Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches.

Reagents

Reagents were obtained from the following sources: mouse anti-GM130 (BD Biosciences), rabbit anti- Chlamydia MOMP (K. Fields, U. of Kentucky), mouse anti-CT813 (G. Zhong, U. of Texas Health and Science Center), mouse anti-vimentin (Invitrogen, clone V6630), rabbit anti-GAPDH (Abcam), mouse anti-EGFP monoclonal antibody (Clontech), rabbit anti-LAP1 antibodies (William Dauer, U. of Michigan), rabbit anti-RpoB/C (M. Tan, UC Irvine), rabbit anti-CPAF (27), mouse anti-phosphotyrosine (Cell signaling), Alexa Fluor 555-conjugated anti-mouse, Alexa Fluor 488-conjugated anti-rabbit, Hoescht 33258 (Invitrogen), FluorSave Reagent (Calbiochem), Staurosporine (Cell Signaling), IL-1β (Biolegend), Cyclohexamide (Sigma-Aldrich), Rifampicin (Sigma-Aldrich), Trimethoprim (Sigma-Aldrich), Slow Fade Gold Antifade Reagent (Invitrogen), JetPrime (Polyplus transfection). Enhanced green fluorescent protein (EGFP)-tagged rat vimentin was provided by Ronald Liem (Columbia University, NY), and tandem dimer Tomato (tdTomato) was provided by Marc Caron (Duke University, NC). Full-length lamin-associated protein 1 (LAP1) was PCR-amplified from MGC Human sequence-verified cDNA clone 3458117 (Thermo Fisher) and inserted into pLEGFP-C1 (Clontech) downstream of EGFP to express an EGFP-LAP1 fusion protein.

Cell Culture and Chlamydia Infections

HeLa cells (CCL-2: ATCC) and Vero cells (CCL-81: ATCC) were maintained in DMEM HG supplemented with 10% FBS (CellGro Mediatech). C. trachomatis LGV-L2 434/Bu and C. trachomatis mutant strains were propagated by infecting HeLa cells with elementary bodies (EBs) that had been stored in sucrose-phosphate-glutamate (SPG) buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid, pH7.0) and purified on Omnipaque (GE Healthcare) density gradients [ 31 ]. EBs were added to HeLa cells at the indicated multiplicities of infection (MOIs), and infections were synchronized by centrifugation at 2500 × g for 30 min at 10°C. Rifampin, spectinomycin, and trimethoprim resistant C. trachomatis LGV-L2 variants were generated as previously described [ 32 ]. The mutations leading to antibiotic resistance in these strains were determined by whole genome sequencing: H471Y in CTL0567 ( rpoB ), G1197 in CTL_r01 / CTL_r02 (16S rRNA copies 1 and 2), and G408R in CTL0369 lead to Rif R , Spc R , and Tmp R , respectively.

Plaque assays

Plaque assays were performed as previously described [ 33 ]. Briefly, monolayers of Vero cells grown in a 6-well plate were infected with a concentration of ~100 inclusion-forming units (IFUs) per well. Cells were incubated for 2 hours at 37 °C and 5% CO 2 , allowing for bacterial internalization. The growth medium in infected cell monolayers was replaced with 6 mL of a DMEM/agarose overlay/well (DMEM HG, 10% FBS, 50 μg/mL Gentamicin, 500 ng/mL cyclohexamide, 1X nonessential amino acids (Gibco), 0.5% SeaKem LE agarose (Lonza)) allowed to solidify for 10 minutes, and dried in a sterile cabinet without lids for 15 minutes. Cells were incubated for 10 to 14 days until plaques were observed. Plaques were isolated using a pipette tip and transferred to Vero cell monolayers for amplification.

Identification and whole genome sequencing of CPAF-deficient LGV-L2 strains

Identification of cpa mutants

LGV-L2 strains containing the null alleles C127T (Q43*) and G882A (W294*) in cpa (CTL0233) were initially identified by whole genome sequencing of pools of chemically-mutagenized and plaque-purified C. trachomatis LVG-L2 434/Bu strains (Bastidas R. J. and Valdivia R. H. unpublished results). Strain CTL2-M532 harboring the cpaC127T allele and strain CTL2-M169 harboring the cpaG882A allele were identified from among two independent pools of mutants and the mutations in the cpa locus ( CTL0233 ) were confirmed by Sanger sequencing.

