The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

Strains and cell culture.The human epithelial cell line HEp-2 was used for the overexpression assays, gDNA and protein extractions, and antibiotic studies. McCoy mouse fibroblasts were used for chlamydial transformation, and human epithelial HeLa cells were used for plaque purification. All of these cell lines were passaged routinely in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Thermo Fisher) and 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and verified to be mycoplasma-free using a LookOut Mycoplasma PCR Detection kit (Sigma). Density gradient-purified Chlamydia trachomatis L2/434/Bu (ATCC VR902B) EBs were used for the antibiotic studies. C. trachomatis serovar L2 EBs (25667R) naturally lacking the endogenous plasmid were prepared and used for transformation (see reference 64).

Bioinformatics analysis.Gene sequences of Chlamydia trachomatis were obtained from STDGen database (http://stdgen.northwestern.edu) or KEGG Genome Browser (65–67). RefSeq protein sequences from Escherichia coli, Bacillus subtilis, Mycobacterium tuberculosis, Staphylococcus aureus, and Pseudomonas aeruginosa were acquired from the NCBI protein database (https://www.ncbi.nlm.nih.gov/guide/proteins/). ClpX pairwise protein alignments to find sequence identity were performed using the NCBI Protein BLAST function (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (68). Multiple-sequence alignments were performed using Clustal Omega (69) with default settings and were presented using Jalview Version 2 (70). PDB files for predicted monomeric three-dimensional (3D) structures were acquired from the Phyre2 website (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (71). Complexes were modeled using SWISS-MODEL available on the ExPASy server (72–75). Protein models and model alignments were rendered using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR-01081) (76). Docking analyses were performed using AutoDock Vina (45). Molecules were prepped using Dunbrack rotamer libraries (77, 78) to replace incomplete side chains and ANTECHAMBER for charge assignment and topology generation (79).

Plasmid construction.A full list of the primers and plasmids used is included in Table S1 in the supplemental material. The Gateway recombination system of cloning was used for plasmids for the bacterial adenylate cyclase two-hybrid (BACTH) system (80). The genes were amplified from Chlamydia trachomatis L2 genomic DNA with added attB recombination sites. The PCR products were then incubated with a pDONR 221 entry vector (containing attP recombination sites) in the presence of BP Clonase II (Invitrogen) to insert the gene via flanking attP recombination sites and remove the ccdB insert, resulting in an entry vector containing the gene of interest flanked by attL sites. These constructs were transformed into DH5α chemically competent E. coli and plated onto kanamycin-containing LB agar. Plasmid was isolated and used for the LR reaction into one of three destination vectors (pST25-DEST, pSNT25-DEST, or pUT18C-DEST). The same entry vector for any given gene was used for all three LR reactions to insert into the destination vector. Entry vector and destination were incubated at a 1:1 ratio. DH5α E. coli cells were transformed with 2 μl of the reaction mix. Purified plasmid from an individual colony was sequence verified prior to use in the BACTH assay (see below).

Constructs for chlamydial transformation were created using the HiFi Cloning (New England BioLabs) protocol. Primers were designed to add a poly-histidine (6×His) tag to the gene of interest with the overlap to insert into the shuttle vector. Primers were generated using the NEBuilder assembly tool available from New England BioLabs (http://nebuilder.neb.com). The backbone used was the pTLR2 derivative of the pASK plasmid (81). For the CRISPRi plasmid, the S. aureus dCas9 was PCR amplified from pX603-AAV-CMV::NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA (a gift from F. Zhang; Addgene plasmid number 61594 [39]) and inserted into a derivative of pBOMB4-Tet::L2 (kind gift of T. Hackstadt, NIH [82]) modified to weaken its ribosome binding site (data not shown). The gRNA cassettes were designed as previously described (38), ordered as gBlock fragments from IDT (Coralville, IA), and inserted into the BamHI site of the pBOMB4-Tet derivative encoding Sa_dCas9 to produce, for example, the plasmid pBOMBLCRia(clpP2X)::L2. HiFi reactions were assembled according to the manufacturer’s protocol. The reaction was transformed into DH10β E. coli, and isolated plasmid was verified by restriction enzyme digest and sequencing by Eurofins Genomics. Sequence-verified plasmids were transformed into Δdam Δdcm E. coli (New England BioLabs) to produce demethylated plasmid, which was verified as described earlier prior to transformation into C. trachomatis (see below).

