Ruffles are a site of cooperative invasion.

<p>(A) 3D-reconstruction of ruffles on HeLa cells infected for 6 min with <i>S</i>.Tm^SopE(pGFP) at an m.o.i. of 250. The actin channel (phalloidin stain) is shown in grey, <i>S.</i> Typhimurium in green (GFP) and extracellular bacteria in red (anti-<i>Salmonella</i> LPS stain before permeabilization). 3 views of a typical, large ruffle (ruffle 1) and details of another ruffle (ruffle 2) are depicted. Scale bar: 10 µm. (B) HeLa cells were infected with a 1∶1 mixture of <i>S</i>.Tm^SopE and <i>S</i>.Tm^Δ4(pGFP)at an m.o.i. of 5 and a movie was acquired via DIC imaging (see also supplementary VideoS4). A snapshot of the movie is depicted. The tracks of 2representative bacteria and their estimated position in the z-axis are indicated by the colored ellipsoids. Both bacteria stop at the ruffle and stay for the remaining observation time (blue track: 4.95 s, green track: 1.24 s). Scale bar: 9 µm. (C) Quantification strategy for analyzing <i>S</i>. Typhimurium docking onto ruffles. HeLa cells were infected for 6 min with a 1∶1 mixture <i>S</i>.Tm^Δ4(pGFP) (green; reporter strain), shown in green and the <i>S</i>.Tm^SopE as a helper strain which did not express <i>gfp</i> at an m.o.i. of 62.5 for each strain. After infection, cells were fixed and stained for actin (TRITC-phalloidin, red) and extracellular <i>S.</i> Typhimurium (indirect immunofluorescence using an anti-<i>Salmonella</i> antibody; blue; stained before permeabilization). Three types of <i>S.</i> Typhimurium can be distinguished: Extracellular reporter <i>S</i>.Tm^Δ4 (labeled green and blue), extracellular helper <i>S</i>.Tm^SopE (only blue), and intracellular reporter <i>S</i>.Tm^Δ4 (only green). Intracellular helper <i>S</i>.Tm^SopE is non-fluorescent and cannot be detected. Scale bar: 10 µm.(D, E) HeLa cells were infected for 6 min with a 1∶1 mixture of a helper strain (either <i>S</i>.Tm^Δ4 or <i>S</i>.Tm^SopE) and the reporter strain <i>S</i>.Tm^Δ4(pGFP) at the indicated m.o.i.. Cells were stained for actin and extracellular bacteria were stained with anti-LPS antibodies. In the control scenario (helper strain <i>S</i>.Tm^Δ4, only non-ruffling cells) bound bacteria were quantified for the area of a whole cell (grey bars); in the ruffling scenario (helper strain <i>S</i>.Tm^SopE) bacteria were quantified over the area of a ruffle as explained in panel (B,C).Even with a complex 3D structure, the surface of a ruffle should be much smaller than the surface of a whole cell. Therefore, if anything, our approach should underestimate the specific recruitment of bacteria onto ruffles. Extracellular bacteria of the reporter strain and the helper strain were quantified separately ((D): reporter strain, expresses <i>gfp</i>; (E): helper strain; no <i>gfp</i>). The bars summarize 170–220 cells/ruffles from two independent experiments. ***: p<0.0001.</p

Strains and plasmids used.

<p>Strains and plasmids used.</p

Simulation of <i>S</i>. Typhimurium targeting to physical obstacles.

<p>(A) To simulate <i>S</i>. Typhimurium targeting to physical obstacles, a round “cell” (light blue sphere) and linearly moving “bacteria” (orange particles) were modeled in an <i>in silico</i> experiment. If during the simulation a bacterium hits the sphere, it is either deflected or has a 10% chance of remaining at that site (“stopping”, red particles). Behavior of particles on the base of the cage ( = “surface”) and upon hitting the sphere if no docking occurred differed according to the respective scenario: Reflection with a random angle in the “random” scenario, with an angle of reflection identical to the angle of the impact in the “billiard” scenario and no reflection but movements following the surface of the object in the “NSS” scenario. The start of the simulation is depicted in the far left. Inserts show an enlarged view of the area around the sphere.</p

Swimming behavior of <i>S.</i> Typhimurium on gelatine coated glass beads.

<p>Gelatine coated glass beads (500 µm diameter) were placed into a glass-bottom dish and the swimming behavior of <i>mCherry</i> expressing bacteria was recorded by time lapse fluorescence microscopy as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002810#ppat-1002810-g003" target="_blank">Fig. 3</a>. (A) Illustration of a bacterium stopping at the glass bottom of a coated glass bead (illustration not to scale). (B) Maximum intensity plot (ImageJ) superimposing all frames of a 15 sec movie acquired at 20 frames per second (300 frames total; suppl. Video S3) illustrating the movement of <i>S</i>. Typhimurium (<i>S</i>.Tm^wt(<i>mCherry</i>)) within the vicinity of the bead. Scale bar: 10 µm.</p

Near surface swimming of <i>S</i>. Typhimurium on cellular surfaces.

