To both Reviewers: We would like to thank the reviewers for their thoughtful and thorough review of the manuscript. We have tried to address every comment to the best of our ability. In response to the reviewers’ suggestions, we have generated novel data, including cell retraction assays with activity domain mutants of ExoS and ExoT, and additional controls. We believe that the revised manuscript is significantly improved. Modifications in the manuscript are indicated in red.

Reviewer #1: It is well established that the P. aeruginosa type III secretion system disrupts the actin cytoskeleton of mammalian epithelial cells, phagocytes, and other non-professional phagocytes. It has also been shown that this disruption involves the effectors ExoS and ExoT via both their GAP and ADPr activities, and that RhoGTPases (Rho, Rac and CDC42), ERMs and Crk proteins are targeted. Here, the authors further investigated the mechanisms behind these events for wild-type bacteria, and various type III secretion mutants expressing only ExoS, T or Y (or no effectors) using primary cultured endothelial cells (human and bovine), and correlated the time course of cell retraction with changes in actin filament dynamics, RhoGPTase-inactivation, focal adhesion kinase dephosphorylation, lim kinase dephosphorylation, cofilin dephosphorylation (activation), and actin filament severing. Constitutively-active forms of Rho or Rac blocked the effects of PA on endothelial cell retraction, and partial effects were observed for active limK and mDia. Some in vivo work in the pneumonia model also showed dephosphorylation of cofilin. ExoY was ineffective in causing cell retraction in these cells despite effective increases in cytosolic cAMP activity. Overall, this is a very nice manuscript with beautiful live kinetic imaging to probe the unexplored dynamic relationship between the actin cytoskeleton and Pseudomonas aeruginosa. The imaging in Figs. 2, 3, and 7 is of very high quality. It is also of interest that endothelial cells seem to behave quite differently from epithelial cells in response to P. aeruginosa and its type III effectors. As the authors suggest, this may reflect use of primary cultures, and this is a strength of this paper. The data is well presented, and in many instances is very clear. The discussion is also well written. I did have a number of questions/concerns for the authors; The following five points relate to the relationship between ExoS or ExoT and actin severing involving lim kinase-cofilin, which seems to be mostly correlative at this stage.

1. In Fig. 5B, pCofilin levels are very different between ExoS (no phosphorylation) and ExoT (phosphorlyation), and wild-type (between ExoS and ExoT). The ExoT data seems inconsistent with Fig. 5C? Also, could this difference between ExoS and ExoT reflect different levels of ADPr activity? I believe they both have similar GAP activity. Also, the data for ExoT does not match the lim kinase dephosphorylation (Fig. 5D)? In Table 1, we compared the phosphocofilin/cofilin ratio derived from Fig. 5b and 5c at 4.5 hpi. The values are similar, moreover considering that data from Fig. 5b and 5c were produced in independent experiments. Table 1: Phospho-cofilin/cofilin ratio comparison between Fig. 5b and 5c

pCof/cof ratio in Fig. 5b pCof/cof ratio in Fig. 5c at 4.5 hpi NI 1 1 WT 0.43 0.30 Pa-T 0.38 0.22 Pa-S 0.032 0.079

We now show cell retraction results with the activity domain mutants of ExoS and ExoT (Fig. 1c,d). The data indicate that the ADPRT domain of ExoS is responsible for the entire toxic activity of the toxin, whereas for ExoT both domains exert cell retraction activities. As discussed in the paper, the ExoS and ExoT ADPRT domains have different substrates, which may explain why they have a slightly different action kinetics. Blots from Fig. 5b and 5d were performed using the same protein extracts. Therefore there is co-existence of inactivated Lim kinase and some remaining phospho-cofilin in Pa-T-infected cells. This suggests that cofilin dephosphorylation does not occur instantly after Lim kinase inactivation.

2. Could lim kinase dephosphorylation be a result of actin destabilization rather than the cause of it since there is a feedback loop? For example, increases in actin tension (clearly seen in early PA infection when stress fibers are induced) could be sufficient to enhance cofilin binding and severing of actin (Hayakawa et al. 2012, Communicative and Integrative Biology)? A time course of lim kinase dephosphorylation during PA infection could help answer this question. As suggested, we performed a kinetic experiment to examine phospho-Lim kinase levels in course of infection (Fig. 5d). We observed a regular decrease of Lim kinase activity; it is thus likely that actin filament severing is caused by progressive Lim kinase inhibition.