For mutation of ClpX Walker B motif, Q5 mutagenesis (New England BioLabs) was used. Primers were designed encoding the E187A mutation for PCR linearization of the plasmid. ClpX BACTH constructs were used as a template for the PCR amplification, and plasmids were recircularized by kinase-ligase-DpnI (KLD) reaction. The resulting reactions were transformed into DH5α E. coli for plasmid production. Plasmids were isolated, and mutations were verified by Sanger sequencing (Eurofins Genomics) prior to use in the BACTH system. These plasmids also served as the template for the PCRs to produce PCR products for insertion of the mutant clpX gene into the pTLR2 plasmid.

Strains created or used in this study are listed in Table S1. Transformed E. coli strains were maintained on LB agar plates, with antibiotics as necessary. To extract chlamydial genomic DNA, EBs were subjected to heat and proteinase K treatment prior to phenol-chloroform extraction (83). Sodium hydroxide lysis was utilized for the extraction of E. coli genomic DNA. For cloning into the pLATE31 plasmid, the aLICator LIC cloning and expression kit 3 (Thermo Scientific) was used according to the manufacturer’s specifications. Plasmids were first cloned into DH5α E. coli for plasmid propagation. Transformants were screened for inserts using colony PCR with Fermentas master mix (Thermo Scientific), and positive clones were grown for plasmid isolation (GeneJet Plasmid Miniprep kit; Thermo Scientific). Sequence-verified plasmids were then transformed into BL21(DE3) ΔclpPAX E. coli (46) for subsequent protein purification.

Purification of recombinant ClpX.His-tagged C. trachomatis ClpX and C. trachomatis ClpX_E187A were purified from 500-ml cultures of BL21(DE3) ΔclpPAX E. coli transformed with the respective plasmid based on the protocol described in reference 17. Samples were induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and incubated with shaking for 20 h at 18°C. Cultures were pelleted and frozen at −80°C prior to purifications. Samples were suspended in buffer A (25 mM Tris base [pH 7.5], 300 mM NaCl, and 10 mM imidazole), sonicated, bound to HisPur cobalt resin (Thermo Scientific), and washed in buffer A. Proteins were eluted from the resin using buffer B (25 mM Tris base [pH 7.5], 300 mM NaCl, and 300 mM imidazole). Buffer exchange for ATPase assay buffer (25 mM HEPES [pH 7.2], 200 mM KCl, 20 mM MgCl₂, and 10% glycerol) was performed using a Millipore Amicon Ultra 15 filtration unit (3-kDa cutoff). ClpX proteins were quantified using the Bio-Rad protein assay, assessed for purity on 10% SDS-PAGE gels with Coomassie staining (see Fig. S9), and identified using anti-His-tag Western blotting. Blotting was performed using a mouse monoclonal anti-6×His antibody (1:1,000, Millipore HIS.H8) and a goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000). Protein samples were aliquoted and stored at −80°C.

In vitro analysis of ClpX homo-oligomerization.Ten micrograms of purified protein was incubated at for 20 min at 37°C in oligomerization buffer (25 mM Tris base [pH 7.5], 5 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol [DTT], and 1% glycerol) prior to mixing with a 5× native sample buffer (5 mM Tris [pH 6.8], 38 mM glycine, 0.06% bromophenol blue). Assays were analyzed on a Bio-Rad MiniProtean 4% to 20% gradient gel for native PAGE. Gels were assessed using Coomassie staining.

Assessment of ClpX ATPase activity in vitro.A 49.5-μl reaction mixture containing 1.5 μg of recombinant wild-type ClpX or ClpX_E187A in ATPase assay buffer (see above) supplemented with 1.7% dimethyl sulfoxide (DMSO) was preincubated for 10 min at room temperature without ATP. Next, ATP dissolved in ATPase assay buffer was added to 1 μM, giving a final volume of 50 μl, and the reaction mixture was incubated at 30°C for 1.5 h. After the 1.5 h, the reaction mixtures were incubated for an additional 30 min at room temperature. Fifty microliters of Kinase-Glo reagent (Promega) was then added and incubated at room temperature for 10 min. Luminescence of the reaction, reflecting ATP not consumed by ClpX or ClpX_E187A, was then measured using a BioTek Synergy H1 plate reader. Reactions were performed in duplicates at least three times with at least two independent protein preparations.

