Volume 13, Issue 6, Pages 746-758 (June 2013) Chlamydia Infection Promotes Host DNA Damage and Proliferation but Impairs the DNA Damage Response  Cindrilla Chumduri, Rajendra Kumar Gurumurthy, Piotr K. Zadora, Yang Mi, Thomas F. Meyer  Cell Host & Microbe  Volume 13, Issue 6, Pages 746-758 (June 2013) DOI: 10.1016/j.chom.2013.05.010 Copyright © 2013 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Ctr Induces Extensive Alterations to Host Chromatin Architecture (A) Heatmap showing four distinct clusters depicting the kinetics of histone PTMs during persistent Ctr infection at the indicated dpi. Whole-cell Laemmli extracts of End1 E6/E7cells were used for immunoblot analysis with antibodies described in Table S1. The band densities were normalized to corresponding band densities of the β-actin loading control. Alterations in histone PTMs compared to uninfected controls are represented as fold change in the heatmap with color scale of −3 (blue) to 3 (red). (B) Human primary fallopian-tube-derived epithelial cells uninfected or infected with Ctr (36 hr) were subjected to immunofluorescence analysis of γH2AX, H3K9me3, and DNA (Draq5). (C) IMR90 cells uninfected or infected with Ctr (48 hr) were analyzed by immunoblotting for γH2AX, H3K9me3, chlamydial Hsp60, and β-actin. (D) End1 E6/E7 cells were uninfected or infected with Chlamydia strains CtrL2 and CtrD (48 hr) and C. pneumonia (Cpn) (96 hr). Total cell extracts were subjected to immunoblot analysis for γH2AX, H3K9me3, chlamydial Hsp60, and β-actin. The heatmap in (A) is from two individual experiments. The images in (B) and blots in (C) and (D) are representative of three independent experiments. See also Figure S1 and Table S1. Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 ROS-Mediated Induction of DSBs upon Ctr Infection Does Not Lead to Apoptotic Cell Death End1 E6/E7 cells were persistently infected with Ctr. (A) Cells were harvested at indicated time points and total cell lysates subjected to immunoblot analysis for γH2AX, chlamydial Hsp60, and β-actin. (B) Neutral comet assays were performed at the indicated time points. Quantity of DSBs is represented as percentage of DNA in tail. Cells treated with etoposide (50 μM) for the indicated time points were used as a positive control. (C) End1 E6/E7 cells uninfected or infected with Ctr (48 hr) were analyzed by immunoblotting for processing of procaspase-3, chlamydial Hsp60, and β-actin. Molecular masses of active caspase-3 proteins are indicated. Cells treated with etoposide for the indicated time points were used as a positive control. (D) Uninfected and Ctr-infected cells with or without DPI (10 nM) added 9 hr before fixation were subjected to immunofluorescence analysis 36 hpi for γH2AX and DNA (Draq5). (E and F) Uninfected and Ctr-infected cells with or without DPI treatment were (E) harvested 48 hpi and analyzed by immunoblotting for γH2AX, chlamydial Hsp60, and β-actin (DPI was added 9 hr before harvesting) or (F) subjected to neutral comet assay 48 hpi. Quantity of DSBs is represented as percentage of DNA in the tails. DPI was added to cells 9 or 24 hr before comet assays. (G) Uninfected and Ctr-infected cells were subjected to immunofluorescence analysis 36 hpi for γH2AX, 8-oxo-dG, and DNA (Draq5). Cells treated with tert-Butyl hydroperoxide (TBHP) were used as a positive control. (H) Ctr-infected cells with or without DPI were lysed at 48 hpi, and lysate was used to infect freshly seeded cells, which were fixed 24 hpi for quantification of infectivity. DPI was added 9 or 24 hr before lysis. Blots in (A), (C), and (E), images in (D), and data in (F) are representative of three independent experiments. Data in (B) and (F) are represented as the mean ± SEM. Data in (H) show the mean ± SD of three experiments normalized to mock-treated, infected cells. A Student’s t test was used to determine p value; ∗∗∗∗p < 0.0001 and ∗∗∗p < 0.001. n is the minimum number of cells analyzed per condition. See also Figure S2. Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Ctr-Induced DSBs Fail to Elicit DNA Repair Activities (A–D) End1 E6/E7 cells (uninfected or Ctr-infected for 36 hr) with or without etoposide treatment for the final 2 hr were subjected to immunofluorescence analysis. (A) Representative confocal images of γH2AX, pATM, and DNA (Draq5) are shown. Mean pixel intensities of γH2AX and pATM or 53BP1 per cell were quantified from confocal images with ImageJ. (B) Data shown are the mean values ± SEM for γH2AX (green) and pATM (red). (C) Representative confocal images of γH2AX, 53BP1, and DNA (Draq5). (D) Data shown are the means ± SEM for γH2AX (green) and 53BP1 (red). n is the minimum number of cells analyzed per condition. (E) End1 E6/E7 cells either uninfected, Ctr-infected (48 hr) or treated with etoposide (2 hr) were harvested and analyzed by immunoblotting for γH2AX, H3K9me3, pATM, ATM, pChk1, Chk1, pChk2, Chk2, chlamydial Hsp60, and β-actin. (F) Uninfected and Ctr-infected END1 E6/E7 cells transfected with siRNAs targeting luciferase, DNAPKc, ATR, and ATM were harvested 48 hpi and analyzed by immunoblotting for γH2AX, chlamydial Hsp60, and β-actin. (G) End1 E6/E7 cells either uninfected or infected with MOI2 or MOI4 of Ctr were additionally treated with 10 Gy ionizing radiation (IR) at the indicated time points, and cells were fixed 48 hr after IR treatment. Nuclei number was determined by automated image acquisition and analysis of cells stained with Hoechst. Data are represented as the mean ± SEM. (H–J) DNA sequences of junctions were obtained after ligation of (H) 3′-3′ linearized plasmid or (I) blunt-blunt linearized plasmid from End1 E6/E7 either uninfected or Ctr-infected for 48 hr. Occurrence of a given sequence is indicated on the right. Nucleotide misincorporations are represented in red (J). The chart shows observed mean nucleotide loss in inaccurate junctions with two experimental conditions. Data are represented as the mean ± SEM. Data in (A)–(F) are representative of three independent experiments, while data in (H)–(J) represent two independent experiments. A Student’s t test was used to determine p values; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05; p > 0.05, not significant (ns). See also Figure S3. Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Ctr-Induced DSBs Selectively Activate SAHF Formation and γH2AX in an ERK-Dependent Manner (A) Uninfected and Ctr-infected End1 E6/E7 cells were subjected to immunofluorescence analysis 36 hpi. Representative confocal images for H3K9me3, HP1β, and γH2AX are shown. (B) End1 E6/E7 cells persistently infected with Ctr were harvested at indicated time points and subjected to immunoblot analysis for H3K9me3, chlamydial Hsp60, and β-actin. (C and D) Uninfected and Ctr-infected End1 E6/E7 cells were subjected to immunofluorescence analysis with γH2AX and H3K9me3 antibodies 36 hpi with or without DPI for the final 9 hr. Mean pixel intensities of (C) γH2AX and (D) H3K9me3 per cell were quantified from confocal images with ImageJ. Data shown are the means ± SEM. (E) Uninfected and Ctr-infected cells with or without DPI treatment were harvested 48 hpi and analyzed by immunoblotting for H3K9Me3, pERK1/2, ERK2, chlamydial Hsp60, and β-actin. DPI was added to the cells 9 hr before harvesting. (F) Uninfected and Ctr-infected cells with or without U0126 treatment at 2 hpi and etoposide-treated cells alone were harvested 48 hpi and subjected to immunoblot analysis for γH2AX, H2AX, H3K9me3, H3, pERK1/2, ERK2, chlamydial Hsp60, and β-actin. (G) Uninfected and Ctr-infected cells with or without U0126 treatment at 2 hpi were subjected to neutral comet assay 48 hpi. The quantity of DSBs is represented as the percentage of DNA in tail. Cells treated with etoposide for 2 hr were used as a positive control. Data are represented as the mean ± SEM. (H) Ctr-infected cells treated with or without U0126 at 2 hpi were lysed 48 hpi, and lysates were used to infect freshly seeded cells. Cells were fixed 24 hpi and infectivity quantified. Data shown are the mean ± SD of three experiments normalized to mock-treated, infected cells. Data in (A)–(G) are representative of three independent experiments. n is the minimum number of cells analyzed per condition. A Student’s t test was used to determine p values; ∗∗∗p < 0.001; p > 0.05, not significant (ns). See also Figure S4. Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Perturbation of SAHF Augments Ctr-Induced DNA Damage and γH2AX Foci, but Not pATM Responses (A and B) Uninfected and Ctr-infected End1 E6/E7 cells treated with or without SUVi were subjected to immunofluorescence analysis. Images were acquired with an automated microscope (Olympus Soft Imaging Solutions). Integrated intensities of (A) H3K9me3 and (B) γH2AX per cell were quantified with ScanR analysis software (Olympus). Black bars indicate the means ± SEM. (C) End1 E6/E7 cells uninfected or Ctr-infected (36 hr) with or without SUVi (5 nM) or treated with etoposide (2 hr) were subjected to immunofluorescence analysis. Representative confocal images of three independent experiments for γH2AX, pATM, and DNA (Draq5) are shown with overlays. (D) The quantity of DSBs was determined in uninfected and Ctr-infected cells with or without SUVi at 48 hpi via neutral comet assays. Data are represented as percentage of DNA in tail. Cells treated with etoposide (2 hr) were used as a positive control. n is the minimum number of cells analyzed per condition. Data are represented as the mean ± SEM. (E) Ctr-infected cells treated with or without SUVi were lysed 48 hpi, and these samples were used to infect freshly seeded cells. Cells were fixed 24 hpi and infectivity quantified. Data shown are the mean ± SD of three experiments normalized to mock-treated infected cells. (F) Uninfected and Ctr-infected END1 E6/E7 cells transfected with siRNAs targeting luciferase or SUV39h1 and SUV39h2 with or without etoposide treatment were harvested 48 hpi and analyzed by immunoblotting for γH2AX, H3K9me3, pATM, ATM, chlamydial Hsp60, and β-actin. Representative blots of three independent experiments are shown. A Student’s t test was used to determine p values; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, and ∗∗p < 0.01. See also Figure S5. Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Ctr-Induces Enhanced Proliferative Signals Despite Persistent DSBs End1 E6/E7 cells uninfected or infected with Ctr were treated with doxycycline 24 hpi to induce persistence and with U0126, SUVi, or etoposide (or left untreated as a control) and cultured until 6 dpi (144 hpi). Cells were pulsed with BrdU for 1 hr before fixation and were subjected to immunofluorescence analysis with Ctr and BrdU antibodies. DNA was counterstained with Hoechst. Images were acquired with an automated microscope. (A) Population doublings over indicated time points were determined by automated counting of nuclei with Scan R analysis software. (B) Single cells positive for BrdU were scored and are represented as percentage of BrdU-positive cells. (C) End1 E6/E7 cells persistently infected with Ctr were harvested at indicated time points and subjected to immunoblot analysis for Cyclin E1/2, pERK1/2, chlamydial Hsp60, and β-actin. (D) Uninfected and Ctr-infected cells with or without U0126 treatment were harvested 48 hpi and subjected to immunoblot analysis for CyclinE1/2, CyclinB1, CyclinD1, CyclinA, pERK1/2, chlamydial Hsp60, and β-actin. Data shown in (A) and (B) are the mean ± SD from triplicates and are representative of three independent experiments. Blots in (C) and (D) are representative of three independent experiments. See also Movie S1 and Figure S6. Cell Host & Microbe 2013 13, 746-758DOI: (10.1016/j.chom.2013.05.010) Copyright © 2013 Elsevier Inc. Terms and Conditions