Bacterial strains, tissue culture cells, and culture conditions. S.
Typhimurium strain IR715 (38
), a fully virulent, nalidixic acid-resistant derivative of American Type Culture Collection (ATCC) isolate 14028, was used as a wild-type isolate. IR715 derivatives carrying mutations in invA
); in sopA, sopB, sopD
, and sopE2
); and in sipA, sopA, sopB, sopD
, and sopE2
) have been described previously. Plasmids carrying the sopD
gene (pMR15), the sopE2
gene (pMR17), the sopB
gene (pMR26), the sopA
gene (pMR28), or the sipA
gene (pMR29) from S
. Typhimurium have been described previously (10
). The sipA425
, and sipA149
fragments of the sipA
gene were amplified without the sipA
promoter from pMR29 and cloned directionally behind the lac
promoter of cloning vector pWSK29 (40
) with BamHI and XbaI. Plasmid pRI203, encoding the Yersinia pseudotuberculosis
invasin protein, has been described previously (22
). Bacteria were grown aerobically at 37°C in Luria-Bertani (LB) broth supplemented with antibiotics. The HeLa 57A cell line stably transfected with an NF-κB luciferase reporter construct (41
) was generously provided by R. T. Hay (the Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, United Kingdom). The HEK293 cell line was purchased from ATCC. HeLa 57A cells and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) at 37°C in a 5% CO2
Construction of expression plasmids.
gene was PCR amplified from the chromosome of S
. Typhimurium IR715, and the obtained product was cloned into the mammalian expression vector pEGFP-C1 (BD Biosciences Clontech). The mutant forms of sipA
were PCR amplified from pEGFP-SipA and cloned in pEGFP-C1. Expression vectors with deletions in the F1 domain (pEGFP-ΔF1ΔABD) and in the F2 domain (pEGFP-ΔF2) were engineered by overlap extension PCR. The two DNA fragments needed for the construction of these sipA
mutant forms were amplified by PCR, gel purified, and fused in one PCR. The full-length sipA
and the mutant forms were expressed as a fusion to the C terminus of the enhanced GFP (EGFP). Construction of Myc-SipA was obtained by cloning the full-length sipA
generated by PCR from pEGFP-SipA into pCMV-myc (Clontech). The full-length human NOD1 and dominant-negative versions of human NOD1 (hNOD1) and hNOD2 were PCR amplified from pUNO-hNOD1 and pUNO-hNOD2 (InvivoGen), respectively. hNOD1DN (amino acids [aa] 127 to 954) lacks the N-terminal caspase recruitment domain (CARD). hNOD2DN (aa 217 to 1041) lacks the two N-terminal CARDs. The obtained PCR products were cloned into a derivative of the mammalian expression vector pTracer-CMV2 (Invitrogen, Life Technologies). The derivative lacks the GFP gene and contains a 3×Flag tag at the C-terminal cloning site (42
). The human RIP2
gene was PCR amplified from cDNA prepared from HEK293 cells. The obtained PCR product was cloned into pTracer-CMV2, yielding pTracer-hRIP2 (aa 1 to 540). The dominant-negative form of hRIP2 (aa 1 to 434), lacking the C-terminal CARD, was PCR amplified from pTracer-hRIP2 and cloned into pCMV-HA (Clontech). The hemagglutinin (HA) tag is fused to the N terminus of hRIP2DN. The plasmid pDeNy-MyD88 expressing a dominant-negative human MyD88
gene was purchased from InvivoGen. All constructs were verified by DNA sequencing.
Gentamicin protection assay.
HEK293 and HeLa 57A cells were seeded in a 24-well tissue culture plate at approximately 50% confluence. At the time of infection, the cells had a confluence of approximately 100%. Overnight cultures of S. Typhimurium strains were diluted 1 in 50 and grown for 3 h at 37°C. HEK293 and HeLa 57A cells were infected with S. Typhimurium strains at approximately 107 CFU/ml. The bacteria were incubated for 1 h at 37°C to allow invasion. The cells were washed three times with Dulbecco’s phosphate-buffered saline (DPBS; pH 7.4) to remove extracellular bacteria, DMEM containing 10% FCS and gentamicin (0.1 mg/ml) was added, and the cells were incubated for 90 min at 37°C. The cells were washed in DPBS and lysed with 0.5 ml of 1% Triton X-100, and the lysates were transferred to a sterile Eppendorf tube. The wells were washed with 0.5 ml PBS and transferred to the Eppendorf tubes. Serial dilutions were plated on LB plates with the appropriate antibiotics to count the intracellular bacteria.
HEK293 cells were grown in 24- or 48-well tissue culture plates in DMEM containing 10% FCS until 40% confluence was reached (~24 h). HEK293 cells were transiently transfected with a total of 250 ng of plasmid DNA, consisting of 25 ng of the reporter plasmid pNF-κB-luc, 25 ng of normalization vector pTK-LacZ, and 200 ng of either empty control vector, pUNO-hMyD88DN (InvivoGen), or pEGFP-SipA constructs. For the cotransfection experiments with pEGFP-SipA constructs and hNOD1DN-3×Flag, hNOD1DN-3×Flag, or HA-hRIP2DN, 100 ng of DNA for each plasmid was used. FuGene HD (Roche) was used as transfection reagent at a lipid-to-DNA ratio of 5 to 1. After 48 h of incubation, the cells were infected with the appropriate bacterial strains or the NOD1 ligand C12-iE-DAP, the NOD2 ligand MDP, or the TLR5 ligand flagellin (InvivoGen).
