Fig. 4.

The intramolecular interactions in IncA_87–246 are critical for its fusogenic activity. a IncA_mutant-FLAG complemented-IncA KO strains express comparable amounts of IncA_mutant-FLAG protein (Supplementary Fig. 10). HeLa cells were infected with the indicated strains for 24 h and then lysed in sample buffer. Samples were analyzed by western blot using anti-FLAG and anti-MOMP primary antibodies. C. trachomatis MOMP served as a loading control for infection. b Immunofluorescence microscopy analysis of HeLa cells infected with the indicated IncA_mutant-FLAG complemented IncA KO strain at 24 hpi. Bacteria were labeled with anti-MOMP (green) antibody and the expression of IncA_mutant-FLAG on the inclusion was revealed with anti-FLAG (red) antibody. DNA was stained with Hoechst (blue). The ring-like FLAG staining (red) shows that the IncA_mutant-FLAG constructs are secreted onto the inclusion surface, indicating that any loss of fusogenic function is not due to their mislocalization. Scale bar = 10 µm. Images are representative of three independent experiments. c and d Quantification of homotypic fusion with the indicated IncA_mutant-FLAG complemented IncA KO strain. Data were normalized to IncA_WT-FLAG (c) or IncA_1–246-FLAG (d) Chlamydia. IncA KO Chlamydia served as a negative control. Graphs display the means of three independent experiments ± the standard deviation. Asterisks indicate statistical significance where, * denotes p-values < 0.05, **denotes p-values < 0.01, and *** denotes p-values < 0.001 (two-tailed student t-test). Source data are provided as a Source Data file