FIG. 8.

Expression of lgt1 and lgt3 during L. pneumophila infection of A. castellanii. Amoeba cells, infected at a multiplicity of infection of 0.1 with agar-grown L. pneumophila Philadelphia-1 at 0 h, were cultivated for different time periods, lysed, and sampled for RT-PCR with primers specific for lgt1 (white columns) and lgt3 (black columns) (a) or for plate counting (b). The corresponding induction ratios, calculated as proportions of mRNA levels at certain time points to that at the time point with the minimal value (24 h for both lgt1 and lgt3), are shown in panel a. Growth of L. pneumophila (b) is shown as changes in log CFU/ml over time. Cultures were extensively washed after 3, 24, and 48 h of incubation before being sampled in order to remove extracellular bacteria. Data obtained demonstrate induction of the genes (a) and changes in numbers (b), predominantly of intracellular bacteria. In contrast, the culture at 72 h was sampled without being washed. This was done due to decreasing numbers of attached amoebae. Therefore, data obtained at 0 h and 72 h demonstrate induction of the genes mainly in extracellular bacteria, which are starting to infect eukaryotes (0 h) or exiting amoebae (72 h). CFU numbers at these time points are shown by single filled circles in panel b. The figure represents data from three independent cell culture experiments.