3. The statement that "cytoskeleton and focal adhesion disruption takes place through the lim kinase-cofilin pathway as a consequence of GTPase" (lines 39 and 40 on page 13 in the discussion) is not fully supported by the data. For example, in Fig. 5E, the lim kinase inhibitor Pyr1 is able to disrupt stress fibers, but has no effect on cell retraction. As indicated above, Lim kinase is inhibited during infection and this will undoubtly lead to cofilin activation, as reported by many articles (as review, see Bernard, Int J Biochem cell Biol 2007, 39:1071). As this molecule is the major actin-severing agent, its total dephosphorylation (Fig. 5c) will result in cytoskeleton collapse, as well as focal contact disruption as these two structures are linked together. Consistent with this, stress fibers collapsed in Pyr1-treated cells although cells did not retract, as opposed to infected cells. We can propose two explanations for this lack of retraction: (i) Pyr1 is known to be highly unstable and we noticed that cells challenged with Pyr1 for 2 hours recovered a full cytoskeleton (not shown); it is thus possible that Pyr-1 action is not long enough to promote cell retraction. (ii) Cell retraction may need other types of action from the bacteria in addition to cytoskeleton collapse: for example, inhibition of actin polymerisation (as suggested from mDia2 transfection data in Fig. 9c) and/or action of Pa-released proteases on intercellular junctions or matrix proteins. Our data collectively show that Pa-induced cytoskeleton disruption is mediated by alteration of Lim kinase-cofilin axis; as suggested above, other mechanisms are probably necessary to achieve cell retraction. Therefore, we believe that the quoted sentence is correct as is, unless Reviewer 1 makes a suggestion.

4. It would also be helpful to show that PA infection-induced dephosphorylation of limK is dependent on a specific GTPase (or all three) by analyzing limK phosphorylation over time in cells overexpressing active form(s) of the GTPase(s). As suggested, we performed this experiment with RacV12 (Fig. 9c). Overexpression of RacV12 alone was sufficient to maintain Lim kinase phosphorylated in infected cells over time. We believe that these novel data clarify the important role of the Lim kinase-cofilin axis in Pa infection.

5. The relationship between cofilin and actin severing in PA infection (with ExoS or ExoT) could be more firmly established by RNAi of cofilin prior to infection which should attenuate cofilin-dependent actin disruption by PA, i.e. could the results present be due to cofilin-independent actin destabilization? This is a good point and we tried quite hard to perform this experiment, however cofilin RNAi was highly deleterious for HUVECs and we failed to obtain reliable data.

Other points. 6. I have doubts about the conclusion that A549 retracts more than H5V or RBE4 because the images of the rodent cells look to be more than a monolayer, i.e. clumps of stacked cells are apparent. This is correct: H5V and RBE4 cells do not have a strong contact inhibition of proliferation and migration like A549 cells or HUVECs. Indeed they have a tendency to make clumps or stacks. However, we consistently noticed that H5V and RBE4 are refractory to intoxication (i.e. cell retraction) compared to the other cell types we studied. To observe cell retraction in these cell types, infection times must be significantly extended. However, as we fixed all cells presented in Online Resource 4 simultaneously, H5V and RBE4 were not retracted in this experiment.

7. The finding that the phosphorylation state of moesin does not change during infection of endothelial cells is important. This is unexpected since moesin is a target of ExoS ADPr activity and constitutively active moesin prevents cell retraction in epithelial cells. Suggest including this data in primary text rather than supplemental files. The supplemental figure is now Fig. 8.

Minor points. 8. Does the data presented in Fig. 1B distinguish cell retraction versus cell loss? Indeed, the data do not distinguish between cell retraction and cell loss. Therefore, the text was modified accordingly and the y-axis is now entitled "Total cell surface" that takes into account both cell retraction and detachment, instead of "Cell spreading".

9. The Fig. 6 legend should state the time of analysis, i.e. 3 h for the right panels. This was modified. 10. In Fig. 6, what is the significance of the elevated CDC42 in ExoY treated cells? The slight increase in cell spreading induced by Pa-Y (Fig. 1b) is probably linked to the Cdc42 elevation observed in Fig. 6d.

11. Are the primary cells seeded onto an extracellular matrix protein(s)? It was not clear. The methods just state that cell culture was as previously described. A brief summary in current paper would be helpful. Cells were seeded on fibronectin-coated glass for all observations in microscopy. This is now included in the Supplemental Methods (Online Resource 2). We also added a brief description of HUVEC isolation from umbilical cords.

12. The missing data that microtubules are destabilized in infected HUVECS should be included. We did not include these data in the first version of the manuscript as we have no mechanism to provide. The immunofluorescent images showing microtubules are now shown in Online Resource 13.

13. Online movie 8 does not play. This movie (now ECM_10) was tested and plays on different computers.

14. An extra proof-reading is needed, i.e. numerous spelling errors throughout the manuscript. The revised manuscript was corrected by a native English speaker.

