Bacterial cultures

Bacterial strains and E. coli isolates were incubated in 25 ml of Lysogenic Broth medium at 37 °C overnight with shaking. Bacterial cultures were centrifuged at 5000×g for 20 min at 4 °C. Bacterial pellets were resuspended in 3 ml of cold PBS and sonicated four-times for 35 s at a 70% amplitude. The sonicated samples were clarified at 6000×g for 25 min at 4 °C. These clarified samples were sterilized throughout 0.22-μm-pore-size cellulose acetate membrane filters (Corning) and protein concentration was quantified by using the micro-Bradford method. Lysates were aliquoted and stored at −20 °C until use.

Cell cultures

HEp-2 cells (ATCC-CCL23) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (GIBCO, USA), 10% fetal bovine serum (ByProductos, Mexico), 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Austria), and cultures were kept in 25 cm2 bottles at 37 °C in 5% CO2.

Cytotoxicity assays

For the cytotoxicity assays, 1.1 × 104 HEp-2 cells were seeded in 48-well microplates and incubated at 37 °C for 24 h in 5% CO2. Then, the old media was removed and a fresh DMEM medium containing bacterial lysates at different concentrations (50, 75, 100, 150, 200, 250, 300 and 350 μg/ml) was added and incubated for 24 h. Lysates from E. coli O86:H34 prototypical strain were used as a positive control and lysates from an 11-85D clinical isolate (cdt−) as a negative control. The culture medium was changed every 24 h by fresh medium without bacterial lysates until reaching 72 h of incubation. After this time, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, washed again and stained with Giemsa for 1 h. Each well was analyzed under light microscopy, and the optimal titer of toxin was taken as the minimal concentration of bacterial lysates (μg/ml) required to cause cell distention of 70–80% of the cell population without causing cell death.

CDT biological activity assays

For the biological activity assays, HEp-2 cells were seeded in 8-well Lab-Tek chamber slides (Nalgene Nunc, USA) at a density of 1.2 × 104 cells/well using the same protocol as that of cytotoxicity assays and the optimal lysate titers previously stablished for each bacterial isolate. After Giemsa staining, the cell size was calculated in each experimental condition. For this purporse, 10 photos were randomly taken with a 3000 × magnification for measuring the length of 50 cells/photo. The length of 500 cells for each bacterial lysate was averaged and these values were plotted using GraphPad Prism version 5.01. In the plot, data are shown as the mean ± SD. Statistical comparison of data was performed with an ANOVA One-Way/Dunn’s multiple comparison test. Values of p < 0.001 were considered statistically significant.

FAS (fluorescent actin staining)

For FAS assays, HEp-2 cells were seeded in 8-well Lab-Tek chambers (Nalgene Nunc, USA) at a density of 1.2 × 104 cells/well using the same protocol as that of biological activity assays. Once fixed, the cells were processed for actin staining by washing 5 times with PBS and blocking for 30 min with 1% bovine serum albumin (BSA) (Research Organics, USA). The actin cytoskeleton (F-actin) was then stained with rhodamine phalloidin, and TO-PRO was used to visualize the cell nuclei (both from Invitrogen, USA), incubated 40 min at room temperature in the dark. Finally, the cells were washed 5 times with PBS, twice with distilled water, and left to dry at room temperature in the dark. The slides were mounted in VectaShield (Vector, USA) medium by covering them with cover slides. Samples were analyzed under a confocal microscopy TCS-SP8 (Leica Microsystems, Wetzlar, Germany) and representative photos of each condition were taken using a 63 × objective and a 1.2 digital zoom.