NF-κB activation was assessed using the NF-κB--luciferase reporter system. HeLa 57A cells stably transfected with an NF-κB--luciferase reporter construct and transiently transfected HEK293 cells were infected with the indicated S. Typhimurium strains (107 CFU/ml) for 3 h, after which the cells were washed with DPBS and incubated at 37°C for an additional 2 h in the presence of DMEM containing 10% FCS. The cells were washed in DPBS and lysed in 0.1 ml of reporter lysis buffer (Promega). Firefly luciferase activity was measured with the luciferase assay system (Promega) using a plate reader. For normalization of the efficiency of transfection, luciferase values were adjusted to β-galactosidase values as determined with the β-galactosidase assay (Promega). Results were expressed as fold increase over the uninfected controls.
Protein expression analysis.
HEK293 cells transfected with fusion proteins were lysed in lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.2% [vol/vol] NP-40, and 1 mM EDTA). An 0.01-mg amount of total protein was separated by SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene fluoride membrane (Millipore) in a semidry transfer process (Bio-Rad). The membranes were incubated in PBS containing 3% nonfat dry milk and 0.05% Tween 20 to block nonspecific binding. The membranes were incubated with polyclonal primary antibodies raised against tubulin (Cell Signaling Technology), mouse anti-Flag (Sigma), or mouse anti-HA (Covance). For GFP detection, a horseradish peroxidase (HRP)-conjugated anti-GFP antibody (Cell Signaling Technology) was used. HRP-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG (Promega) were used as the secondary antibodies for tubulin, Flag, and HA detection. Horseradish peroxidase activity was visualized by adding Immobilon Western chemiluminescent substrate (Millipore) to the membrane. Images were recorded and processed by a BioSpectrum imaging system (UVP).
HEK293 cells were transiently transfected for 48 h and lysed with lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% [vol/vol] NP-40, 1 mM EDTA, and protease inhibitors). Samples of the whole-cell lysate were collected for analysis by SDS-PAGE and immunoblotting. The whole-cell lysate was incubated for 2 h at 4°C with Dynal protein G beads (Invitrogen) coated with anti-Flag M2 antibody (Sigma). After 10 washes with the lysis buffer and 2 washes with 50 mM ammonium bicarbonate, samples of the immunoprecipitate were collected and subjected to SDS-PAGE and immunoblotting. The proteins of interest were detected with anti-Flag M2 antibody (Sigma), anti-Myc antibody (Cell Signaling), and anti-GFP antibody (Sigma).
HEK293 cells were transfected with GFP-SipA and NOD1-Flag. After 48 h, the cells were washed and fixed with 3% paraformaldehyde. The cells were permeabilized with 0.1% saponin-10% goat serum (blocking reagent) in PBS for 30 minutes at room temperature. After blocking, cells were incubated with anti-Flag M2 antibody (1:1,000 dilution; Sigma) in 0.1% saponin and 10% goat serum in PBS for 1 h at room temperature. The cells were rinsed twice in 0.1% saponin in PBS and then twice in PBS and incubated (1 h) with Alexa Fluor 647 goat anti-mouse IgG (1:1,000 solution; Invitrogen). After the cells were rinsed twice in 0.1% saponin in PBS, twice in PBS, and once in H2O, they were embedded in Fluorsave (Calbiochem) and analyzed with an LSM700 confocal microscope (Zeiss).
Streptomycin (20 mg/mouse) (Sigma)-pretreated C57BL/6 mice (Jackson Laboratory) and NOD1/NOD2-deficient mice (generously provided by D. Portnoy) were mock infected with 0.1 ml of sterile LB broth or infected orally with 1 × 109 CFU (in 0.1 ml of LB broth) of an S. Typhimurium sopABDE2 mutant (ZA20) or a sipA sopABDE2 mutant (ZA21) carrying the plasmid pHP45Ω to confer streptomycin resistance. At 48 h after infection, mice were sacrificed, and samples of the cecum were snap-frozen in liquid nitrogen for isolation of mRNA.
Formalin-fixed, hematoxylin- and eosin-stained cecal tissue sections were blinded for evaluation by a veterinary pathologist. The following pathological changes were scored: (i) neutrophil infiltration, (ii) infiltration by mononuclear cells, (iii) submucosal edema, (iv) epithelial damage, and (v) inflammatory exudate. The pathological changes were scored on a scale from 0 to 4 as follows: 0, no changes; 1, detectable; 2, mild; 3, moderate; 4, severe. Images were taken using an Olympus BX41 microscope.
Samples of the cecum were collected, immediately snap-frozen in liquid nitrogen, and stored at −80°C. RNA was extracted using TriReagent (Molecular Research) according to the instructions of the manufacturer. Reverse transcription was performed on 1 µg of DNase-treated RNA with TaqMan reverse transcription reagent (Applied Biosystems). For each real-time reaction, 4 µl of cDNA was used. Real time transcription-PCR (RT-PCR) was performed using Sybr green (Applied Biosystems) and an ABI 7900 RT-PCR machine (Applied Biosystems). The fold change in mRNA levels was determined using the comparative threshold cycle (CT) method (Applied Biosystems). Target gene transcription was normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.
A paired Student t test was used to confirm statistical significance in the tissue culture experiments. To determine statistical significance of the relative mRNA transcription between treatment groups in the animal experiments, an unpaired Student t test was used. To determine the statistically significant differences in the total histopathology scores, a Mann-Whitney U test was used. A two-tailed P value of <0.05 was taken to be significant.