tagged GBD H208D point mutant were infected with EHECΔ<i>dam</i> or EPEC and treated with DAPI to identify bacteria and phalloidin to detect F-actin. Pedestal formation indices were determined as in (B). (D) Plasmids encoding the N-WASP GBD fused to the LexA DNA-binding domain and EspF_U fragments fused to the Gal4 transcriptional activation domain were co-transformed into a yeast two-hybrid reporter strain. Data represent the mean+/−SD of β-galactosidase activity for three co-transformants for each pairwise combination.</p

Increasing the number of EspF_U repeats does not alter affinity for the GBD, but promotes the formation of an Arp2/3-containing complex.

<p>(A) Isothermal titration calorimetry analyses of the interactions between WASP GBD and EspF_U fragments are shown. The GBD was titrated into R′5 (left), R′4-5 (middle), or R′1-5 (right). Raw and integrated heats of injections are shown in upper and lower panels, respectively. Black lines in the lower panels show fits of data into a single-affinity, multi-site binding model. Fits of the data for R′4-5 and R′1-5 to models with two different affinities were not statistically improved over the single-affinity model. (B) Interactions between Arp2/3 complex and N-WASP_C in complex with Alexa647-labeled EspF_U R′5 or R′4-5 were examined by gel filtration chromatography. The A₆₅₀ profile is shown. (C) Actin (4 µM) was polymerized by itself (black curve), or in the presence of 10 nM Arp2/3 complex plus either 0.5 µM WASP GBD-VCA (purple, control), 0.5 µM GBD-VCA+1 µM R′5 (yellow), or 0.5 µM GBD-VCA+0.5 µM R′4-5 (blue), or 0.5 µM GBD-VCA+0.2 µM R′1-5 (red). F-actin fluorescence was measured in arbitrary units (AU). Note that R′5, R′4-5, and R′1-5 were used at the same total repeat concentration.</p