Figure 6.

C. burnetii infection does not alter the rate of Tf recycling and PV-associated Tf puncta persist after extensive recycling. (A) HeLa cells infected with C. burnetii for 3 days or left uninfected were loaded with Tf488 for 45 min and the decrease in intracellular Tf488 was measured by quantitative fluorescence microscopy. Plots depict percent Tf488 intensity calculated relative to uninfected cell cultures (Uninfected) at 0 min. CellProfiler was used to select only cells that emitted high C. burnetii signal (Infected—Large PV) from infected cell cultures (Infected). Statistical significance (P < 0.01) was determined by two-way ANOVA using Tukey's test for multiple comparisons with the mean number of cells counted per sample equal to 945 ± 301. A green asterisk indicates the mean Tf intensity of the subset of cells containing large PV is significantly greater than for uninfected cells, while a black asterisk indicates the mean Tf intensities of the subset of cells containing large PV and infected cell cultures are significantly greater than for uninfected cells. However, regression analysis of the linear first 10 min of the assay indicate the apparent rates of recycling for uninfected cells, infected cells, and infected cells containing large PV are similar. Results are representative of 3 independent experiments and error bars indicate the standard errors from the means. (B) Representative confocal fluorescence micrographs depicting the intracellular localization of Tf488 (green) in HeLa cells infected with C. burnetii (red) for 3 days, and loaded with Tf488 for 45 min, then incubated in medium lacking Tf488. After a 0, 10, or 30 min incubation, samples were fixed and immunostained with rabbit anti-C. burnetii serum and Alexa Fluor 594 conjugated anti-rabbit secondary antibody. PV-associated Tf puncta persist after the 30 min chase. Scale bar, 10 μm. (C) Tf puncta remaining in HeLa cells (5 days post-infection) after Tf488 (green) labeling and a 30 min chase were immunostained for CD63 or EEA.1 using monoclonal antibodies and Alexa Fluor 568 conjugated anti-mouse secondary antibody (red). Bacterial and host DNA (blue) were stained with Hoescht. Tf puncta co-localize with EEA.1, and to a lesser extent, CD63. Scale bar, 10 μm.