Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Thomas F. Meyer ( meyer@mpiib-berlin.mpg.de ).

Animal experiments were approved by the Local District Government (LAVES; permit numbers: 33.19-42502-04-13/1223), and performed following the German regulations of the Society for Laboratory Animal Science (GV-SOLAS) for protection of animal life and the European Health Law of the Federation of Laboratory Animal Science Associations (FELASA). Ten to 12-week old male C57BL/6J mice (wildtype; RRID: IMSR_JAX:000664) were acquired from Charles River Laboratories and adapted to specific-pathogen-free (SPF) conditions at the L2/S2 animal facilities at Hannover Medical School for a minimum of two weeks. Groups to be directly compared with each other, e.g. mice infected with one serovar of Ctr and treated either with buffer or drug, were run repetitively in parallel, with similar groups sizes of n = 2 to n = 7 animals in each experimental run. Animals were assigned to the different experimental groups by chance. Before and during infection experiments, 2 to 6 mice were kept in a type 2L cage. Corresponding groups were kept in the same cage to minimize differences due to housing conditions.

Infections with mycoplasma-free (PCR) Chlamydia were conducted using Ctr serovars L2 (CTL2, ATCC VR-902B) and D (CTD, ATCC VR-885). For propagation and purification of Ctr, HEp-2 cells (ATCC CCL-23; RRID:CVCL_1906) were infected at MOI 1 in RPMI 1640 medium containing 5% FCS and 1 μg/ml cycloheximide and incubated at 35°C. After 2 d, infected cells were disrupted by vortexing with glass beads. Host cell debris was removed by centrifugation (5 min at 2,000 g and 4°C) and the supernatant was centrifuged at 48,000 g for 40 min at 4°C. The pellet was washed with SPG buffer (220 mM sucrose, 3.8 mM KHPO, 7 mM KHPO, 5 mM L-glutamic acid, pH 7.4), centrifuged and resuspended in SPG buffer using a syringe. Aliquots of 20 to 100 μl were frozen at -80°C. For mouse lung infection experiments, bacterial stocks were prepared in BHK-21 cells (ATCC CCL-10; RRID: CVCL_1915) as recently described ().

HeLa cells (ATCC CCL2; RRID: CVCL_0030; authenticated via short tandem repeat (STR) profiling by the DSMZ, Braunschweig, Germany) were maintained in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Biochrom AG). NIH3T3 and HEK 293T cells were maintained in DMEM (Gibco) supplemented with sodium pyruvate (Gibco) and glutamine (Gibco).

Anatomically normal human ectocervical tissue from a 56-year old patient was obtained from the Department of Gynecology, Charité University Hospital, Berlin, Germany and used within 3 h after removal. Scientific usage was approved by the ethics committee of the Charité University Hospital, Berlin (EA1/059/15). Informed patient consent was obtained. Cells were maintained in supplemented media as outlined below.

Method Details

Infections and Infectious Progeny Assay Infections were conducted using Ctr serovars L2 (CTL2, ATCC VR-902B) and D (CTD, ATCC VR-885) at the indicated MOIs. Unless otherwise stated, 72 h post transfection cells were infected for 2 h in RPMI growth medium containing 5% FCS at 35°C. Medium was then replaced with fresh growth medium and cells were cultured for the indicated times at 35°C. Infectious progeny assays were conducted as follows. Cells seeded in 384-well plates were infected with CTL2 and CTLD as described above. Infected cells were lysed at 48 h p.i. by adding Nonidet P40 (NP40) at a final concentration of 0.06% per well. Lysates were diluted in infection medium and transferred to fresh HeLa cells at a final dilution of 1:40. After 2 h medium was exchanged to 5% FCS RPMI containing amphothericin B (2.5 μg/ml; HyClone), gentamycin (50 μg/ml; Calbiochem) and vancomycin (50 μg/ml; Serva). Plates were incubated for 24 h at 35 C and 5% CO 2 and processed for immunolabelling. CTD-containing lysates were centrifuged at 920 x g for 30 min onto the cell monolayers to improve reinfection efficiency. For immunolabelling, Cy3-conjugated goat anti-mouse antibodies (1:200 Jackson Immunoresearch) and mouse monoclonal species-specific KK-12 IgG2a Ctr antibodies (1:10,000) obtained from D. Grayston, University of Washington, Seattle, WA, USA were used. Hoechst 33342 was obtained from Sigma. Infection experiments were performed in three independent biological experiments, unless stated otherwise in the respective figure legend.

