Fig. 5.

Effect of F. nucleatum internalization on P. gingivalis invasion. (a) Ca9-22 cells were co-infected with F. nucleatum TDC 100 and P. gingivalis 33277 for 2 h. It was common to find both P. gingivalis and F. nucleatum intracellulary, but some cells exclusively contained P. gingivalis (data not shown). Internalized bacteria were stained red, while extracellular bacteria were shown green-yellow. The host cell cytoskeleton stained with phalloidin appeared blue. (b) Quantification of intracellular bacteria. Pg: P. gingivalis 33277, Fn: F. nucleatum TDC 100. Values are shown as means ± standard deviations. At least five fields and ~200 cells were analyzed for each experiment, and three independent experiments were done. Statistical significance between invasion levels was measured by using Mann–Whitney U test. *p < 0.05. (c) Ca9-22 cells were first infected with F. nucleatum TDC 100 for 1 h, then P. gingivalis 33277 was added and further incubated for another hour. In this sequential infection experiment, intracellular bacteria were predominantly F. nucleatum. Internalized bacteria were stained red, while extracellular bacteria were shown green-yellow. Anti-P. gingivalis and anti-F. nucleatum antisera were used. The host cell cytoskeleton stained with phalloidin appeared blue. (d) Quantification of intracellular bacteria. Values are shown as means ± standard deviations. *p < 0.05. (For interpretation of color referred in this figure legend, the reader is referred to web version of the article.)