Determining protein-protein interactions with the BACTH system.The bacterial adenylate cyclase two-hybrid (BACTH) assay was utilized to test the interaction between ClpX wild-type and the mutant (84). The genes of interest are translationally fused to one of either subunit, denoted as T18 and T25, of the Bordetella pertussis adenylate cyclase toxin, which can complement adenylate cyclase deficient (Δcya) DHT1 E. coli. Wild-type and mutant clpX genes cloned into one of the pST25, pSNT25, or pUT18C Gateway vectors was tested for both homotypic and heterotypic interactions (9, 80). Plasmids from each background were cotransformed into chemically competent DHT1 E. coli cells, which were plated on a double antibiotic minimal M63 medium selection plate supplemented with 0.5 mM IPTG for induction of the protein, 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal), 0.04% casein hydrolysate, and 0.2% maltose. Leucine zipper motifs were used for controls in pKT25 and pUT18C backgrounds on the appropriate antibiotic selection plates because these have been previously shown to interact (85). Blue colonies, indicative of positive interaction, were screened using the β-galactosidase assay. Random positive colonies were selected and grown in M63 minimal medium with the appropriate antibiotics; 0.1% SDS and chloroform were used to permeabilize the bacteria prior to addition of 0.1% o-nitrophenol-β-galactoside (ONPG), and 1 M NaHCO₃ was used to stop the reaction after precisely 20 min of incubation at room temperature. Absorbance at the 405 nm wavelength was recorded and normalized to bacterial growth (optical density of 600 nm [OD₆₀₀]), dilution factor, and time (in minutes) of incubation prior to stopping the reaction. Totals were reported in relative units (RU) of β-galactosidase activity.

Chlamydial transformation.The protocol followed was a modification of the method developed by Mueller et al. (86) and as previously described (17). For transformation, 10⁶ C. trachomatis serovar L2 EBs (25667R) naturally lacking the endogenous plasmid were incubated with 2 μg of unmethylated plasmid in a volume of 50 μl CaCl₂ at room temperature for 30 min. Reaction volume was sufficient for one well of a six-well plate of McCoy mouse fibroblasts. Transformants were mixed with 1 ml of Hanks balanced salt solution (HBSS) and added to 1 ml of HBSS in a six-well plate. The plates were centrifuged at room temperature for 15 min at 400 × g. The plate was then incubated at 37°C for 15 min. After incubation, the HBSS was aspirated and replaced with antibiotic-free DMEM plus 10% FBS. Eight hours postinfection, the medium was replaced with DMEM containing 1 μg/ml cycloheximide and 1 U/ml penicillin. Cells infected with transformants were passaged every 48 h until a population of penicillin-resistant bacteria was established. EBs were harvested and frozen in sucrose-phosphate (2SP) (64) solution at −80°C.

Determining the effect of overexpression of wild-type and mutant Clp proteins via immunofluorescence and inclusion-forming unit analysis.C. trachomatis transformants containing plasmids encoding the 6×His-tagged protein of interest were used to infect a confluent monolayer of HEp-2 cells. Penicillin treatment was maintained throughout the duration of the infection. At 10 hpi, samples were induced or not with 10 nM anhydrotetracycline (aTc). At the designated time points, three wells of a 24-well plate were scraped in 2SP, vortexed with three 1-mm glass beads, and frozen at −80°C. At the same time point, a coverslip was fixed in 3.25% formaldehyde and 0.025% glutaraldehyde for 2 min, followed by permeabilization with cold 90% methanol for 1 min. Coverslips were labeled with primary goat anti-major outer membrane protein (MOMP; Meridian, Cincinnati, OH), rabbit anti-6×His (Abcam, Cambridge, MA), and 4′,6-diamidino-2-phenylindole (DAPI). Appropriate donkey secondary antibodies were used (Invitrogen, Carlsbad, CA). Images were acquired on an Axio ImagerZ.2 equipped with Apotome.2 optical sectioning hardware and X-Cite Series 120PC illumination lamp. Frozen IFU samples were titrated onto a fresh monolayer of HEp-2s without antibiotics. At 24 hpi, samples were fixed with methanol for 10 min, stained for MOMP, and enumerated.

