Cellular and Molecular Life Sciences

Cellular and Molecular Life Sciences

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Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown

Huber P.1,2,3,4 *, Bouillot S. 1,2,3,4, Elsen S. 1,2,3,4 and Attree I. 1,2,3,4

1 INSERM, U1036, Biology of Cancer and Infection, Grenoble, France, 2 CNRS, ERL 5261, Bacterial Pathogenesis and Cellular Responses, Grenoble, France, 3 Université Joseph Fourier-Grenoble I, Grenoble, France, 4 CEA, DSV/iRTSV, Grenoble, France

*Corresponding author: E-mail:

Nota bene: Movies (ESM_6-12) are in separate files

Online Resource 1

ESM_1 Strains and plasmids used in this work

Strains or plasmid / Description / Abbreviations used / Refs
Strains
P. aeruginosa
PAO1F (RP1831)
PAO1FΔPscD (RP1871)
PAO1FΔST (RP1947)
PAO1FΔSY (RP1924)
PAO1FΔTY (RP1948)
PAO1FSTY (RP576)
PAO1FΔ3Tox/ExoS
PAO1FΔ3Tox/ExoSGAP-
PAO1FΔ3Tox/ExoS ADPR -
PAO1FΔ3Tox/ExoT
PAO1FΔ3Tox/ExoTGAP-
PAOIFΔ3Tox/ExoTADPR -
Escherichia coli
Top10 / Wild-type PAO1 (exoS, exoT, exoY)
PAO1F ΔpscD
PAO1F ΔexoSΔexoT
PAO1F ΔexoSΔexoY
PAO1F ΔexoTΔexoY
PAO1F ΔexoSΔexoTΔexoY
PAO1F ΔexoSΔexoTΔexoYattB::exoS
PAO1F ΔexoSΔexoTΔexoYattB::exoS-R146K
PAO1F ΔexoSΔexoTΔexoYattB::exoS-E379D/E381D
PAO1F ΔexoSΔexoTΔexoYattB::exoT
PAO1F ΔexoSΔexoTΔexoYattB::exoT-R149K
PAO1F ΔexoSΔexoTΔexoYattB::exoT-E383D/E385D
Chemically competent cells / Pa-WT
pscD
Pa-Y
Pa-T
Pa-S
3Tox
ExoS
ExoSGAP-
ExoSADPRT-
ExoT
ExoTGAP-
ExoTADPRT- / [1]
[2,3]
[2,3]
[2,3]
[2,3]
[2,3]
This study
This study
This study
This study
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This study
InVitrogen
Plasmids
pCR-Blunt II-TOPO
pUCP20
pRK2013
mini-CTX1
pFLP2
pCTX-ExoS E/B
pCTX-ExoS RK
pCTX-ExoS 2ED
pUCP-ExoT E/B
pUCP-ExoT RK
pUCP-ExoT 2ED / Knr; Cloning vector
Apr; broad-host-range plasmid vector
Knr; Helper plasmid with conjugative properties
Tcr; Vector for single-copy integration onto the
P. aeruginosa chromosomal attB site
Apr; Flp-recombinase expressing plasmid
mini-CTX1 vector carrying wild-type exoS gene
mini-CTX1 vector carrying exoS-R146K gene
mini-CTX1 vector carrying exoS-E379D/E381D gene
mini-CTX1 vector carrying wild-type exoT gene
mini-CTX1 vector carrying exoT-R149K gene
mini-CTX1 vector carrying exoT-E383D/E385D gene / InVitrogen
[4]
[5]
[6]
[7]
This study
This study
This study
This study
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This study

Online Resource 2

ESM_2: EXPERIMENTAL PROCEDURES

Reagents

The anti-phospho-Fak-Y576, phospho-moesin-T558, MYPT and myc antibodies were from SantaCruz Biotechnologies; the anti-cofilin, phospho-cofilin-S3, Limk and phospho-Limk1/2-T508/T505 were from Cell Signaling; the anti-Fak and phospho-MYPT-T696 were from Millipore; the anti-actin antibody and phalloidin-FITC were from Sigma-Aldrich; the anti-phospho-Fak-Y397 antibody was from Upstate Biotechnology, the anti-paxillin was from BD Biosciences, the ß-tubulin antibody was from Sigma. The polyclonal antibody against ezrin-radixin-moesin was a gift from Anne-Sophie Ribba. The secondary antibodies coupled with Alexa 488 were purchased from Invitrogen, and those coupled with cyanine 3 and cyanine 5 were from Jackson Immunoresearch Laboratories. Peroxidase-conjugated antibodies were purchased from Biorad laboratories. Pyr1 was a gift from Laurence Lafanechère.

Bacterial growth conditions

P. aeruginosa was grown in liquid Luria Broth (LB) medium at 37°C with agitationuntil the cultures reached A600 values of 1.0.

