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

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

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

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