Article Figures & Data
Figures
- Fig. 1.
Kinetics of intracellular multiplication of AIEC LF82 in J774-AI macrophages. The numbers of viable LF82 bacteria were determined in attached (■) and detached (□) macrophage fractions after different times (hours) of gentamicin treatment. Results are expressed as the number of intracellular bacteria relative to that obtained at 1 h after gentamicin exposure, taken as 100%. Means and standard errors of the means are indicated and correspond to six different experiments.
- Fig. 2.
Survival of AIEC LF82 (A) and S. entericaserovar Choleraesuis (B) in murine peritoneal macrophages. Means and standard errors of the mean are indicated and correspond to four different experiments in duplicate wells. Bacterial CFU per well containing 5 × 105 macrophages (yaxis) and the time after addition of gentamicin (xaxis) are shown. Numbers of viable bacteria were determined in the attached (■) and detached (□) macrophage fractions.
- Fig. 3.
Survival of AIEC LF82 (A) compared to those of serovar Dublin SL2260 (B) and nonpathogenic E. coli K-12 C600 (C) in HMDM. Each time point represents the mean of three independent experiments in triplicate wells. Bacterial CFU per well containing 5 × 105 macrophages (y axis) and the time after addition of gentamicin (x axis) are shown. Numbers of viable bacteria were determined in the attached (■) and detached (□) macrophage fractions.
- Fig. 4.
DNA electrophoresis of J774-A1 cells at 24 h postinfection. Lanes: L, ladder of molecular size markers in base pairs (SmartLadder SF; Eurogentec); 1, uninfected cells; 2, cells infected with the AIEC strain LF82; 3, cells infected with S. flexneri M90T. Only M90T-infected cells generated ladders of 180 to 200 bp, characteristic of apoptosis. LF82-infected cells were negative for DNA degradation.
- Fig. 5.
TEMs of J774-A1 macrophages infected at an MOI of 10 bacteria per cell. At 1 h postinfection, AIEC LF82 bacteria were observed in small vacuoles (A). After 8 h of gentamicin treatment, the size of the vacuoles was increased (B). Note phagosome fusion leading to the formation of a large vacuole observed after 24 h of gentamicin treatment (C). Magnification: ×7,200.
Tables
- Table 1.
Uptake and ability of E. coli strains isolated from patients with CD to evade killing by J774-A1 cells after 1, 4, 8, 24, and 48 h of gentamicin treatment
MOI and strain Uptake of bacteria (104CFU)a after 1 h of treatment % Survival of bacteriab after the indicated treatment period 4 h 8 h 24 h 48 h MOI = 10 K-12 C600 2.4 ± 0.2 122 ± 5 124 ± 11 6 ± 1 0.6 ± 0.1 LF82 12.6 ± 1.1 217 ± 31 280 ± 40 504 ± 63 1122 ± 297 LF9 32.5 ± 20.3 132 ± 22 201 ± 85 969 ± 261 1743 ± 798 LF15 17.9 ± 6.7 74 ± 7 98 ± 6 624 ± 212 700 ± 57 LF16 18.5 ± 4.8 162 ± 25 205 ± 20 3537 ± 418 7421 ± 1246 LF28 8.5 ± 2.2 213 ± 92 212 ± 62 563 ± 120 291 ± 85 LF31 8.1 ± 3.6 130 ± 6 146 ± 21 850 ± 137 1676 ± 317 LF43 15.1 ± 7.5 258 ± 71 455 ± 124 820 ± 164 910 ± 319 LF46 0.5 ± 0.04 364 ± 160 670 ± 221 2782 ± 582 4286 ± 633 LF50 7.5 ± 1.0 156 ± 19 346 ± 13 2071 ± 490 5506 ± 417 LF65 12.1 ± 2.1 286 ± 42 423 ± 81 1239 ± 31 3267 ± 44 LF71 4.8 ± 0.6 259 ± 28 600 ± 213 646 ± 278 782 ± 277 LF100 13.8 ± 2.9 191 ± 16 248 ± 46 251 ± 47 265 ± 24 LF106 33.1 ± 1.0 156 ± 24 184 ± 18 365 ± 169 225 ± 106 LF107 2.2 ± 0.8 147 ± 11 420 ± 186 572 ± 237 591 ± 212 LF120 10.5 ± 1.2 158 ± 21 167 ± 67 495 ± 137 1451 ± 274 MOI = 10 K-12 C600 6.7 ± 1.8 145 ± 4 ND 2.18 ± 1.10 0.8 ± 0.1 S. entericaserovar Chloeraesuis 253.0 ± 61.2 134 ± 29 ND 0.04 ± 0.02 ND S. flexneri M90T 10.4 ± 1.2 57 ± 11 ND 0.40 ± 0.30 ND LF82 75.0 ± 26.0 237 ± 41 268 ± 84 450 ± 59 851 ± 264 ↵a Numbers of CFU recovered from lysed monolayers; means ± standard errors of the means determined from three to five independent experiments in duplicate.
↵b Mean percentages ± standard errors of the means of intracellular bacteria relative to the number after 1 h of gentamicin treatment, defined as 100%. ND, not determined.