Genomic sequencing

Strains were harvested from infected Vero cells grown in a 6-well cell culture plate by hypotonic lysis of host cells with 800 μl of dH 2 O per well (for 20 minutes) followed by addition of 200 μl of 5X SPG buffer. Lysates were sonicated (2 × 10 seconds in ice water) and bacterial cells spun down at 14,000 rpm, for 15 minutes at 4 °C. Bacterial pellets were pooled and resuspended in 1X DNAse I buffer (New England Biolabs, Ipswich, MA, USA). Depletion of host DNA was achieved by treating cell suspensions with 4 units of DNAse I (New England Biolabs) for 1 hour at 37 °C. Bacterial pellets were washed with PBS buffer and total DNA isolated with a DNA isolation kit (DNeasy tissue and blood kit, Qiagen) following the manufacturers instructions. M169 (1 μg) and M532 enriched DNA (1 μg) were each sheared with an Adaptive Focused Acoustics S220 instrument (Covaris). DNA sequencing libraries were prepared with a library construction kit (TruSeq DNA Sample Preparation Kit v2, Illumina, Inc. San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were sequenced in a MiSeq DNA Sequencing Platform (Illumina) at the Duke University IGSP DNA Sequencing Core facility. Genome assembly and single nucleotide variant (SNV) identification was performed with Geneious Software version 6 (Biomatters - http://www.geneious.com ). The C. trachomatis LGV L2 434/Bu genome (GenBank no. NC_010287 ) was used as reference sequence. All non-synonymous single nucleotide variants (SNVs) identified in M169 and M532 (Table 1 and Table S1 ) were verified by Sanger dideoxy DNA sequencing.

Generation of M169 RST recombinant strains Recombinant

LGV-L2 strains were generated as previously described [ 33 ]. Briefly, confluent Vero cells grown on a 24-well plate were co-infected with M169 (Rif R ) and a Spc R mapping strain at a MOI ratio of 3:3. Recombinant progeny were selected from among plaques that formed in the presence of 200 ng/mL Rif and 200 μg/mL Spc. Plaque-purified recombinants were further expanded in Vero cells and genotyped to assess the segregation of mutations present in the CTL2-M169 parental strain. Rif R and Spc R M169 recombinants harboring the cpaG882A allele were further backcrossed to a trimethoprim resistant (Tmp R ) mapping strain as described above and progeny selected from plaques that formed in the presence of Spc and 150 μg/mL Tmp. These second-generation, plaque-purified recombinants were further expanded in Vero cells and a M169-derived recombinant strain harboring the cpaG882A allele (M169 RST17 (CPAF − )) was identified by genotyping with SNV specific primers. A co-isogenic recombinant strain (RST5 (CPAF + )) that shares the same background SNVs as RST17 (CPAF − ) and that inherited a wild type cpa allele was also identified. The genomes of both RST recombinant strains were re-sequenced and the relevant SNVs identified (Table 2 and in Table S2 ). A list of C. trachomatis strains described in this study can be found in Table S3 .

