Increased Expression of Periplasmic Cu,Zn Superoxide Dismutase Enhances Survival of Escherichia coli Invasive Strains within Nonphagocytic Cells

Reagents.Ampicillin, bafilomycin A₁, bovine serum albumin, diethyldithiocarbamate, cytochrome c, gentamicin, kanamycin, penicillin, pyrogallol, streptomycin, trypsin, xanthine, and xanthine oxidase were purchased from Sigma. Restriction endonucleases, DNA-modifying enzymes, and catalase were obtained from Boehringer Mannheim. All other chemicals were purchased from BDH and were of the highest grade available. Oligonucleotides were synthesized by Genset. Culture tissue media were obtained from Seromed.

Plasmids, bacterial strains, and culture conditions.The plasmids, E. coli strains, and oligonucleotides used in this work are listed in Tables 1 and2. Plasmid pRI203 was kindly provided by S. Falkow. This plasmid, which carries the inv gene ofY. pseudotuberculosis, renders E. coli cells able to invade animal cells (23). A DNA fragment containing the whole E. coli sodC gene (22) was obtained by PCR amplification of E. coli chromosomal DNA carried out with the oligonucleotides prom5′ and prom3′. The approximately 920-bp amplified DNA was digested with EcoRI andHindIII and subcloned in the corresponding sites of pRI203 to obtain pRIEcSOD. In order to study the transcriptional regulation of sodC, its promoter region (corresponding to the 223 bp before the translation start site) was amplified with the oligonucleotides prom5′ and lacZ. The amplified DNA fragment was digested with EcoRI and BamHI and inserted into promoter probe plasmid pMC1403 (11) to obtain pMCPromEcSOD. In this vector, the sodC promoter is cloned upstream of thelacZ coding region, allowing the possibility of analyzingsodC expression following the accumulation of β-galactosidase (β-Gal).

E. coli HB101 (8) and E. coli GC4468 (10) were from our laboratory collection. TherpoS mutant E. coli QC1110 was obtained by P1 transduction of the katF13::Tn10 allele (28) in the parental strain GC4468. Regions (about 1,150 bp) of DNA flanking and entering into the sodC structural gene were amplified by PCR and ligated in order to generate a 350-bp internal deletion of the sodC gene. The primers used to amplify the upstream fragment (ending at position 93 of the structuralsodC gene sequence) were sodCb2, generating anEcoRV restriction site at its 5′ end, and sodCb1, generating a SalI restriction site at its 5′ end. The primers used to amplify the downstream fragment (starting at position 438 of the structural sodC gene) were sodCa1, generating aSalI site at its 5′ end, and sodCa2, generating anEcoRV restriction site at its 5′ end. The PCR products were cloned into the pCR2-1-topo cloning vector (Invitrogen) to create pDT32 and pDT31. After appropriate digestion, the fragments were successively transferred into pUC19 and ligated at the SalI restriction site, generating a deleted sodC gene (pDT34). The deleted region was substituted by a kanamycin resistance gene block (Pharmacia) inserted at the SalI site. The construction was verified by sequencing. The resulting plasmid (pDT35) was digested withEcoRV and the 3.6-kb linear fragment containing thesodC mutated region was used to transfer the mutation to the chromosome, transforming a wild-type strain bearing plasmid pTP223, which avoids nucleolytic digestion of linear DNA (32). A Kan^r Amp^s transformant was selected. The strain was further tested for correct integration of the kanamycin-containingsodC gene by PCR with different primers, including oligonucleotides internal to both the kanamycin and sodCgenes, and by bracketing the insertion fragment. Amplifications with sodC3-sodC4, sodC1-sodC4, sodC2-sodC3, sodC4-kan1, and sodC3-kan2 oligonucleotide pairs (see Table 2) were carried out. In all cases, a band of the expected molecular weight was obtained. The sodCmutation was P1, transduced into GC4468 and selected for Kan^r (QC2725). The sodC mutation was 56% cotransducible with sodB, in good agreement with the distance between the two genes (∼10 kb). Activity assays carried out on a periplasmic extract of QC2725 confirmed the lack of Cu,ZnSOD in this strain.

E. coli strains were grown on Luria-Bertani (LB) medium containing 100 μg of ampicillin per ml. E. coli cells bearing pRI203 and pRIEcSOD exhibited identical growth rates and viability. Bacterial cells were grown overnight until the stationary phase was reached; a bacterial suspension in exponential phase was obtained by subculturing the overnight cultures in fresh medium at 37°C. The cultures, in different growth phases, were pelleted and washed with phosphate-buffered saline (PBS) without Ca²⁺and Mg²⁺ and then resuspended in minimal essential medium (MEM) for the invasion assay at a concentration of about 10⁷ cells/ml. The minimal bactericidal concentration of gentamicin for each strain was determined by counting the number of CFU after incubation of 10⁷ bacterial cells/ml in gentamicin-containing MEM for 2 h at 37°C.

Host cells.HeLa S3 cells (from an epithelioid carcinoma of the human cervix) and Caco-2 cells (from a human colonic carcinoma) were grown as monolayers at 37°C in MEM supplemented with 1.2 g of NaHCO₃ per liter, 2 mM glutamine, 100 U of penicillin per ml, 0.1 mg of streptomycin per ml, and 10% heat-inactivated fetal calf serum (FCS) in a 5% CO₂ incubator. During the infection experiments, FCS was added at a concentration of 2%.