<p>(A) Scheme describing the four stages of <i>S</i>. Typhimurium movement observed at cellular surfaces. Inserts (I) and (II) indicate two possible mechanisms for trapping <i>S</i>. Tymphimurium in proximity to the surface: hydrodynamic entrapment (I) and DLVO interactions (II), respectively. A “stop” on the surface can be due to an obstruction hindering the path of the bacterium (3a) or reversible binding/irreversible docking (3b), see text for details. (B) Snapshot of a movie acquired using DIC imaging. HeLa cells were infected with <i>S</i>.Tm^Δ4 and the interactions of bacteria with cells were followed in real time. The tracks of 2 representative <i>S</i>.Tm^Δ4bacteria are indicated, their estimated positions in the Z- layer while moving along the cellular surface are indicated by the shade of color. The bacterium indicated in green encounters a mitotic cell and stops until the end of the movie while the bacterium indicated in blue crosses and leaves the field of view. Compare supplementary Videos S1 and S2. Scale bar: 18 µm. (C) HeLa cells were infected with <i>S</i>.Tm^Δ4(pGFP) and 5-minute fluorescence microscopy movies were acquired. Representative frames illustrating key stages of <i>S</i>. Typhimurium NSS and our quantification strategy are depicted (see text as well as Materials and Methods for details). In each frame, the star indicates the position of the corresponding bacterium in the previous frame. Note the fluorescent ring in time points 0 s and 42.1 s, indicating the start (“landing”) and end (“take off”) of the contact with the host cell. (D) Quantitative analysis of the different stages of NSS (see A and C) for <i>S</i>.Tm^Δ4(pGFP). 5 independent experiments were analyzed (n = 122 bacteria). The box plot represents the median, interquartile range, the 5%–95% range as well as outliers. (E) Quantification of the fraction of <i>S</i>.Tm^Δ4(pGFP) making no stops (“NSS only”), or the fraction making the indicated number of stops on the cell surface during NSS (“NSS and stopping”; further analysis of the experiment in D). Error bars:standard deviation. (F) Quantitative analysis of the time a bacterium spends stopped at the surface. The data is plotted either in seconds (left panel) or as a fraction of the total contact time of the respective bacterium (right panel; further analysis of a subset of the experiment in D). (G) HeLa cells were infected with <i>S</i>.Tm^Δ4(pGFP) and 5-minute fluorescence movies were acquired focusing either >100 µm above the cells (“swimming in solution”) or on the cell layer. For 40 time points from 2 independent experiments the number of <i>S</i>.Tm^Δ4(pGFP) in a field of view was quantified. Either the whole population (“NSS and stopping”) or only moving bacteria (“NSS”, immotile bacteria excluded) were counted. **: p<0.01; ***: p<0.0001.</p

Cellular morphology affects docking of <i>S</i>. Typhimurium.

<p>(A) Cells were incubated with the indicated concentration of cytochalasin D for one hour prior to infection with<i>S</i>.Tm^Δ4(pGFP) at an m.o.i. of 125 for 10 min. Afterwards, cells were fixed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002810#ppat-1002810-g006" target="_blank">Fig. 6</a> and stained with DAPI and TRITC-phalloidin (stains actin). Stacks of confocal images were acquired in the actin channel (grey) and the bacteria channel (green). An extended focus projection and a reconstruction of a zx-layer are shown. Scale bar, upper images: 20 µm, zx-layer: 5 µm. (B)Cells were pre-treated with the indicated concentration of cytochalasin D for 1 hour and infected with the respective <i>S.</i> Typhimurium strain for 12 min. After washing, fixing and staining <i>S.</i> Typhimurium docking was quantified by an automated microscopy based docking assay <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002810#ppat.1002810-Misselwitz1" target="_blank">[9]</a>. The curves shown summarize 3 independent experiments. Error bars: standard deviation.</p

Cooperative invasion at membrane ruffles.

<p>(A) Scheme explaining our quantification strategy for cooperative invasion. (B) HeLa cells were incubated with <i>S</i>.Tm^SopE(pGFP) at the indicated m.o.i. for 9 min, followed by washing, fixation and staining of actin and extracellular bacteria (anti-LPS antibody; staining before permeabilization). Left panel: quantification of the fraction of cells carrying ruffles. Middle panel: quantification of the number of “inside” and “outside” <i>S</i>.Tm^SopE(pGFP) per individual ruffle. Right Panel: number of invaded <i>S</i>.Tm^SopE(pGFP) per cell, calculated by multiplying the number of intracellular bacteria per ruffle with the fraction of ruffling cells. The black line indicates the estimated number of invaded <i>S</i>.Tm^SopE(pGFP) assuming independent invasion events, extrapolated from experimental data of ruffles with only one associated bacterium at the lowest m.o.i.. Each data point summarizes 4×150 cells for the analysis of cellular ruffling and 4×25 cells for inside and outside bacteria from 2 independent experiments. Error bars: standard deviation.</p