Western blot analyses

HeLa cells were grown in 6-well plates to confluency and infected with C. trachomatis LGV-L2 Rif R parent or its derived mutant strains at an MOI of 1. At the indicated hours post infection, cells were washed with 1X PBS (Invitrogen), lysed with 1% SDS buffer (1% SDS, 150 mM NaCl, 50 mM Tris-HCl pH 7.5) heated in boiling water bath immediately before addition to cells. Lysates were incubated at 65 °C for 10 minutes to solubilize and denature proteins and sonicated 2×10 seconds to shear DNA. Protein concentrations were determined by the DC protein assay (Bio-Rad). Equal amounts of lysate were loaded into SDS-PAGE 4–15% gradient gels (Bio-Rad), transferred to 0.45 μM nitrocellulose membranes using a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad), blocked in Odyssey blocking buffer (LI-COR) and incubated in primary antibodies, followed by incubation with goat anti-rabbit IRDye 680LT (LI-COR) or goat anti-mouse IRDye 800CW (LI-COR). Membranes were imaged with the LI-COR Odyssey infrared imaging system. In Vitro CPAF cleavage assays HeLa cells grown in 6-well plates were mock-infected or infected with the C. trachomatis LGV-L2 Rif R strain or with the indicated mutant strains at an MOI of 1. At 40 hours post infection (hpi) crude protein extracts were prepared by lysing infected cells in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40) supplemented with a protease inhibitor cocktail (Roche). Purified recombinant CPAF (40 μg) [ 34 ] or crude protein extracts were prepared as described above and incubated with 20 μg recombinant GST-CT695 for 1hr at 37 °C, and cleavage was assessed by colloidal blue Commassie Blue staining (Invitrogen). Figures were compiled and intensities adjusted for display using Photoshop CS6. To test the effectiveness of 1% SDS buffer in preventing post-lysis degradation by CPAF, the activity of recombinant CPAF in this denaturing buffer was assessed. Crude protein extracts were prepared from HeLa cells by rinsing monolayers with PBS, adding 1% SDS buffer pre-warmed to 100 ° C, transferring to a microfuge tube, heating at 65 ° C for 10 minutes, and then clarifying the lysate by centrifugation (10,000 × g , 15 minutes, room temperature). For the in vitro CPAF cleavage assays, 200 μg of total HeLa protein extract with or without 100 μM of the CPAF-inhibitory peptide Pep2 [ 34 ] was mixed with 0.5, 2.5, or 5 μg recombinant CPAF in a final volume of 100 μl. The reactions were assembled at room temperature, incubated at 37 ° C for 20 minutes, and then inactivated with SDS-PAGE sample buffer and incubation at 65 ° C for 10 minutes. As a positive control, reactions were also performed under non-denaturing conditions. Crude protein extracts were generated from HeLa cells by rinsing monolayers with PBS, adding ice-cold TNEX buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, complete protease inhibitor cocktail (Roche)), incubating at 4 ° C for 10 minutes and then clarifying the lysate by centrifugation (10,000 × g , 15 minutes, 4 ° C). Reactions were assembled on ice, incubated at 37 ° C for 20 minutes, and subsequently processed as described for the reactions in 1% SDS buffer. CPAF activity was determined by monitoring vimentin cleavage via western blot analysis. In vitro cleavage assays were used to demonstrate that CPAF cleaves EGFP-LAP1. HeLa cells transfected with the EGFP-LAP1 construct were harvested in TNEX buffer, and crude protein extracts were generated as described above. For the reactions, 100 μg of transfected or non-transfected extract was incubated with either recombinant CPAF or TNEX protein extracts prepared from mock-infected or LGV-L2-infected (44 hours post-infection) HeLa cells. The reactions were assembled on ice, incubated at 37 ° C for 30 minutes, and then inactivated with SDS-PAGE sample buffer and incubation at 65 ° C for 10 minutes. Cleavage was assessed by western blot analysis with mouse anti-EGFP) and rabbit anti-LAP1 antibodies.

IFU burst assays

Vero cells were seeded onto 96-well plates (density of 15,000 cells/well) and infected with each of the strains analyzed (six biological replicates per each timepoint) at a MOI = ~0.6. To determine the input inclusion forming units (IFUs), a set of infected wells were fixed with 100% methanol (EMD Millipore) for 10 minutes on ice and stained with a polyclonal anti-LGV-L2 sera followed by Alexafluor-conjugated secondary antibodies and cells samples mounted for fluorescence microscopy using the FluorSave reagent. Images were acquired in a Zeiss Axioskop 2 upright epifluorescence microscope and the number of inclusions in at least 5 different fields per replicate were counted. Output IFUs at 30 hpi and 48 hpi were determined by lysing infected cells as described in [ 33 ] and infecting Vero cells with serial dilutions of harvested cell lysates. After 40–42 hpi, cells were fixed, immunostained, and the number of inclusions determined as described above. To determine the infectious progeny generated per input bacteria (“IFU burst”), the total number of output IFUs was divided by the total number of input IFUs.

Immunofluorescence microscopy

For routine indirect immunofluorescence, HeLa cells were grown on glass coverslips and infected at the indicated MOIs. Cells were fixed with 3% formaldehyde/0.025% glutaraldehyde or 4% paraformaldehyde or methanol in phosphate-buffered saline (PBS) for 20 minutes and permeabilized with 0.1–0.2% Triton X-100 for 10 minutes. After blocking with 5% bovine serum albumin (BSA) in PBS for 20 minutes, cells were stained with specific antibodies followed by Alexa-conjugated secondary antibodies at room temperature at 20 minutes. Host and bacterial DNA were stained with 1 μg/mL Hoechst. Coverslips were mounted with FluorSave, or Slow Fade.