Inhibition of cell cycle analyses

To record the arrest of the cell cycle induced by CDT, HEp-2 cells were seeded in 35-mm Petri dishes (Corning, USA) at a density of 2.2 × 105 cells and incubated at 37 °C for 24 h in 5% CO2. The old media was removed and a fresh DMEM medium containing bacterial lysates at the optimum concentration for each isolate was added and incubated for 48 h (medium was changed each 24 h by fresh culture medium without lysates). After the incubation time, the cells were washed twice with PBS, detached using 1 ml of 0.05% trypsin–EDTA, and then diluted in 2 ml of cold PBS and centrifuged at 240×g for 15 min at 4 °C. The cells were fixed overnight at −20 °C in 70% ethanol. Ethanol was then removed by centrifugation at 400×g for 10 min at room temperature and cells were resuspended in 0.25 ml of 0.06 mg/ml RNase A (Sigma, St. Louis, MO) in PBS and incubated at 37 °C for 1 h. Cells were stained with 30 μg/ml propidium iodine at 37 °C for 1 h in the dark. Cells (2 × 104) were analyzed by a FACSCalibur flux cytometer (Becton Dickinson). Cell cycle analysis was performed by a univariate analysis using FlowJo_V10 software (Tree Star Inc., USA), where 2N values represent the cell population percentage in phase G0/G1, S represents the percentage in phase S and 4N the percentage in phase G2/M. In the plot, the averages from four independent experiments ± standard deviation (SD) are shown. The data were plotted using GraphPad Prism version 5.01software and the statistical data comparison was performed using ANOVA One-Way/Bonferroni’s multiple comparison test. Values of p < 0.001 were considered statistically significant.

Bacterial adherence assay to HEp-2 cells

To evaluate the adherence pattern to epithelial cells, HEp-2 cells were seeded in 48-well plates (JetBiofil, China) at a density of 4 × 104 cells in DMEM and incubated at 37 °C for 24 h in 5% CO2. Before infection, the cells were washed three times with DMEM without supplements. Previously, bacteria were grown in 2 ml of LB overnight at 37 °C at 150 rpm. An OD600nm of culture was recorded to calculate a MOI of 10 for control bacteria, and a MOI of 25 for isolates to infect the cells. The cells were infected with these different MOI and incubated at 37 °C for 4 h in 5% CO2. The infected cells were washed twice with DMEM without supplements and fixed with 4% PFA at room temperature for 20 min and then washed twice with PBS. The cells were stained with a 1:10 ratio of Giemsa:PBS for 2.5 h and photos were taken at 600x.

Typification of the cdt +E. coli isolates

All bacterial isolates and the prototypical O86:H34 strain (used as a positive control) were typed according to the types cdt-I, cdt-II, cdt-III and cdt-IV by PCR [4]. Briefly, a bacterial colony in 20 µl sterile water was boiled for 10 min and then centrifuged 14,000 rpm for 5 min. For the PCR, 3 µl of bacterial supernatants were used as well as the following components: 0.2 mM DTPs, each one, (Invitrogen), 1X Taq DNA polymerase buffer, 0.4 µM forward and reverse oligonucleotides, 1.5 mM MgCl2 and 1 U Taq DNA polymerase (Invitrogen) at a final volume of 25 µl. The amplification reaction was as follows: an initial denaturation (94 °C for 5 min), 30 cycles of denaturation (94 °C for 1 min), annealing (55 °C for 1 min), extension (72 °C for 1 min), and with a final extension (72 °C for 10 min). The PCR products were detected in 1.5% agarose and stained with ethidium bromide.

Oligonucleotides were previously described [4]. For cdt-I type, the primers cdt-IF and cdt-IR were used and produce an amplicon of 411 bp (Table 1). For cdt-II type, the primers cdt-IIF and cdt-IIR were used and produce an amplicon of 556 bp. For cdt-III the primers cdt-IIIF and cdt-IIIR were used and produce an amplicon of 555 bp. For cdt-IV the primers cdt-IVF and cdt-IVR were used and produce an amplicon of 350 bp (Table 1).