High-throughput Screening of the Human Genome Small interfering RNAs (siRNAs, 200 nM) were arrayed in 384-well plates at 4 μl per well. To each well 8 μl of RPMI 1640 medium containing HiPerfect (0.16 to 0.24 μl per well, depending on the reagent lot) were added. Following shaking for 1 min and 15 min incubation at room temperature, 30 μl of cell suspension, containing 900 cells were added per well to give a final siRNA concentration of 19 nM. Cells were incubated at 37°C and 5% CO 2 for 72 h, infected with CTL2 at MOI 0.3 to 0.5 and further incubated at 35°C and 5% CO 2 until fixation of primary infection at 24 h post infection (p.i.) and lysis and reinfection along with cytotoxicity determination at 48 h p.i., respectively. Fixed cells were stained and immunolabelled for nuclei and Chlamydia MOMP antigen and analysed using automated microscopy and image analysis software from Olympus (ScanR). Automated liquid handling pipetting steps were performed using a Biomek FXp Laboratory Automation Workstation (Beckman Coulter). The human genome-wide siRNA library (Qiagen) consisting of Human Druggable genome V2.0 (6,992 genes covered with four siRNAs per gene), Human Supplement V1.0 (∼10,000 genes covered with two siRNAs per gene) and Human predicted Genes siRNA Set V1.0 (∼6,000 genes covered with two siRNAs per gene) was used for screening. In total ∼22,950 target genes were screened three times independently. RNA interference oligonucleotide siAllstars (scrambled, neutral control for screen), siGMPS (inhibitory control, 5’-AACAGAGAACTTGAGTGTATT-3’), siPLK1 (toxic control, 5’-CCGGATCAAGAAGAATGAATA-3’) and transfection reagent HiPerfect were purchased from Qiagen.

Indirect Immunofluorescence Labelling Cells were fixed with ice-cold MetOH and stored at 4°C until immunofluorescence labelling. After 30 min blocking with PBS containing 0.2% BSA and 0.05% Tween 20, cells were incubated with the primary antibody against Ctr species-specific KK-12 IgG2a (1:10,000) for 1 h, washed twice with PBS and incubated for 1 h with Cy3-conjugated goat anti-mouse secondary antibody (1:200) and 10 μg/ml Hoechst. After washing twice with PBS, the labelled plates were stored at 4°C until image acquisition.

Automated Microscopy and Image Analysis The numbers of Chlamydia-infected host cells were determined using automated microscopy (Olympus Soft Imaging Solutions): Images were taken with DAPI and Cy3 filter sets (AHF-Analysetechnik). ScanR Analysis Software (Olympus Soft Imaging Solutions) was used to automatically identify and quantify Chlamydia inclusions and cell nuclei.

Determination of Cytotoxicity For determination of cytotoxicity upon siRNA knockdown in host cells the CytoTox-ONE assay (Promega) was performed to determine overall LDH activity in cell lysates. 7 μl of CytoTox-ONE substrate were mixed with 7 μl of cell lysate obtained in the infectious progeny assays and incubated for 20 min at room temperature. Fluorescence was then measured using an Envision plate reader with excitation wavelength of 560 nm and emission wavelength of 590 nm. Host cell viability upon compound treatment was determined by measuring glycolytic production of NAD(P)H in viable cells with WST-1 assay (Roche). 48 h after addition of compounds 4 μl WST-1 reagent was added to cells in 40 μl culture medium. After incubation for 2 h at 37°C the OD was measured at 450 nm.

Metabolite Profiling Pietzke et al., 2014 Pietzke M.

Zasada C.

Mudrich S.

Kempa S. Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics. Kuich et al., 2014 Kuich P.H.

Hoffmann N.