Invasion assay.Invasion of cultured cells was assayed by a modification of the technique of Isberg and Falkow (23). Briefly, semiconfluent monolayers of HeLa S3 or Caco-2 cells grown without antibiotics in 12-well plates (Costar) were infected with invasive E. coli strains, in either exponential or stationary phase, at a multiplicity of infection (MOI) of 100 (defined as 100 bacteria per cell). The infection was performed for 1 h at 37°C. Then, cells were washed extensively with PBS without Ca²⁺ and Mg²⁺, and 1 ml of fresh medium containing 200 or 100 μg of gentamicin per ml was added to each well, which was infected with E. coli GC4468(pRI203) or GC4468(pRIEcSOD), QC2725(pRI203) or QC2725(pRIEcSOD), and HB101(pRI203) or HB101(pRIEcSOD). After a further 2-h incubation period at 37°C, infected cells were washed extensively and subsequently treated with trypsin-EDTA (a mixture of 0.05% trypsin [1/250] and 0.02% EDTA) for 5 min at 37°C and lysed by the addition of 1.0 ml of cold 0.1% Triton X-100. Cell lysates were diluted in PBS and plated on LB medium containing 100 μg of ampicillin per ml to quantify the number of viable intracellular bacteria.

Intracellular survival assay.After bacterial infection, HeLa or Caco-2 cells were washed and fresh medium containing 50 μg of gentamicin per ml, 2% FCS, 1.2 g of NaHCO₃ per liter, and 2 mM glutamine was added. The monolayers were then incubated for 4, 6, 24, and 48 h at 37°C. At these times, cells were washed and lysed and the number of viable intracellular bacteria was evaluated by CFU counts.

Effect of ammonium chloride and bafilomycin on bacterial invasion and survival.Prior to the invasion assays, HeLa cell monolayers were preincubated for 30 min with or without 20 mM NH₄Cl or 100 nM bafilomycin A₁, either of which is known to inhibit vacuolar acidification (34). After bacterial infection, gentamicin- and NH₄Cl- or bafilomycin-containing medium was added to the cell monolayers. At different times, cultured cells were washed with PBS and lysed, and viable intracellular bacteria were counted, as described above.

Electron microscopy.Twenty-four-well tissue culture plates were seeded with 1 × 10⁶ HeLa cells/well and pulsed with ferritin (0.2 mg/ml) for labeling of lysosomes as described by D'Arcy et al. (14). After 3 h of incubation at 37°C, monolayers were washed five times with MEM to remove free ferritin; then, they were infected with E. coli strains bearing pRI203 and pRIEcSOD at an MOI of 100 (see “Invasion assay,” above). At different time intervals (2, 24, and 48 h), cells were incubated with a 0.05 trypsin–0.02% EDTA solution, washed gently with PBS, and pelleted at 600 × g for 10 min. Pellets were fixed with 2.5 mM glutaraldehyde in cacodylate buffer for 1 h at room temperature and postfixed in 1% unbuffered OsO₄. Cells were then dehydrated with increasing concentrations of ethyl alcohol and embedded in Agar 100. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 208S transmission electron microscope.

Activity assays.Cu,ZnSOD activity was assayed by the pyrogallol method (30). The periplasmic fraction was obtained by a procedure described previously (3), with the only difference that cells were resuspended at an optical density at 600 nm (OD₆₀₀) of 100. The low expression level of Cu,ZnSOD and the presence of small amounts of cytoplasmic FeSOD and MnSOD in the periplasmic extracts prevent accurate measurements of Cu,ZnSOD activity in E. coli. Therefore, to characterize our model we have determined the Cu,ZnSOD activity in periplasmic extracts before and after a 15-min incubation with 2 mM diethyldithiocarbamate, a copper chelator which inactivates the Cu,ZnSOD enzyme without affecting the activity of MnSOD and FeSOD (7). Protein content was determined by the method of Lowry et al. (29). β-Gal activity was measured by a previously described procedure (35).

Characterization of the sodC strain.Susceptibility to extracellular superoxide was evaluated by monitoring bacterial survival upon exposure to superoxide generated by the action of xanthine oxidase on xanthine. Bacteria grown until stationary phase were washed and suspended at a density of about 10⁵cells/ml in PBS containing 0.1 to 1 mM xanthine and 1 U of catalase (Boehringer Mannheim) to remove hydrogen peroxide generated by xanthine oxidase and ensure that bacterial death was due to superoxide and not to the hydrogen peroxide formed by the spontaneous dismutation of the superoxide anion under assay conditions (37). Superoxide generation was initiated by the addition of xanthine oxidase to a final concentration of 0.1 to 0.5 U/ml. Effective superoxide formation under the above-mentioned conditions was checked by monitoring the rate of reduction of cytochrome c (previously purified by gel filtration) at 550 nm (31). Aliquots of the reaction mixture were diluted and plated at different times to determine the number of CFU per milliliter.

To detect the bacterial survival in stationary phase, E. coli GC4468 and E. coli QC2725 cells were grown in a 250-ml Erlenmeyer flask containing 50 ml of LB medium at 37°C. Each day, the OD of the cultures was checked and cell survival was evaluated by plating serial dilutions of the bacterial suspensions.