PMC

Invasion of HTE cell monolayers by B. pertussis isolates. Each B. pertussis strain or isolate (7 × 10⁶ CFU) was added to a separate well of a 24-well tissue culture plate each of which contained 7 × 10⁴ epithelial cells. The invasion of HTE cells by B. pertussis was assayed as described in Materials and Methods. The values shown are means ± SEM in thousands of CFU recovered from gentamicin-treated monolayers in three experiments. The diamond symbol indicates P < 0.05 compared to B. pertussis strain 18323.

Invasion of HTE cell monolayers by B. pertussis mutants. Each B. pertussis strain (7 × 10⁶ CFU) was added to an individual well of a 24-well tissue culture plate, each of which contained 7 × 10⁴ epithelial cells. The invasion of HTE cells by B. pertussis was assayed as described in Materials and Methods. The values shown are means ± SEM in thousands of CFU recovered from gentamicin-treated monolayers in three to six experiments. The diamond symbol indicates P < 0.05 versus the parental B. pertussis strain, Tohama I (A) or 18323 (B).

Invasion of HeLa 229 cell monolayers by B. pertussis parental strains and mutants. Each B. pertussis strain (7 × 10⁶ CFU) was added to a separate well of a 24-well tissue culture plate, each of which contained 7 × 10⁴ epithelial cells. The invasion of HeLa 229 cells by B. pertussis was assayed as described in Materials and Methods. The values shown are means ± SEM in thousands of CFU recovered from gentamicin-treated monolayers in three to six experiments. The diamond symbol indicates P < 0.05 versus the parental B. pertussis strain, Tohama I (A) or 18323 (B).

Transmission electron micrographs showing B. pertussis within HTE cells after 1 or 3 h of coincubation. Panels: A, adherent parental strain B. pertussis Tohama I; B and C, intracellular B. pertussis Tohama I; D, adherent AC-Hly-deficient mutant B. pertussis 348; E and F, intracellular AC-Hly-deficient mutant B. pertussis 348. The lack of AC-Hly expression did not appear to render these bacteria more susceptible to damage by lysosomal enzymes. In panels C and F, note the close proximity of the bacterial outer membrane to the membrane of the endocytic vacuole. For panels A and D, the coincubation period was 1 h. For panels B, C, E, and F, the coincubation period was 3 h.

Effect of cAMP on the invasion of HTE cells by B. pertussis. Each B. pertussis strain (7 × 10⁶ CFU) was added to a separate well of a 24-well tissue culture plate each of which contained 7 × 10⁴ epithelial cells. The invasion of HTE cells by B. pertussis was assayed as described in Materials and Methods. Forskolin was incubated with monolayers for 1 h before the addition of the AC-Hly-deficient mutant (B. pertussis 18HS19) to stimulate cAMP production. The effects of forskolin are reversible, and the concentration used (100 mM) was maintained throughout the experiment. (A) As a control, epithelial cells were incubated with (cells & forskolin) or without (control) forskolin. Results are expressed in picomoles of cAMP per 10⁵ cells and are the means ± SEM of three independent determinations. The diamond symbol indicates P < 0.05 versus the control (cells). (B) Values are means ± SEM in thousands of CFU recovered from gentamicin-treated monolayers in three experiments. The diamond symbol indicates P < 0.05 versus B. pertussis 18323.

Intracellular survival of B. pertussis parental and mutant strains in human respiratory epithelial HTE cells. Each B. pertussis strain (7 × 10⁶ CFU) was added to a separate well of a 24-well tissue culture plate, each of which contained 7 × 10⁴ epithelial cells. The invasion of HTE cells by B. pertussis was assayed as described in Materials and Methods. A value of 100% corresponds to the absolute number of viable intracellular bacteria of each strain in a coincubation period of 5 h. For the 24- and 48-h points, the percentages of surviving intracellular bacteria of each strain compared to the 5-h point, independently of the parental strain, are shown. The persistence experiment was performed twice with the parental strain B. pertussis Tohama I and HTE cells and only once with the parental strain B. pertussis 18323 and the mutants. The values above the histograms for the 5-h time point correspond to the means ± SEM of the absolute number of intracellular bacteria of each strain per cell in all of the four to six experiments performed for this time point.