Kempa S. Maui-VIA: a user-friendly software for visual identification, alignment, correction, and quantification of gas chromatography-mass spectrometry data. HeLa cells were seeded and infected with CTL2 at MOI 1 for the indicated time points (12, 24, 36 and 48 h). For cell harvesting, cells were quickly flushed with washing buffer (140 mM NaCl, 5 mM HEPES, 2.5 g/l glucose + 4 mM glutamine). Cells were then immediately quenched with 5 ml ice-cold 50% methanol containing 2 μg/ml cinnamic acid (Sigma) for internal control and scraped into the solvent. The lysate was stored at −20°C. For metabolite extraction, 1 ml chloroform was added and samples were shaken for 1 h at 4°C, followed by centrifugation for 10 min at 5000 rpm and drying overnight in a speedvac. Then samples were dissolved in 600 μl 20% methanol, followed by 1 h shaking at 4°C, centrifugation for 10 min at 5000 rpm and drying of samples overnight in a speedvac. Samples were derivatised by adding 40 μl of methoxyamine hydrochloride solution (Sigma, 40 mg/ml in pyridine, Roth) and shaking for 90 min at 30°C. N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA; Machery-Nagel; 120 μl/sample) was added and samples were shaken at 37°C for 1 h followed by 10 min centrifugation at maximum speed. Gas chromatography-mass spectrometry (GC-MS) measurement and the analysis were carried out as previously described (). The GC-MS chromatograms were processed with the ChromaTOF software (LECO). Mass spectra data were extracted using the software tool Maui-VIA ().

Isolation of Human Primary Cells and Propagation Human ectocervical tissue was washed with PBS, minced with surgical scissors and incubated in 0.5 mg/ml collagenase type II (Calbiochem) for 2.5 h at 37°C followed by TrypLE (Gibco) digestion for 15 min at 37°C. Dissociated tissue was pelleted and resuspended in Advanced DMEM/F12 (Gibco) and single cells were isolated by passing through a cell strainer. Cells were pelleted by centrifugation, resuspended in human ectocervical primary cell medium consisting of Advanced DMEM/F12, 12 mM HEPES (Invitrogen) and 1% GlutaMax (Invitrogen), supplemented with 1% B27 (Invitrogen), 1% N2 (Invitrogen), 0.5 μg/ml hydrocortisone (Sigma), 10 ng/ml human EGF (Invitrogen), 100 ng/ml human noggin (Peprotech), 100 ng/ml human FGF-10 (Peprotech), 1.25 mM N-acetyl-L-cysteine (Sigma), 10 mM nicotinamide (Sigma), 2 μM TGF-β R kinase Inhibitor IV (Calbiochem), 10 μM ROCK inhibitor Y-27632 (Hölzel), 10 μM forskolin (Sigma) and 1% penicillin/streptomycin (Gibco) for cell expansion in 75cm2 flasks coated with collagen (Sigma). At 70-80% confluence, cells were passaged using TrypLE and seeded onto lethally irradiated NIH3T3 fibroblasts (ATCC CRL1658; RRID: CVCL_0123) in ectocervical primary cell medium. For Chlamydia infection and lentivirus transduction experiments human primary ectocervical cells were subjected to differential trypsinization to separate fibroblasts from epithelial cells and epithelial cells were seeded on plastic dishes coated with collagen.

shRNA-Based Stable Knockdown in Human Primary Cells Naldini et al., 1996 Naldini L.

Blomer U.

Gallay P.

Ory D.

Mulligan R.

Gage F.H.

Verma I.M.

Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Zufferey et al., 1997 Zufferey R.

Nagy D.

Mandel R.J.

Naldini L.

Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Stewart et al., 2003 Stewart S.A.

Dykxhoorn D.M.

Palliser D.

Mizuno H.

Yu E.Y.

An D.S.

Sabatini D.M.

Chen I.S.

Hahn W.C.

Sharp P.A.