Table 1 Characteristics of primers used in this studyDeletion of cdtABC in E. coli E6468, O86:H34, (cdt-I) and the 08-184 isolate (cdt-II)

Deletion of cdtABC genes was performed using the technique for inactivating genes using PCR products [20]. cdt genes were replaced by a kanamycin resistance gene, which was generated by PCR. This resistance gene was synthetized using homologous sequences to the internal end regions of the genes being deleted (Table 1). The pKD4 was used as a template for the kanamycin resistance gene. The PCR conditions were as follows: 5 cycles of denaturalization (94 °C for 1 min), annealing (50 °C for 1 min), and extension (72 °C for 90 min), then 30 cycles of denaturation (94 °C for 1 min), annealing (65 °C for 1 min), and extension (72 °C for 2 min and 30 s), with a final extension (72 °C for 5 min).

The oligonucleotides used were cdt∆ABC-F and cdt∆ABC-R to amplify the kanamycin resistance gene for the prototypical E. coli E6468 (O86:H34) strain and cdt II-FRT-F and cdt II-FRT-R for the 08-184 lysate (Table 1). The first set of primers was designed using the known sequence of the cdtABC genes (access U03293.1). For the unknown sequence of cdt isolate 08-184, 27 cdt homologous sequences (BLAST, NCBI) were analyzed using CLUSTAL W alignment software. The oligonucleotide candidates were analyzed using Oligo Analyzer 1.1.2 software (University of Kuopio, Finland) to choose those representing the best characteristics: cdtII-FRT-F and cdtII-FRT-R (Table 1).

Two PCRs were used to verify the lack of the cdtB gene. The first one was performed using the cdt-IF and cdt-IR for the O86:H34 strain (cdt-I), and cdt-IIF and cdt-IIR for the clinic isolate 08-184 (cdt-II) (Table 1). Amplification was performed with 30 cycles of denaturation (94 °C for 1 min), annealing (55 °C for 1 min), and a final extension (72 °C for 10 min). The PCR amplicons were analyzed in 1.5% agarose gel stained with ethidium bromide. The second was performed using k2 and kt primers for the kanamycin resistance gene (Table 1) [20]. Amplification was performed with one cycle of denaturation (94 °C for 2 min), 30 cycles of denaturation (94 °C for 1 min), annealing (63 °C for 45 s), extension (72 °C for 1 min and 30 s), and a final extension (71 °C for 10 min). The PCR amplicons were analyzed in 1.5% agarose gel stained with ethidium bromide.

Cloning of cdtAB IIC from 08-184 isolate

For cloning the cdtABC genes from the clinical isolate 08-184 in pTrcHis2B, 13 cdtABC type II sequences were obtained from NCBI BLAST, and analyzed with alignment software CLUSTAL W. Two oligonucleotides were obtained: a degenerate forward primer cdtCII-BamH1-F for introducing a BamHI site and a reverse primer cdtABC-PstlI-R for introducing a PstI site. This primer set was used to amplify cdtABIIC genes from the clinical isolate 08–184 by using the following protocol: an initial denaturation (94 °C for 3 min), 30 cycles of denaturation 95 °C for 30 s), annealing (60 °C for 30 s) extension (72 °C for30 s), and a final extension (72 °C for 10 min). The PCR products were detected in 1% agarose stained with ethidium bromide.

The PCR products (2,153 bp) were purified from a 1% agarose gel using QIAquick gel Extraction Kit (Qiagen) and were digested by BamHI and PstI. cdtABIIC genes were cloned by ligation in pTrcHis2B (Invitrogen). The construction was used to transform E. coli BL21[DE3]pLysS. This construction was verified by detecting cdt type II by PCR (as described above) as well as by detecting the released insert (2,153 bp) using BamHI and PstI, and finally by sequencing the whole construction was done by Plasmidsaurus (Eugene, OR).

For complementation assays, cdtABIIC construction was introduced in a cdtABIIC mutant of isolate 08–184 or the cdtABIC mutant of strain O86:H34 by electroporation. Each bacterial complementation was verified by amplifying cdtABIIC by PCR (as described above).