Volume 19, Issue 7, Pages 1406-1417 (May 2017) Absence of Specific Chlamydia trachomatis Inclusion Membrane Proteins Triggers Premature Inclusion Membrane Lysis and Host Cell Death  Mary M. Weber, Jennifer L. Lam, Cheryl A. Dooley, Nicholas F. Noriea, Bryan T. Hansen, Forrest H. Hoyt, Aaron B. Carmody, Gail L. Sturdevant, Ted Hackstadt  Cell Reports  Volume 19, Issue 7, Pages 1406-1417 (May 2017) DOI: 10.1016/j.celrep.2017.04.058 Copyright © 2017 Terms and Conditions

Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 1 Multiple Inclusion Membrane Proteins Are Required for Intracellular Replication, Inclusion Development, and In Vivo Infection (A) HeLa cells were infected with wild-type L2 or Inc mutant at an MOI of 1, and at 0, 4, 12, 24, 36, or 48 hr, cells were lysed and replated on fresh monolayers to enumerate progeny IFUs. Data are representative of two independent experiments. Statistical analysis was tabulated using one-way ANOVA and generated a statistical difference of p < 0.0001 for all mutants compared to wild-type L2 at 48 and 36 hr. Error bars represent SEM. (B) Immunofluorescence was conducted on HeLa cells infected at an MOI of 2 for 18, 24, or 36 hr. Cells were fixed with methanol and probed with anti-MOMP (green) and anti-IncA (red) antibodies, respectively. Images were acquired by confocal microscopy. Scale bar represents 10 μm. Data are representative of three independent experiments with at least 100 infected cells observed per experiment. (C) Five female C3H/HeJ mice were intra-vaginally infected with EBs for each Inc mutant and wild-type L2. Bacterial shedding was determined by enumerating infectious progeny EBs by plating on HeLa cells for IFU assay. Statistical analysis was tabulated using one-way ANOVA and generated a statistical difference of p < 0.01 (∗∗) or p < 0.05 (∗) compared to wild-type L2. Error bars represent SEM. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 2 Infection of Host Cells with CT229::bla, incC::bla, and CT383::bla Triggers Programmed Cell Death (A) LDH release into the supernatant of infected cells was monitored at 18 and 24 hr post-infection. Statistical analysis was tabulated using t test and generated a statistical difference of p < 0.0001 (∗∗∗) or p < 0.05 (∗) compared to wild-type L2. Error bars represent SEM. (B) TUNEL staining of C. trachomatis-infected HeLa cells. DAPI (blue) was used to visualize the nucleus, bromodeoxyuridine (BrdU) staining (red) stained nuclei with nicked DNA, and bacteria were stained with anti-MOMP (green) antibodies. Data are representative of two independent experiments. Scale bars, 10 μm. (C) At 18 hr post-infection, infected cells were stained with Annexin V (FITC) and counter-stained with propidium iodide (PerCP). Stained cells were analyzed on a FACS Aria II Cell Sorter. Data are representative of two independent experiments. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 3 CT229, incC, and CT383 Mutants Trigger Cytochrome c Release and Caspase Cleavage (A and B) HeLa cells were infected with EBs for 18 hr or treated with 1 μM staurosporine for 4 hr. Host cells were lysed and immunoblots probed with (A) anti-PARP or (B) pro- or cleaved caspase-3, -7, or -9 antibodies. The black lines in the panels for procaspase 9 and cleaved caspase 7 represent regions from the same gel and exposure spliced for presentation purposes. (C) Uninfected or infected cells or staurosporine-treated cells were fractionated into cytosolic or mitochondrial fractions and probed with anti-cytochrome c antibodies. Data are representative of at least two independent experiments. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 4 Host Protein Synthesis and Retrograde Trafficking Is Required for CT229::bla, but not incC::bla or CT383::bla, Inclusion Lysis HeLa cells were infected at an MOI of 2 with each strain in the presence or absence of 1 μg/mL cycloheximide or 1 μg/mL brefeldin A. (A) Immunofluorescence microscopy of cells infected for 18 hr in the presence or absence of cycloheximide or brefeldin A. Bacteria were stained with anti-MOMP (green) or anti-IncA (red). Scale bars, 10 μm. (B and C) At 18 hr post-infection, LDH release was monitored (absorbance 450) and infectious progeny EBs were enumerated by IFU assay in the absence or presence of (B) cycloheximide or (C) brefeldin A. Data are representative of at least two independent experiments. Statistical analysis was tabulated using t test and generated a statistical difference of p < 0.0001 (∗∗∗), p < 0.001 (∗∗), or p < 0.05 (∗). Error bars represent SEM. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 5 Cytosolic Chlamydiae Associate with Lysosomes and Autophagosomes (A) HeLa cells were infected at an MOI of 2, and at 18 hr post-infection, cells were fixed with formaldehyde and stained with anti-MOMP (green) and anti-LC3, Lamp1, or STING (red). Scale bar, 10 μm. (B) HeLa cells were infected at an MOI of 2, and at 18 hr post-infection, cells were lysed in SDS and analyzed by western blotting. Membranes were probed with anti-LC3 or anti-p62. Data are representative of two independent experiments. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 6 Inhibition of Autophagy Prevents Host Cell Death for Host Cells Infected with CT383::bla or incC::bla (A) HeLa cells were infected at an MOI of 2 in the presence or absence of 5 mM 3MA. LDH release was assessed at 18 hr post-infection. Error bars represent SEM. (B) HeLa cells were infected at an MOI of 1 with or without 3MA. At 44 hr post-infection, host cells were lysed and infectious progeny EBs were enumerated by IFU assay. Error bars represent SEM. (C) HeLa cells were infected with mutant EBs with or without 5 mM 3MA, and at 18 hr post-infection, samples were lysed with SDS and membranes were probed with pro- or cleaved caspase-9. (D) Beclin-1 and (E) STING knockdown cells were infected at an MOI of 2, and LDH release was monitored. Data are representative of at least two independent experiments. Statistical analysis was tabulated using one-way ANOVA and generated a statistical difference of p < 0.0001 (∗∗∗), p < 0.001 (∗∗), or p < 0.05 (∗). Error bars represent SEM. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions

Figure 7 Model for the Triggering of Apoptosis in C. trachomatis-Infected Cells The absence of specific C. trachomatis Incs triggers premature inclusion lysis and host cell death. The absence of specific inclusion membrane proteins results in a fragile inclusion that ruptures as it begins to expand. Damaged inclusion membranes, lacking CT383 or IncC, are recognized by STING and LC3. Activation of autophagy triggers intrinsic apoptosis resulting in truncation of the chlamydial developmental cycle. Cell Reports 2017 19, 1406-1417DOI: (10.1016/j.celrep.2017.04.058) Copyright © 2017 Terms and Conditions