(A) HeLa cells were infected with C. trachomatis (Ctr) wild-type (WT) or Tn-cdu1 and the proteasome inhibitor MG-132 was added 8 hpi. Cells were lysed under denaturing conditions 24 hpi and Mcl-1 was precipitated. Precipitated Mcl-1 (left panel) as well as the whole proteome (right panel) were analyzed for their ubiquitination pattern by immunoblot using an anti-ubiquitin antibody. (B) HeLa cells were infected with Ctr WT or the Tn-cdu1 mutant for 24 hr and fixed with 4% PFA/Sucrose. Indirect immunofluorescence staining against ubiquitin (FK2, red channel) and Mcl-1 (green channel) was performed. DAPI staining (blue channel) marks the host cell nucleus and chlamydial DNA. (C) Quantification of ubiquitin co-localization with Mcl-1 by comparison of Pearson’s co-localization coefficient using the COLOC2 plugin from FIJI. 10 ROI were randomly selected from five individual experiments. The significance was calculated with the Student’s T-test ***p<0.001. (D) HeLa cells were infected with Ctr WT or Tn-cdu1. Apoptosis was induced 20 hpi with 50 ng/ml TNFα and 5 µg/ml CHX. Apoptosis induction was analyzed by PARP cleavage in an immunoblot. Depicted are mean values from the ratio of cleaved PARP/PARP calculated from four individual experiments ± SD. The significance was calculated with the student’s T-test *p<0.05, **p<0.01. (E) Infectivity assay of Ctr WT and Tn-cdu1 in primary Fimb cells. Infected cells were challenged with 5 ng/ml IFNγ upon 8 hpi. Depicted are mean values of the bacterial load calculated from three individual experiments ± SD. The significance was calculated with the student’s T-test *p<0.05. (F) Female C57BL/6 mice (n = 9) were infected transcervically with either Ctr WT, Tn-cdu1 or Tn control Tn-IGR. Five days post-infection, genital tracts were harvested, homogenized and DNA isolated. DNA from uterine horns was subjected to ddPCR to determine detectable Chlamydia copies/µL. Samples are normalized to host DNA (copies/µL). WT and Tn-IGR-infected animals showed no significant difference while both WT vs Tn-cdu1 and Tn-IGR vs Tn-cdu1 showed a significant difference between detectable copies/µL (*p<0.01, Kruskall-Wallis test with Dunn’s multiple comparison post-test). See also Figure 7—source data 1.