- Table 2.
Absence of LDH release by J774-A1 cells infected with AIEC strain LF82
Gentamicin treatment period (h) LDH activitya (% cytotoxicityb) at an MOI of: 10 100 AIEC LF82 K-12 C600 S. flexneri M90T Serovar Choleraesuis AIEC LF82 K-12 C600 S. flexneri M90T Serovar Choleraesuis 1 20.8 ± 4.7 (2.2) 16.1 ± 4.2 (0.4) 43.4 ± 15.6 (10.8) 27.0 ± 7.0 (4.6) 19.8 ± 4.8 (1.9) 21.7 ± 4.4 (2.6) 82.8 ± 19.2 (25.7) 29.5 ± 4.5 (5.5) 4 22.2 ± 5.2 (2.8) 23.2 ± 3.1 (3.2) 83.8 ± 37.1 (26.8) 31.0 ± 1.0 (6.1) 19.8 ± 5.2 (1.9) 23.0 ± 5.7 (3.1) 131.5 ± 32.0 (44.2) 56.5 ± 2.5 (15.89) 8 29.5 ± 4.5 (5.5) 24.5 ± 9.5 (3.6) 177.0 ± 29.5 (61.6) 35.5 ± 13.5 (7.8) 26.0 ± 8.0 (4.2) 27.5 ± 2.5 (4.8) 202.0 ± 5.0 (70.9) 125.5 ± 61.5 (41.9) 24 41.8 ± 5.3 (10.2) 43.8 ± 9.6 (10.9) ND 148.5 ± 0.4 (50.6) 42.8 ± 3.8 (10.6) 42.3 ± 4.4 (10.4) ND 208.5 ± 11.5 (73.3) ↵a Determined in supernatants by using NADH as a substrate. Release of LDH was measured at different gentamicin treatment times after a 2-h infection period and expressed as units per liter of supernatant (means ± standard errors of the means n = 3 independent experiments in duplicate). ND, not determined.
↵b Calculated as (experimental release − spontaneous LDH release at 1 h)/(total LDH release at 1 h − spontaneous release at 1 h) × 100. Spontaneous LDH release by uninfected cells were 14.9 ± 4.2 and 43.2 ± 5.2 after 1 and 24 h of gentamicin treatment, respectively.
- Table 3.
Enumeration of apoptotic and necrotic J774-A1 macrophages after bacterial infection or treatment with gliotoxin
% Apoptotic cellsa % Necrotic cellsa 8 h postinfection 24 h postinfection 8 h postinfection 24 h postinfection Untreated cells 10.1 ± 0.9 11.2 ± 1.2 1.6 ± 1.1 2.0 ± 0.7 AIEC LF82 7.8 ± 0.2 10.0 ± 1.6 1.4 ± 0.6 4.0 ± 1.4 Gliotoxin (5 μM) 36.6 ± 1.0 ND 4.5 ± 0.4 ND S. flexneriM90T 26.6 ± 2.8 19.5 ± 4.0 5.0 ± 1.2 12.4 ± 1.8 Serovar Cholereasuis 25.0 ± 2.3 28.0 ± 0.8 2.0 ± 1.2 5.5 ± 2.6 K-12 C600 10.9 ± 2.5 11.7 ± 0.4 1.1 ± 1.1 0.9 ± 0.0 ↵a Mean percentages ± standard errors of the means of apoptotic or necrotic cells visualized by using the annexin V-FITC assay (Sigma). The percentage of stained cells was scored by analysis of 100 cells of three independent experiments in duplicate. ND, not determined.
- Table 4.
Production of proinflammatory cytokines IL-1β and TNF-α by J774-A1 cells after AIEC LF82 infection
Gentamicin treatment period (h) IL-1β concn (pg/ml)ain: TNF-α concn (pg/ml)b Uninfected cells Uninfected lysed cells AIEC LF82- infected cells S. flexneri M90T infected cells Uninfected cells AIEC LF82- infected cells LPS-stimulated cells 1 <10 (0%) 97.0 ± 2.8 (100%) <10 (0%) 40.5 ± 0.7 (41.7%) 214 ± 48 99 ± 6 110 ± 5 24 <10 (0%) ND <10 (0%) ND 302 ± 13 3,533 ± 111 1,300 ± 86 ↵a J774-A1 cells were prestimulated with LPS and then infected with S. flexneri M90T or AIEC LF82. Medium containing gentamicin was added, and IL-1β concentrations in culture supernatant were measured by ELISA at the indicated times of gentamicin treatment. Results are means ± standard deviations from two independent experiments performed in duplicate, expressed in picograms per milliliter and as percentage of total IL-1β. ND, not determined.
↵b J774-A1 cells were exposed to AIEC LF82 or to LPS for 2 h. Medium containing gentamicin was added, and TNF-α concentrations in culture supernatant were measured by ELISA at the indicated times of gentamicin treatment. Results are means ± standard errors of the means of two independent experiments performed in duplicate, expressed in picograms of secreted TNF-α per milliliter.