Golgi Fragmentation Analysis

HeLa cells were seeded onto coverslips to visualize the Golgi apparatus in cell infected with Rif R or M532 (CPAF-) C. trachomatis strains. A set of infected cells were treated with Z-WEHD-fmk (75 μM, Enzo Life Sciences) for 9h after infection as a positive control for blocking Golgi fragmentation. At 24h after infection, cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized, and blocked with BSA-PBS. Bacteria, the Golgi apparatus, and DNA were detected by incubating cells with rabbit anti- Chlamydia MOMP and mouse anti-GM130 antibodies for 20 minutes followed by incubation with fluorescently conjugated secondary antibodies and Hoescht for 20 minutes at room temperature. Cells were mounted with FluorSave and allowed to cure at room temperature overnight before imaging. Images were acquired with a Zeiss 780 scanning confocal microscope with the same settings for each sample with a 100× objective and processed using ImageJ. Confocal images of specific samples were used to quantify Golgi fragmentation. The number and area of Golgi elements in 18 cells at minimum per condition were counted after a applying a threshold calculated with the Otsu algorithm for each image and using the Analyse Particles function in ImageJ software excluding particles smaller than 0.1 μm. Three independent experiments were performed and imaged to analyze fragmentation. Figures were compiled and intensities adjusted for display using Adobe Photoshop CS6. All raw data is available upon request.

Detergent Extraction Assays

For immunofluorescence assays after detergent extraction, HeLa cells were seeded in 96-well plates, with three biological replicates per condition (+Triton X-100 and −Triton X-100), and infected at an MOI of 1 with the indicated strains. At 44 hpi, 52 hpi, or 60 hpi cells in the untreated condition were washed twice with ice cold PBS and fixed with 3% formaldehyde/0.025% glutaraldehyde at room temperature for 20 minutes prior to permeabilization and blocking with BSA-PBS. For live cell samples extracted with Triton X-100, infected cells were first washed twice with ice cold PBS before incubation with pre-chilled 0.5% Triton X-100 in PBS supplemented with 100 μM anti-CPAF peptide [ 34 ] on ice for 5 minutes. Cells were then fixed, blocked with BSA-PBS and immunostained with mouse monoclonal anti-vimentin antibodies, followed by fluorophore conjugated secondary anti-mouse antibodies. Samples were mounted with Slow Fade Gold Antifade reagent and images from at least 5 fields for each replicate were acquired with a Zeiss Axioskop 2 upright epifluorescence microscope using Axiovision v3.0 software. The number of infected cells with altered vimentin staining was calculated for each replicate in 0.5% Triton X-100 treated and untreated samples. The average percentage of infected cells with altered vimentin (6–8% of total cells) staining in the untreated samples was subtracted from the treated control for each replicate. Figures were compiled and intensities adjusted for display using Photoshop CS6. Two-way ANOVA with Bonferroni’s post test was performed using GraphPad Prism for Windows, GraphPad Software, San Diego California USA. NF-κB Reporter Assays The NF-κB luciferase HeLa reporter cell line used in this study was generated by stably transducing HeLa cells with an NF-κB-Luciferase reporter system (SABiosciences) following the manufacturer’s instructions. NF-κB activity was assayed by infecting reporter cells in triplicate for each condition in 96-well plates with the indicated strains at an MOI of 10, 30, or 50. Experiments were performed in duplicate. At the time of infection, cells were treated with 10 ng/mL of IL-1β (BioLegend). After 24 hours, cells were lysed and luciferase activity measured using the britelite plus reagent (PerkinElmer) according to manufacturer’s instructions and luminescence values determined using an EnSpire 2300 Multilabel reader (PerkinElmer). Luminescence values obtained for each sample were normalized to the IL-1β treated, mock-infected control. Data was analyzed and figures generated using GraphPad Prism (GraphPad Software, San Diego California USA).

Apoptosis Induction Assay

HeLa cells grown on glass coverslips were infected with a MOI of 0.5 with the indicated strains (two biological replicates and two technical replicates per condition). Six hours prior to fixation cells were treated with 2μM staurosporine (Cell Signaling). At 24 hpi, 36 hpi, and 48 hpi infected cells were fixed with methanol and stained with anti-LGV-L2 antibodies followed by Hoechst and Alexa-conjugated secondary antibodies. The number of infected or mock-infected cells with condensed nuclei was counted using a Zeiss Axioskop 2 upright epifluorescence microscope with Axiovision v3.0 software for 200 infected cells in each condition. Data was analyzed and figures generated using GraphPad Prism.