et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. 2 . Two days post transfection, lentiviral particles present in the medium were harvested, filtered (0.45 μm) and used for transduction of human primary ectocervical cells. Cells were seeded in 10 cm dishes one day before lentiviral particles were ready for use. At 30-40% cell confluence, lentiviral particles were added onto cells together with 8 μl of polybrene (10 mg/ml; Sigma) followed by medium exchange after overnight incubation. After four days of lentivirus transduction, 0.5 μg/ml of puromycin (Gibco) was added to select positively transduced cells. The expanded pools of transduced cells were used for determination of Chlamydia infectivity and knockdown efficiency by real-time PCR. All shRNAs used in this study were purchased from Sigma. Recombinant lentiviruses expressing shRNAs were produced according to standard protocols using a three-plasmid lentivirus packaging system (). shRNA-containing pLKO lentiviral vectors (a gift from Bob Weinberg; Addgene plasmid # 8453 ()) were dissolved in Opti-MEM medium (Gibco) together with Fugene 6 (Promega) transfection reagent and psPax2 packaging plasmids (a gift from Didier Trono; Addgene plasmid # 12260, Trono Lab Packaging and Envelope Plasmids, unpublished) and pMD.2G (VSVG) envelope plasmids (a gift from Didier Trono; Addgene plasmid # 12259, Trono Lab Packaging and Envelope Plasmids, unpublished) and incubated for 20-30 min at RT. After incubation, liposomes formed were added to the cells in growth medium. Next day, medium was replaced and left for an additional 24 h at 37°C in 5% CO. Two days post transfection, lentiviral particles present in the medium were harvested, filtered (0.45 μm) and used for transduction of human primary ectocervical cells. Cells were seeded in 10 cm dishes one day before lentiviral particles were ready for use. At 30-40% cell confluence, lentiviral particles were added onto cells together with 8 μl of polybrene (10 mg/ml; Sigma) followed by medium exchange after overnight incubation. After four days of lentivirus transduction, 0.5 μg/ml of puromycin (Gibco) was added to select positively transduced cells. The expanded pools of transduced cells were used for determination of Chlamydia infectivity and knockdown efficiency by real-time PCR.

CRISPR/Cas9 Knockout Cell Line Generation Heckl et al., 2014 Heckl D.

Kowalczyk M.S.

Yudovich D.

Belizaire R.

Puram R.V.

McConkey M.E.

Thielke A.

Aster J.C.

Regev A.

Ebert B.L. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. 2 . Two days post-transfection lentiviral particles present in the medium were harvested, filtered (0.45μm) and used for HeLa cell transduction. HeLa cells were seeded in 10 cm plates one day before lentiviral particles were ready for use. At 30-40% confluence lentiviral particles were added onto cells together with 8 μl of polybrene (10 mg/ml), followed by medium exchange after overnight incubation. After seven days of lentivirus transduction, GFP-positive cells were FACS-sorted and seeded as single cells into 96-well plates. Single cell clones of transduced cells were expanded, determined to be mycoplasma-free and used for infectivity and Western blot experiments. Guide RNAs (gRNAs) targeting IMPDH2 were designed using the CHOP CHOP tool ( http://chopchop.cbu.uib.no/ ) and cloned into pL-CRISPR.EFS.GFP plasmid (a gift from Benjamin Ebert; Addgene plasmid # 57818 ()) after digesting the vector with Esp3I restriction enzyme. HEK 293T cells (ATCC CRL3216; RRID: CVCL_0063) were transfected with gRNAs containing pL-CRISPR.EFS.GFP plasmids together with packaging vectors to produce lentiviruses for transduction of HeLa cells. Briefly, HEK 293T cells were grown to 60-70% confluency in 10 cm plates and transfected with lentiviral constructs containing gRNA and lentiviral packaging plasmids (psPax2 and VSVG). The lentiviral vectors were dissolved in Opti-MEM medium together with Fugene 6 transfection reagent and packaging plasmids psPax2 and pMD.2G (VSVG) and incubated for 20-30 min at RT. After incubation, liposomes formed were added to the cells in growth medium. Next day, medium was replaced and left for another 24 h at 37°C in 5% CO. Two days post-transfection lentiviral particles present in the medium were harvested, filtered (0.45μm) and used for HeLa cell transduction. HeLa cells were seeded in 10 cm plates one day before lentiviral particles were ready for use. At 30-40% confluence lentiviral particles were added onto cells together with 8 μl of polybrene (10 mg/ml), followed by medium exchange after overnight incubation. After seven days of lentivirus transduction, GFP-positive cells were FACS-sorted and seeded as single cells into 96-well plates. Single cell clones of transduced cells were expanded, determined to be mycoplasma-free and used for infectivity and Western blot experiments.

