Figure 3.

The C. burnetii T4SS effector AnkF is essential for intracellular replication. (A, B) HeLa cells were infected with wild-type C. burnetii (WT) and ankF::Tn (ankF::Tn) at an MOI of 50. (A) 4 h post-infection the number of intracellular bacteria was determined from 100 infected cells. Error bars represent mean standard deviations of three independent experiments. (B) 48 h post-infection, the cells were fixed and stained with antisera against C. burnetii (green). Nuclei and bacterial DNA were stained with DAPI (blue). Representative LSM images are shown. (C) Human monocyte-derived macrophages were infected with wild-type C. burnetii (WT) and ankF::Tn (ankF::Tn) at an MOI of 10 for 4, 96, and 120 h. The bacterial numbers were determined by colony-forming unit (CFU) counts. Error bars represent the mean standard deviation of three independent experiments. (D, E) U2OS cells were challenged either with wild-type C. burnetii or the ankF::Tn mutant strain, both expressing GFP, at an MOI of 100. Six days post-infection, cells were fixed and labeled with an anti-LAMP1 antibody (red) and Hoechst (blue) to visualize CCVs and host cell nuclei, respectively. (D) Images were acquired with an Arrayscan VTI Live epifluorescence automated microscope equipped with a 20× objective and an ORCA ER CCD camera. Representative images are shown. (E) An average of 50,000 cells were then automatically imaged and analyzed from triplicate experiments for each condition and the phenotypic profile of the ankF::Tn mutant was compared to that of wild-type C. burnetii and expressed as z-scores over 11 independent features.