Strain construction

To complement PAO1F3Tox mutant, a fragment comprising the exoS coding sequence, 150 base pairs (bp) upstream from the start codon and 71 bp downstream from the stop codon, was amplified by PCR using CHA genomic DNA as template and the ExoS/F-EcoRI and ExoS/R-BamHI primers. A second fragment, comprising the exoT coding sequence, 150 bp upstream from the start codon and 100 bp downstream from the stop codon, was also PCR amplified using ExoT/F-EcoRI and ExoT/R-BamHI primers. The resulting 1592-bp fragment and 1640-bp, respectively, were cloned into pCR-BluntII-TOPO and sequenced. Generation of exoS and exoT mutations was performed using the QuickChange site-directed mutagenesis kit (Stratagene) and the pairs of the primers listed in ESM_1. After cleavage with EcoRI and BamHI, the different exoS and exoT fragments were cloned into mini-CTX1 and pUCP20, respectively, cut by the same enzymes. The three pUCP20-derived plasmids were introduced into PAO1FΔ3Tox using transformation, whereas the three mini-CTX-derived plasmids were introduced by triparental conjugation, using the conjugative properties of the pRK2013 helper plasmid. The transconjugants were selected on PIA plates containing Tc. The pFLP2 plasmid (harboring the counter-selectable sacB marker) was used to excise the Flp-recombinase target cassette: single colonies were thus plated on PIA medium containing 5% sucrose to select for the loss of the plasmid and the sensitivities to Tc and Cb were verified. The complementations were checked by comparing the secreted ExoS and ExoT toxins in the different strains upon T3SS induction, either by Western blotting using anti-ExoS antibodies or silver nitrate staining for ExoT (Online Resource 3).

DNA constructs for transfection

Plasmids coding for Lifeact-EGFP and Lifeact-RFP were obtained from Roland Wedlich-Soldner. Vinculin-GFP was from Hélène Delanoë-Ayari. GFP-Rho-V14 and GFP-Rac-V12 were from Cécile Gauthier-Rouvière; plasmid encoding myc-LimK-T508EE was from Kenzaku Mizuno and that coding for EGFP-mDia2-FH1FH2 from Gregg Gundersen. The vector pEGFP-C1 (Clontech) was used as control.

Cell culture, transfection and infection

Human umbilical vein endothelial cells (HUVECs) were isolated according to previously described protocols [9]. Briefly, the umbilical cord vein was canulated, washed with Hank's buffer saline and PBS, and perfused with collagenase A (Sigma) for 10 min at 37°C. Recovered cells were cultured in Endothelial-Basal-Medium (EBM-2, Lonza) supplemented as recommended by the manufacturer. Human microvascular endothelial cells (HMVEC) were purchased from Lonza and grown in EBM-2 MV (Lonza). Bovine aortic endothelial cells (BAEC) were produced as previously described [10] and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). The mouse endothelioma cell line H5V was provided by Annunciata Vecchi and grown in DMEM with 10% FCS. The rat brain endothelial cell line (RBE4) was provided by Pierre-Olivier Couraud and grown in 45% minimum essential medium, 45% H10 medium and 10% FCS. The human carcinoma cell line A549 was grown in RPMI medium with 10% FCS. For infection comparison, confluent cells were all infected in EBM2 medium without supplement.

HUVECs were transfected with 5 µg of plasmid using nucleofection (Amaxa Biosystems) according to manufacturer’s instructions. Cells were infected 24 or 48 h post-transfection by bacteria in exponential growth with a MOI (multiplicity of infection) of 10. For real-time imaging, cells were observed under a CellR videomicroscope (Olympus) with regulated temperature and CO2. For microscopy experiments, cells were grown on glass coverslips or Lab-Tek chambers (Thermo Scientific), previously coated with fibronectin.

Western blots

Cells were lysed in Triton X-100 and protein concentration of the lysates was determined with a Micro-BCA kit (Pierce, Rockford, IL) using BSA as standard. Protein extracts were then separated by SDS-polyacrylamide gel electrophoresis, transferred onto Hybond ECL membrane (Amersham Biosciences, Saclay, France) and revealed with the appropriate antibody using standard procedures. Signals were revealed with a ChemiDoc system (BioRad). Band intensities were quantified on non-saturated images using imageJ software.

REFERENCES

1. Bleves S, Soscia C, Nogueira-Orlandi P, Lazdunski A, Filloux A (2005) Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1. Journal of bacteriology 187 (11):3898-3902

2. Cisz M, Lee PC, Rietsch A (2008) ExoS controls the cell contact-mediated switch to effector secretion in Pseudomonas aeruginosa. Journal of bacteriology 190 (8):2726-2738

3. Vance RE, Rietsch A, Mekalanos JJ (2005) Role of the type III secreted exoenzymes S, T, and Y in systemic spread of Pseudomonas aeruginosa PAO1 in vivo. Infection and immunity 73 (3):1706-1713

4. West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ (1994) Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148 (1):81-86

5. Figurski DH, Meyer RJ, Helinski DR (1979) Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro. Journal of molecular biology 133 (3):295-318

6. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212 (1):77-86

7. Hoang TT, Kutchma AJ, Becher A, Schweizer HP (2000) Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 43 (1):59-72

8. Sun Y, Karmakar M, Taylor PR, Rietsch A, Pearlman E (2012) ExoS and ExoT ADP ribosyltransferase activities mediate Pseudomonas aeruginosa keratitis by promoting neutrophil apoptosis and bacterial survival. J Immunol 188 (4):1884-1895

9. Garnier-Raveaud S, Usson Y, Cand F, Robert-Nicoud M, Verdetti J, Faury G (2001) Identification of membrane calcium channels essential for cytoplasmic and nuclear calcium elevations induced by vascular endothelial growth factor in human endothelial cells. Growth factors (Chur, Switzerland) 19 (1):35-48

10. Gory S, Vernet M, Laurent M, Dejana E, Dalmon J, Huber P (1999) The vascular endothelial-cadherin promoter directs endothelial-specific expression in transgenic mice. Blood 93 (1):184-192

Online Resource 3-13

FIGURE AND MOVIE LEGENDS

ESM_3 Secretion of ExoS and ExoT mutants in complemented 3Tox strain

a The 3Tox strain was complemented with either ExoS wild type (S), ExoSADPRT- or ExoSGAP-. Strains were compared for ExoS secretion in low-calcium culture conditions. Bacteria supernatants were analyzed for their ExoS contents by Western blotting with anti-ExoS antibodies. b The 3Tox strain was complemented with either ExoT wild type (T), ExoTADPRT- or ExoTGAP-. Strains were compared for ExoS secretion in low-calcium culture conditions. Bacteria supernatants were analyzed for their ExoT contents by electrophoresis and silver staining.

ESM_4 Susceptibility of various endothelial cell types to P. aeruginosa-induced cell retraction

Confluent monolayers of human primary cells, HUVECs and HMVECs, or bovine primary cells, BAECs, as well as the mouse endothelioma cell line H5V, the rat brain endothelial cell line RBE4 and the human carcinoma cell line A549 were infected with PAO1F wild-type (Pa-WT) (MOI = 10) in EBM2 medium. For comparison, cells were fixed simultaneously and labeled with anti-ß-actin antibodies to visualize the entire cell body (in red). Non-infected cells are shown above. Infection of HUVECs, HMVECs and BAECs induced extensive cell retraction (arrowheads), while this effect was absent in H5V cells or weak in RBE4. Infected A549 cells exhibited rounding (arrows), but little retraction, as the cell surface was similar to the uninfected cells.

ESM_5 Transendothelial albumin flux in infected HUVECs

Confluent HUVECs cultured on porous membranes were infected at MOI of 10 with Pa-WT, PaPscd, Pa-S or Pa-T. Controls were uninfected cells (NI) and membranes devoid of cells (Empty). Albumin-TRITC was simultaneously loaded in the upper compartments and TRITC fluorescence was measured at 4 hours p.i. Data are presented as mean fluorescent units (n = 3) + SD. Statistical differences with NI were established with Student's test: * p<0.01; ** p<0.001.

ESM_6 Actin cytoskeleton stability in HUVECs mock-infected by LB

HUVECs were transfected with Lifeact-EGFP and treated 24 h later with LB. Videomicroscopy images of EGFP labeling were taken every 2 minutes post-infection and are shown at 6 frames per second (fps). Movie is representative of at least 20 different cells that were observed under these conditions.

ESM_7 Actin cytoskeleton disruption in HUVECs infected by P. aeruginosa wild-type

HUVECs were transfected with Lifeact-EGFP and infected 24 h later with P. aeruginosa WT. Videomicroscopy images of EGFP labeling were taken every 2 minutes post-infection and are shown at 6 frames per second (fps). Movie is representative of at least 40 different PAO1-infected cells that were observed under these conditions. Retraction was consistently preceded by cytoskeleton collapse.

ESM_8 Actin cytoskeleton disruption in HUVECs infected by Pa-S

Same procedure as in ESM_5. Images were captured every 2 min. Movie is representative of at least 20 different cells that were observed under these conditions. Retraction was consistently preceded by cytoskeleton collapse.

ESM_9 Actin cytoskeleton disruption in HUVECs infected by Pa-T

ESM_10 Simultaneous disruption of actin filaments and focal adhesions during infection

HUVECs were transfected with Lifeact-RFP and vinculin-EGFP and infected 24 h later with P. aeruginosa WT. Videomicroscopy images of actin in red and vinculin in green were taken every 10 minutes post-infection and are shown at 6 fps.

ESM_11 Lamellipodial activities are inhibited in infected HUVECs

Same procedure as in ESM_5 but on sparse cells. Images were captured every 2 min.

ESM_12 Filopodial activities are inhibited in infected HUVECs

Same procedure as inESM_5 but on sparse cells. Images were captured every 5 min.

ESM_13 Destabilization of microtubules in P. aeruginosa-infected HUVECs

At 4 hours p.i. with PAO1, cells were treated with OPT buffer to eliminate free tubulin and then fixed and labeled with anti-tubulin antibodies (green). Control with uninfected cells (NI) is shown. In infected cells, the microtubules were remodeled and some cells exhibited major regression of the tubule network (arrowheads).