Rescue Experiments with GMP and AMP Human primary ectocervical cells were infected with CTL2 at MOI 0.3. Four hours post-infection MMF (0.1 and 10 μM) was added either in the presence or absence of 10 μM GMP and AMP (Sigma). After 48 h infectious progeny assay was performed to determine infectivity.

Western Immunoblotting Cells were lysed in SDS lysis buffer (100 mM Tris/HCL pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, 10% 2-mercaptoethanol) and boiled at 90°C for 10 min. Equal amounts of protein were separated using SDS-PAGE and immunoblotted using rabbit monoclonal IMPDH2 antibodies (ab131158, 1:1,000 from Abcam) and mouse monoclonal β-actin antibodies (AC-15, 1:4,000–16,000, Sigma). Secondary HRP-linked ECL anti-rabbit (NA934, 1:3,000) and HRP-linked anti-mouse (NA931, 1:3,000) antibodies were obtained from Amersham.

Determination of Compound Efficacy Compounds were serially diluted at a 1:3 ratio in 5% FCS RPMI to achieve eight different concentrations ranging from 62.5 μM to 29 nM and added to CTL2- and CTD-infected cells 5 h post infection. Infectivity and cytotoxicity were assayed and analysed as described above. Dose response curve fitting was performed using the Genedata Screener software.

Pharmacokinetics Zhang et al., 2010 Zhang Y.

Huo M.

Zhou J.

Xie S. PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. MMF (CellCept®, Roche) was administered at a single dose of 2 mg/kg intravenously in 10% DSMO/80% saline and 40 mg/kg PO in 100% saline to three male C57BL/6J mice each. Plasma samples were obtained at 5, 15, 30, 60, 120, 240, 480 and 1440 minutes after administration and analyzed for the active metabolite. MPA was extracted from plasma by protein precipitation. Extracts were analyzed by liquid chromatography tandem-mass spectrometry using a Prominence UFLC system (Shimadzu) coupled to a QTrap 5500 instrument (ABSciex). MPA was separated using a C18 column and an acetonitrile/water gradient containing 0.1% formic acid as solvent. Chromatographic conditions and mass spectrometer parameters were optimized prior to measurement. MPA plasma concentrations were calculated by means of a standard curve. Pharmacokinetic parameters were calculated using PKSolver ().

PK/PD Modeling MPA plasma concentrations obtained from oral administration were best fitted to the two-compartment model. Percent activity for given concentrations was calculated using the Hill equation (Hill slope = 1) and an in vitro IC50. All calculations were performed using unbound MPA concentrations (mouse plasma protein binding 90%).

In Vivo Studies Dutow et al., 2016 Dutow P.

Wask L.

Bothe M.

Fehlhaber B.

Laudeley R.

Rheinheimer C.

Yang Z.

Zhong G.

Glage S.

Klos A. An optimized, fast-to-perform mouse lung infection model with the human pathogen Chlamydia trachomatis for in vivo screening of antibiotics, vaccine candidates and modified host-pathogen interactions. To test the effectiveness of the drug in vivo, a mouse lung infection model with CTL2 for in vivo screening of antibiotics was used as recently described (). Ten to 12-week old male C57BL/6J mice were intranasally infected with 8 × 105 CTL2 chlamydial IFU per mouse. MMF at a dosage of 200 mg/kg body weight was administered orally twice a day starting at 6 h p.i. (see arrows in the graph). Infected control mice received buffer instead. Body weight was determined daily. Four days p.i. and 12 h after the final application of MMF all mice were sacrificed; infectious burden and other laboratory parameters were analyzed in homogenates from right lung lobes. Thawed, diluted tissue homogenates were centrifuged onto monolayers of HeLa T cells (received from R. Heilbronn, FU Berlin, Germany; subclone of HeLa229, ATCC CCL-2.1; RRID:CVCL_1276) growing in 96 well plates. After 24 h of culture at 37°C cells were fixed with ice-cold MetOH and the bacterial load (MOI) was determined by immunofluorescence, automated microscopy and quantitative image analysis as described above. Mouse INF-γ and TNF-α were quantified using ELISA Kits (Biolegend) according to the manufacturer’s instructions. The granulocyte marker enzyme myeloperoxidase was determined using the mouse MPO ELISA (HyCult Biotechnology). When an experiment was conducted with CTD serovar, an IFU of 8 × 106 per mouse was applied.