15. How was fluorescence measured? As we did not measured fluorescence levels in the manuscript, Reviewer 1 probably meant the luminescence system (ECL) used to quantify signals in Western blot. Signals were acquired with an image acquisition equipment (Chemidoc from BioRad). The acquisition time was adapted to be in the linear range.

Reviewer #2: This paper studies the role of ExoS and ExoT in cell retraction and cytoskeletal alterations during infection of vascular endothelial cells by P. aeruginosa. The authors find that these T3S toxins are mostly responsible for the disappearance of stress fibers and adhesion structures as well as cell retraction in these endothelial cells. A comprehensive picture is provided whereby, following the inactivation of RhoGTPases, activation of LIM kinase together with the disappearance of adhesion structrures lead to cell retraction. Activation of LIM kinase in infected lungs was also observed. This is an interesting piece of work, presenting important data on cells that may provide a mechanistic basis for the translocation of P. aeruginosa across the vascular endothelium. It seems to me that the work would gain in significance if the authors could clarify the respective role of the GAP and ADP-ribosyltransferase domains of ExoS and ExoT in their cell systems. I have a other suggestions concerning mostly controls and statistics. Data with activity domain mutants of ExoS and ExoT are now presented (Fig. 1c,d) and discussed in the paper. Briefly, ExoS ADPRT domain was solely responsible for ExoS-induced cell retraction, whereas the GAP and ADPRT domains of ExoT had both toxic activity. 1. It is very difficult to see details in Fig. 1A. A higher magnification with better resolved actin structures should be shown. In Fig. 1a, we did not use phalloidin that only labels filamentous actin, but anti-actin antibodies that label both free and filamentous actin, thereby providing a uniform staining of the entire cell body. This was the best uniform cell labeling we obtained after having tested several compounds. In these conditions, we could easily binarize the images with ImageJ software and measure the surface occupied by the cells in each image. Therefore, even at higher magnification there is no observable subcellular structure with this labeling. We kept the low magnification images in Fig. 1a, as they illustrate the type of images that were quantified to produce the data in Fig. 1b. To observe the actin cytoskeleton of infected cells, please see the images in Fig. 2.

2. l. 42: " ...which was even more striking than the wild-type in the case of Pa-S. Is the difference in Fig. 1B between WT and the Pa-S mutant strain statistically significative? Can the authors comment on that? The difference was indeed significative (p = 0.006) between the WT and Pa-S. There are at least two explanations: (i) ExoS is the most potent exotoxin and there is no competition with the other T3SS substrates in the Pa-S mutant for its injection into cells, or (ii) another toxin, potentially ExoY, has an activity that antagonises that of ExoS when co-injected with the WT. These explanations remain highly speculative; this is why we did not include this discussion in the paper.

3. Ideally, in Fig.1B, the effects of complemented mutants should be shown. In Fig. 1c,d we performed cell retraction assays on cells infected with the D3Tox mutant complemented with either ExoS or ExoT.

4. To claim that ExoS is more potent than ExoT, the authors should check the levels of expression / translocation during infection. It is extremely difficult to monitor the real (probably tiny) amounts of toxin indeed injected in cells because of the possible contamination from exotoxin-containing bacteria remained attached to the dish, even after extensive washes. We thus removed this claim from the paper.

5. In Fig. 1c, the absence of cAMP production by WT relative to the Pa-Y strain is interesting but raises many questions. It also calls for additional controls to show that ExoY is expressed and injected in WT. The adenylate cyclase activity should be tested for cells infected with the Pa-S and Pa-T mutants? As suggested, the experiment was performed with the Pa-S and Pa-T mutants (Fig. 1e). Data show that cAMP levels were comparable with uninfected and wild-type conditions (ie, different from Pa-Y). This suggests a possible interplay among the toxins.

6. For the cell counting assays in Fig. 1D, were kinetics longer than 4.5 hours tested? It is surprising that the data do not correlate with those in Fig. 1B for Pa-T. There is a slight decrease in Fig. 1d (now 1f) data, but the differences were not statistically significative at 4.5 hpi. Pa-T-infected cells significantly detached at longer time points. 7. What is the time frame for the effects depicted in Fig.2? Were other kinetics analyzed? The number of experiences, events of cell retraction / showing disappearance of actin fibers should be mentioned. In this experiment, cells were fixed at 4 hpi. As cell intoxication is T3SS-dependent and thus contact-dependent, morphology changes were not simultaneous in the monolayer during the infection process, but stochastic. Therefore kinetic studies are relevant for global experiments (Western blot, cell retraction assay, GTPase activity assay,…), but not for microscopy, as cell alteration levels will change from one area to another during the infection process. Numerous experiments of this type (immunolabeling of F-actin and paxillin) were performed as well as time-lapse recording of Lifeact-transfected cells. Altogether, we can conclude that (i) actin cytoskeleton disruption consistently occurred prior to cell retraction, (ii) cytoskeleton disruption is a required step for cell retraction and (iii) the time between cytoskeleton disruption and cell retraction is very short (within minutes). Images showing total absence of cytoskeleton but still no cell retraction, as shown in Fig. 2 WT for example, are rare, because this step is very transient. We did observe progressive cytoskeleton loss in every infected cell (see Online Resources 7-9). Once cells are retracted, one cannot see the cytoskeleton anymore, either with phalloidin or Lifeact labeling, as cells become a fluorescent sphere (see the movies). Therefore, it is difficult to quantify this phenomenon. We included in Fig. 2 legend a sentence indicating that images are representative of at least 40 imaged cells.