Assessment of secondary infections

HeLa cells were seeded on coverslips in 24-well plates (50,000 cells/well) and incubated overnight. For primary infections, cells were infected with purified EBs from either C. trachomatis L2 434/Bu or its mutant Rif R derivatives, as indicated at an MOI of 1. At 29 hpi, secondary infections were performed using a C. trachomatis L2 434/Bu strain transformed with the GFP-expressing plasmid pGFP-SW2 [ 35 ]. After 1h, cells were fixed (3% formaldehyde-0.025% glutaraldehyde, 20 min, RT) and immunostained with mouse anti-phospho tyrosine monoclonal antibodies without permeabilization and counter-stained with anti-mouse Alexafluor 555-conjugated secondary antibodies (Invitrogen). Coverslips were mounted in Slow Fade Gold Antifade media and images were acquired using a Zeiss Axioskop 2 upright epifluorescence microscope with Axiovision v3.0 software. Phosphotyrosine-containing foci were counted in 35 cells for each of two biological replicates, in two independent experiments performed by two independent observers. One-way ANOVA with Bonferroni’s multiple comparison test was performed using GraphPad Prism.

EGFP-Vimentin and LAP1-EGFP transfection and live cell microscopy

Cells were seeded onto #1.5 glass-bottom plates, infected with Rif R L2 434/Bu, M532 (CPAF − ), M169 (CPAF − ), RSTE4 (TS2 − ) strains, and co-transfected with either EGFP-Vimentin or EGFP-LAP1 and tdTomato using the lipid-based JetPrime reagent 4h after infection. Cells were imaged every seven minutes from 54–76hpi after infection at 37°C under 5% CO 2 using a motorized Zeiss Axio Observer Z1 widefield fluorescence microscope equipped with a 40× air objective. Fifteen stage positions at minimum were recorded with 6–23 instances of inclusion rupture in transfected cells observed for each condition. Only cells expressing moderate levels of the fluorescent reporters were included in the analysis. Images were viewed with Metamorph to manually assess inclusion rupture by tdTomato diffusion into the inclusion lumen and the structured or diffuse nature of the EGFP signal. Images were deconvolved using Huygens Essential, and processed with ImageJ and Photoshop for presentation.

Supplementary Material Supp Table S2 Supp TableS1 Supp TableS3 Supp VideoS1 Video S1. Vimentin cleavage in live cells occurs after inclusion rupture and requires CPAF and T2S. HeLa cells were infected with the indicated strains and transfected with N-terminally EGFP-tagged vimentin and tdTomato vectors and imaged using widefield deconvolution live-cell microscopy every seven minutes after 54hpi for 14 hours. Images of representative cells from each condition ten frames prior to and following inclusion rupture were selected and processed into aligned and synchronous video which plays at 2 frames/sec. The frame immediately following loss of inclusion membrane integrity is annotated. Inclusion rupture was assessed by the influx of tdTomato signal into the inclusion lumen and occurs prior to 01:17:00. In cells infected with Rif R (WT) LGV-L2, the filamentous EGFP-vimentin signal (cyan arrowheads) became diffuse immediately after inclusion rupture. EGFP-vimentin remained in a filamentous form after loss of inclusion integrity (magenta arrowheads) in cells infected with CPAF or T2S-deficient strains. For the cell shown, loss of plasma membrane integrity occurs at 01:31:00 for WT and CPAF-strains and at 01:45:00 for the T2S-deficient strain. Elapsed time is displayed as hr:min:sec. Supp VideoS2 Video S1. LAP1 cleavage in live cells occurs after inclusion rupture and requires CPAF and T2S. HeLa cells were infected with the indicated strains and transfected with N-terminally EGFP-tagged LAP1 and tdTomato vectors and imaged using widefield deconvolution live-cell microscopy every seven minutes after 54hpi for 14 hours. Images of representative cells from each condition ten frames prior to and following inclusion rupture were selected and processed into aligned and synchronous video which plays at 2 frames/sec. The frame immediately following loss of inclusion membrane integrity is annotated. Inclusion rupture was assessed by the influx of tdTomato signal into the inclusion lumen and occurs prior to 01:17:00. Loss of EGFP-LAP1 localization to the nuclear membrane (cyan arrowheads) occurs rapidly following inclusion rupture in cells infected with Rif R (WT) but not in cells infected with CPAF or T2S-deficient strains (magenta arrowheads). Elapsed time is displayed as hr:min:sec.