Genomic DNA isolation and qPCR enumeration of genomic equivalents.At 24 or 48 hpi, one well of a six-well plate was scraped into the medium overlay and pelleted at 17,000 × g at 4°C for 15 min. Each sample was resuspended in 500 μl of cold phosphate-buffered salin (PBS), frozen three times at −80°C, and processed using the Qiagen DNeasy blood and tissue kit according to the manufacturer’s specifications. DNA concentrations were assessed using a spectrophotometer prior to dilution down to 5 ng/μl. Five microliters of the resulting dilution was used for a 25-μl quantitative PCR (qPCR) volume using SYBR green PCR master mix (Applied Biosystems). Each reaction was performed in triplicates. A standard curve using C. trachomatis L2 genomic DNA was generated for interpolation of sample threshold cycle (C_T) values. This experiment was performed three times for three biological replicates.

Analysis of HctB levels upon Clp overexpression.At 24 or 48 hpi, one well of a six-well plate per test condition was rinsed twice with HBSS. To lyse the cells, 500 μl of denaturing lysis buffer (8 M urea, 10 mM Tris, 2.5% 2-mercaptoethanol, 1% SDS) was added to each well and incubated for 15 min at room temperature. Three hundred units of universal nuclease (Pierce) per ml of lysis buffer was added immediately prior to addition to the wells. Following incubation, samples were centrifuged at 17,000 × g at 4°C for 15 min to remove any insoluble material. Samples were quantitated using the EZQ protein quantitation kit (Pierce). Fifty micrograms of each sample was run in a 4% to 20% gradient SDS-PAGE gel (Bio-Rad) and transferred to a polyvinylidene difluoride (PVDF) 0.45-μm-pore-size membrane for 1 h at 300 mA. The membrane was probed using goat anti-MOMP (Meridian) and rabbit anti-HctB (generously provided by T. Hackstadt, NIH) primary antibodies followed by staining with donkey anti-goat 680 and donkey anti-rabbit 800 (LI-COR) secondary antibodies. The membrane was imaged on an Azure c600 imaging system. The channels were gray scaled and equally contrast corrected, and the resulting images were used for integrated density measurement with FIJI software (87). To assess relative HctB levels, the HctB integrated density of each sample was normalized to its respective MOMP integrated density to avoid bias due to lower overall organism numbers. The ratios were then used to compare induced versus uninduced relative HctB levels. These experiments were performed three times for a total of three biological replicates.

Transmission electron microscopy assessment of the effect of overexpression of mutant Clp isoforms.Samples were infected and induced as previously discussed (see above). At 48 hpi, samples were fixed using 2% glutaraldehyde, 2% formaldehyde in 0.1 M Sorensen’s phosphate buffer, pH 7.2. Samples were then stained postfixation in 1% osmium tetroxide in water for 1 h. Samples were dehydrated in an ethanol series (50%, 70%, 90%, 95%, and three changes of 100% ethanol), all steps 15 min each, and then soaked in propylene oxide (100%, 3 changes for 15 min each). Samples were left overnight in a fume hood in a 1:1 mixture of propylene oxide and Embed 812. The following day, the samples were placed in molds with fresh Embed 812 and polymerized overnight in an oven set at 65°C. Blocks were thin sectioned 90-nm thick on a Leica UC6 Ultramicrotome using a Diatome diamond knife. Sections were placed on uncoated 200 mesh copper grids and stained with 2% uranyl acetate and Reynold’s lead citrate. Sections were examined on a FEI Tecnai G2 transmission electron microscope (TEM) operated at 80 kV.

