Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening (original) (raw)
Figure 6
Coxiella OmpA is involved in host cell invasion and intracellular replication.
A431 (A) and THP-1 (E) cells were incubated with wt Coxiella, the control transposon mutant Tn1832 or the OmpA mutant Tn208. 60 minutes after infection (top panels) cells were fixed and labeled with an anti-Coxiella antibody coupled to Alexa Fluor 555 (blue) and with Atto-647N phalloidin (red) prior to cell permeabilization. Internalized bacteria were detected by GFP fluorescence (green) in the case of Tn208 and Tn1832 whereas for wt Coxiella infections, cells were permeabilized and bacteria were stained with the anti-Coxiella antibody as above, coupled to Alexa Fluor 488 (green). Alternatively, cells were fixed 6 (A, bottom panels) or 5 (E, bottom panels) days after infection, DNA (red) was labeled with Hoechst 33258 and wt Coxiella (green) with the specific antibody as above. The automated image analysis software CellProfiler was used to calculate the percentage of internalized bacteria (B and F), the number of colonies/cell (C and G) and the area (in microns2) of intracellular Coxiella colonies identified for each condition (D and H). Values are means ± standard deviations of triplicate experiments where an average of 8000 bacteria (B and F) or 400 vacuoles (C, D, G, H) were analyzed for each condition (values were compared to wt Coxiella infections. ns = non-significant; *** = P<0.001 2way ANOVA for B and F and t test for C, D, G, H). The difference between the percentage of internalized wt Coxiella (or the control mutant Tn1832) and the Tn208 mutant was statistically significant in B (P<0.001, 2way ANOVA) and non-significant in F. Arrows indicate internalized bacteria (green); arrowheads indicate extracellular bacteria (green and blue). Scale bars 10 µm.