8. This analysis is important, because it would allow to analyze cells establishing cell-cell junctions. Also, it would allow to better estimate the effects on the actin cytoskeleton since the effects on Lifeact transfected cells appear much less pronounced. The lesser effects on Lifeact transfected cells may not be too surprising, since this construct may stablize actin filaments. The number of experiences, events of cell retraction / showing disappearance of actin fibers should be mentioned. This is correct: cell-cell junctions may retain cell retraction during progressive cytoskeleton disruption, as shown in Fig. 2 WT. It is likely that final cell retraction suddenly occurs when cell-cell junctions are abolished by a mechanism that remains to be determined. It is indeed possible that Lifeact maintains actin fibers for longer time. However in each movie that we recorded, cells retracted after progressive cytoskeleton loss, without any exception. As mentioned above, it is difficult to quantify these events on fixed cells. We reported in the revised version the number of cells that were analyzed in Movies ESM_6,7 legends.

9. Fig. 3A could be improved. The effects are not very clear. It is also not clear what the arrowheads point to. Panels showing controls cells should be included, which would probably imply to limit the number of time frames. The number of experiences, events of cell retraction / showing disappearance of actin fibers should be mentioned. The movie shown in Fig. 3a (now 3b) was indeed too contrasted and there was quality loss when images were transformed in .mpg movies. We present now a version of better quality. The arrowheads pointed to initial positions of actin fibers. As they were indeed confusing, they were removed. Control cells were added (Fig. 3a, Movie ESM_6). The number of cells observed by this technique is now indicated in Fig. 3 legend. 10. I do not really see the point of the cells plated on patterned substrates shown in Fig. 3B. It seems that the authors would need to add additional controls to show the specificity of the effects, or that what they call "stress fibers" are indeed stress fibers connected to adhesion plaques. In my opinion, Fig.1B could be removed. As suggested, Fig. 3b was removed.

11. p.8, l. 22: It is unclear that dephosphorylation of pFAK-Y576 follows that of Y397 from Fig. 4B. To make this point, a graph showing the intensity of Y397 should be superimposed to that of Y576 in Fig.C. We decided to remove this point from the text.

12. The biphasic effects on Rho activation need further clarification, since such effects are not anticipated from the action of ExoS and ExoT. One possible explanation to these unexpected results is that Rac is a primary target for ExoS/ExoT during the first hour of infection. Rho activation then would just merely result from Rac inactivation. To test this, the levels of active Rho could be tested in cells transfected with V12Rac. As suggested, Rho activity was measured in RacV12- or EGFP-transfected cells (Fig. 9b). Overexpression of Rac-active indeed prevented the transient upregulation of Rho, which suggests that it was induced by early Rac downregulation.

13. Membrane ruffles are very difficult to see in Fig. 7A -NI. We provide novel images for Fig. 7a-NI showing a cell with more ruffles.

14. The number of experiences, events of cell showing disappearance of membrane ruffles / filopodia should be mentioned. The number of cells observed showing this phenomenon is now indicated in the legend.

15. p.10, l. 31-33: The sentence : " Altogether, our data suggest that impediment of cell retraction requires both inhibition of actin filament severing and activationof actin polymerization." is highly speculative. There could be several explanations in the difference in amplitude in the inhibition of cell retraction mediated by active Rho GTPases and LimK or mDia2 FH1FH2. In my opinion, these sets of experiments are simply consistent. The sentence has been changed.

Minor comments: * p.2, * l. 38: "As most Gram-negative.." any evidence for that? It is indeed difficult to evaluate. "most" was replaced by "many"

* l. 51: should read "the presence of ExoS and ExoU..." * p. 3 * l. 1: "...when exotoxin ADPRT domain is inactivated" is misleading. Perhaps, "The GAP domain of these toxins is sufficient to..." * p. 6 * l. 37: " Deletion of all three type 3 toxins, (Δ3Tox; Fig. 1b) did not significantly affect cell spreading,..." sentence is not clear. Rather, " a mutant strain deleted for ExoS, T and U did not induce cell retraction..". These modifications have been introduced in the text.