TABLE 4.
Tissue culture model and in vivo characteristics of phospholipase mutants
| PC-PLC phenotype | Value (mean ± SD) for tissue culture model of infectiona
|
In vivo LD50e | ||||
|---|---|---|---|---|---|---|
| Escape primary vacuoleb (%) | Apparent doubling timec (min)
|
Plaque size (%)d
|
||||
| 2.5–5.5 h | 5.5–8.5 h | Wild type | PI-PLC− | |||
| Wild type | 36 ± 4 (6) | 80 ± 24 (12) | 99 ± 14 (12) | 100 (20) | 90.6 ± 3.6 (10) | (1–3) × 104 |
| PC-PLC− | 0 (4) | ND | ND | 62.3 ± 6.4¶¶¶ (20) | 32.7 ± 4.8*** (19) | 2 × 105 |
| D4E | 63 ± 2*** (4) | 65 ± 25 (3) | 94 ± 38 (3) | 97.9 ± 2.7 (5) | 90.2 ± 3.0 (5) | ND |
| H56Y | 11 ± 3*** (4) | 183 ± 71*** (7) | 98 ± 36 (7) | 95.3 ± 4.0¶¶ (16) | 89.9 ± 2.9 (16) | ND |
| D4E H56Y | 21 ± 2*** (4) | 238 ± 104*** (3) | 99 ± 42 (3) | 91.8 ± 2.6¶¶¶†† (5) | 85.4 ± 4.2*† (5) | ND |
| PLCBc | 56 ± 12** (6) | 64 ± 9 (7) | 93 ± 24 (7) | 84.2 ± 4.5¶¶¶§§§ (10) | 60.4 ± 5.9***§§§ (10) | (7–8.5) × 104 |
Numbers of separate experiments are indicated in parentheses. ND, not determined. Two-tailed P values of unpaired t tests are classified as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to values obtained with wild-type PC-PLC; †, P < 0.05; ††, P < 0.01 compared to H56Y mutant; §§§, P < 0.001 compared PC-PLC− mutant. Two-tailed P values of paired t tests are classified as follows: ¶¶, P < 0.01; ¶¶¶, P < 0.001 compared to plaque measurements (in millimeters) obtained with the wild type (see text).
Percent total intracellular bacteria staining with rhodamine-phalloidin at 2 h after infection of Henle 407 human epithelial cells with mutants in an LLO− (DP-L2161) background (see text).
Calculated from slopes of growth curves from 2.5 to 5.5 h and 5.5 to 8.5 h, respectively, after infection of Henle 407 human epithelial cells with mutants in an LLO− (DP-L2161) background (see text).
Plaque size in L2 mouse fibroblast monolayers 4 days after infection with mutants in wild-type and PI-PLC− (DP-L1552) background compared to wild-type plaques (100% by definition).