CAB strains were derived from POR1 (RIMD 2210633 ΔtdhAS
), generously provided by Drs. Tetsuya Iida and Takeshi Honda (Park et al., 2004b
). They were made by deleting the transcription factors ExsA and/or VtrA that regulate the two T3SSs (Kodama et al., 2010
; Kodama et al., 2007
; Zhou et al., 2008
). To induce T3SS2, media was supplemented with 0.05% bile salt and bacteria were grown at 37°C for 2 hours (Gotoh et al., 2010
). V. cholerae
strains 1587 were generously provided by Dr. John Mekalanos and it was grown in LB at 37 °C.
LDH release at each indicated time point was measured in triplicate using a Cytotoxicity Detection kit (Takara) according to the manufacturer’s instructions. Results are presented as cytotoxicity as calculated from percent of total lysis.
HeLa cells were infected at a multiplicity of infection (MOI) of 10 for the indicated time points. At each time point, 100 µg/ml gentamicin was added to kill extracellular bacteria. Cells were lysed with 0.5% Triton X-100 and the intracellular bacteria were assessed. For the gentamicin incubation experiments, cells were infected for 2 hours, washed with DMEM and incubated in DMEM containing 100 µg/ml gentamicin. At each time point, cells were washed and lysed and the intracellular bacteria were assessed.
HeLa cells were infected for two hours and fixed in 2.5% glutaraldehyde in 0.1M sodium cacodylate, followed by 1% osmium tetroxide in 0.1M sodium cacodylate. Cells were embedded in epoxy resin (Electron Microscopy Sciences) and polymerized at 60 °C. Ultrathin sections were cut at 80 nm and stained with uranyl acetate and lead citrate. Sections were examined at 120KV using a Tecnai G2 Spirit transmission electron microscope (FEI Company) and images recorded on a Gatan USC1000 2k CCD camera (Gatan Inc).
VopC deletion strain
The nucleotide sequence 1kb upstream and 1kb downstream of VopC were cloned into pDM4, a CmR OriR6K suicide plasmid (kindly provided by Dr. Doug Call). The resulting plasmid was conjugated into CAB2 and transconjugants were selected on media containing 25µg/ml chloramphenicol. Bacteria were counter-selected by growing on media containing 15% sucrose.
Reconstitution of CAB2ΔvopC
The ΔvopC deletion strain was reconstituted using pBAD (Invitrogen) containing vopC FLAG or vopC C220S FLAG preceded by 1kb of its upstream sequence and a kanamycin resistance gene.
-FLAG or pBAD vopC C220S
-FLAG were mated into different strains. Bacteria were induced with 0.05% bile salt at 37 °C (Gotoh et al., 2010
). 3h after induction, the culture was centrifuged to separate supernatants and pellets. The supernatant was filtered and ice-cold trichloroacetate was added to a final concentration 17 of 10%. Samples were kept on ice overnight and then centrifuged. The precipitates and pellets were analyzed by western blotting using mouse anti-FLAG antibody (Sigma).
Tissue culture and transfection
HeLa cells were cultured in DMEM (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Sigma) at 37 °C with 5% CO2. Cells were transfected using Fugene HD (Roche) transfection agent according to manufacturer’s protocol.
HeLa cells were fixed in 3.2% paraformaldehyde after treatment. Nuclei were stained with Hoechst (Sigma) and actin cytoskeleton was stained with rhodamine-phalloidin (Molecular Probes). Cells were viewed on a Zeiss LSM 510 scanning confocal microscope and images were converted using ImageJ and Adobe Photoshop.
GTPase activation assay in infected and transfected cells
HeLa cells were infected at an MOI of 10. At each time point, cells were washed and lysates were collected by scraping into Mg2+ lysis buffer (20mM Tris HCl pH 7.5, 10mM MgCl2, 150mM NaCl, 1% Triton X-100). Cellular debris was spun down at 12,000g for 20 minutes and 500µg of cleared lysates was added to 30µg of GST-PAK PBD on glutathione agarose beads and incubated for one hour at 4°C. Samples were separated by SDS-PAGE and immunoblotted with anti-CDC42 (Cell Signaling). As a loading control, total cell lysates were immunoblotted with anti-aldolase (Santa Cruz Biotechnology). Transfected HeLa cells were collected and treated in a similar fashion as described above. Activated RhoA was pulled down using a RhoA activation kit (Cytoskeleton) according to manufacturer’s instructions.
pGex-KG-PAK-PBD (provided by Dr. Neal Alto), pGex-KG-RhoA, pGex-KG-Rac, pGex-KG-Rac Q61L, pGex-KG-CDC42 (provided by Dr. Paul Sternweis), pET28a-MBP-VopCΔ67 and pET28a-MBP-VopCΔ67 C220S were transformed into BL21 (DE3) (Novagen). His-tagged proteins (MBP-VopCΔ67 and MBP-VopCΔ67 C220S) were purified using Ni2+ affinity purification (Qiagen). GST-tagged proteins (GST-PAK-PBD, GST-RhoA, GST-Rac, GST-Rac Q61L and GST-CDC42) were purified using glutathione agarose beads (Sigma).
3 mg/ml of GST-RhoA, GST-Rac, GST-Rac Q61L or GST-CDC42 were incubated with 1:20 molar ratio of MBP-VopCΔ67 or MBP-VopCΔ67 C220S and 5 mM dansylcadaverine (Sigma) at 37 °C for 2 hours. Samples were boiled and separated on 12% SDS-PAGE. Gels were examined with UV exposure using an AlphaImager 2200 (Alpha Innotech Corporation) and images were recorded. The gels were stained with Coomassie Brilliant Blue (Biorad) for protein bands.
120 µM of GST-Rac or GST-Rac Q61L were incubated with a 1:20 molar ratio of MBP-VopCΔ67 or MBP-VopCΔ67 C220S at 37°C for 2 hours. The released NH4+ from glutamine deamidation was measured using an ammonia assay kit (Sigma) according to the manufacturer’s instructions.
pGex-KG-Rac alone or pGex-KG-Rac and pET28a-MBP-VopCΔ67 together were transformed into BL21 (DE3) (Novagen) for protein expression. GST-Rac was purified using glutathione agarose beads (Sigma). Proxeon nano-tips (Denmark) were used to inject the samples into a QStar XL Q-TOF mass spectrometer (Applied Biosystems, Framingham, MA). Spectra were acquired with mass range m/z 500–2000. The molecular weights of proteins were calculated using the Bayesian Protein Reconstruct tool of the Analyst QS1.1 software.
Ten micrograms of each sample were fractionated by SDS-PAGE and bands containing GST-Rac were cut out, treated with DTT and iodoacetamide and digested with chymotrypsin. Samples from the digests were analyzed by nano-LC/MS/MS using a system in which a Dionex LC-Packings HPLC (Sunnyvale, CA) was coupled with an LTQ Orbitrap Velos (Thermo Scientific).
VopC deletion in V. cholerae 1587
The V. cholerae
strain was generated essentially as described (Merriam et al., 1997
; Walker and Miller, 2004
). Briefly, regions 1 kb upstream and downstream of VopC were cloned into pSR47S, a KanR
OriR6K suicide plasmid (kindly provided by Dr. Virginia Miller). pSR47s was then conjugated into V. cholerae 1587
and transconjugants were selected by replating twice on Vibrio
-selective medium (VSM) containing 60 µg/ml kanamycin. Bacteria were counter-selected by growing on media containing sucrose.