Confirmation of clpP2X and incA knockdown.Briefly, two wells of a six-well plate per condition were infected with pBOMBLCRia(clpP2X)::L2 transformed C. trachomatis L2 at a multiplicity of infection (MOI) of 0.8. At either 4 or 10 hpi, samples were or were not induced with 10 nM aTc. At each designated time point, total RNA was collected using TRIzol reagent (Invitrogen) and was extracted with chloroform as described previously (17, 83, 88–90). The aqueous layer was precipitated using isopropanol, as per the manufacturer’s instructions. Samples were DNase treated using the TURBO DNA-free kit (Ambion), and 1 μg of the resulting RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen). Equal volumes of cDNA were loaded for each qPCR. To extract genomic DNA, one well per condition was harvested and processed using the DNeasy blood and tissue kit (Qiagen) according to the manufacturer’s instructions as noted above. Samples were diluted to 5 ng/μl, and 5 μl of the resulting dilution was used per qPCR. cDNA and gDNA samples were quantified using 25-μl reaction mixtures with 2× SYBR PowerUP green master mix (Invitrogen) analyzed on a QuantStudio 3 (Applied Biosystems) thermal cycler using the standard cycling conditions. A standard curve using purified wild-type C. trachomatis L2 genomic DNA was generated for sample quantification. Data are displayed as the ratio of cDNA to gDNA normalized to the 10-h uninduced sample. For incA knockdown, HEp-2 cells were infected with the pBOMBLCRia(incA)::L2 transformant, induced with 10 nM aTc as described above, and fixed at 24 hpi with methanol. Cells were labeled with primary guinea pig anti-major outer membrane protein (MOMP; kind gift of E. Rucks, UNMC), rabbit anti-Sa_dCas9 (Abcam, Cambridge, MA), and sheep anti-IncA (E. Rucks) antibodies and DAPI. Appropriate donkey secondary antibodies were used (Invitrogen). Coverslips were mounted on glass slides using ProLong Glass Antifade mounting medium (Thermo Fisher). Images were acquired on a Zeiss Axio ImagerZ.2 equipped with Apotome.2 optical sectioning hardware and X-Cite Series 120PC illumination lamp.

Determination of the effect of clpP2X knockdown on C. trachomatis.Twenty-four-well plates of HEp-2 cells were infected at an MOI of 0.8 with either pBOMBLCRia(clpP2X)::L2 or pBOMBLCRia(incA)::L2 transformed into C. trachomatis L2. Samples were induced or not at 4 hpi and were harvested and fixed, and the titer was determined as previously described. To monitor the expression of dCas9 and, if present, the complemented gene of interest, immunofluorescence samples on glass coverslips were fixed and permeabilized using ice-cold 90% methanol at 24 or 48 hpi. Each sample was then stained using goat anti-MOMP (Meridian BioScience), rabbit anti-Sa_dCas9, and mouse anti-FLAG (Sigma-Aldrich) primary antibodies. Samples were then stained with the appropriate donkey secondary antibodies and were imaged as discussed above. Each titration was fixed using 4% formaldehyde and 0.025% glutaraldehyde to preserve GFP fluorescence. IFU counts of GFP-positive inclusions are displayed as a percentage of the uninduced sample at the designated time point. Plasmid retention for each condition is displayed as the percent GFP positive to total number of inclusions for each condition.

GFP_SsrA stability assay.Chlamydial transformants harboring either pBOMBmC-GFP(VAA) or pBOMBmC-GFP(VDD) plasmids were used to infect a fresh monolayer of McCoy mouse fibroblasts on coverslips in a 24-well plate. Samples were induced or not at 8 hpi. At 16 hpi, all media were replaced with fresh DMEM with or without the designated compound. GFP expression was either continued through 20 hpi or was removed from 16 to 20 hpi for pulse-chase samples. At 20 hpi, all samples were formaldehyde-glutaraldehyde fixed as previously discussed. Coverslips were then mounted and imaged as previously discussed using a 40× lens objective. GFP and mCherry integrated densities were measured for approximately 60 fields of view on two separate coverslips per condition. These experiments were repeated for two biological replicates. To determine the ratio of GFP to mCherry, GFP integrated density measurements were divided by those for mCherry.

Effect of ClpX-targeting compounds on chlamydial growth and host cell viability.Stocks of ClpX-specific inhibitor 334 and its derivative, 365, were synthesized as previously reported (44), resuspended at 25 mg/ml in DMSO, and frozen at −20°C. Methods for the synthesis, purification, and analysis of these compounds are available upon request. A dose curve for treatment was assessed to determine an inhibitory concentration of the compounds on C. trachomatis, and 25 μg/ml was chosen (data not shown). For the 24- and 48-h samples, 500 μl of DMEM containing 25 μg/ml of the compounds was added at 8 hpi, and samples were harvested at the designated time point. For the time of infection samples, compounds were added at 15 min postinfection and removed at 8 hpi. For the reactivation samples, DMEM containing the respective compound was added at 8 hpi, washed out three times with HBSS at 24 hpi, and then replaced with DMEM only for 24 additional hours prior to harvest. To determine the effect on preformed EBs, compound was added at 24 hpi, and samples were harvested at 48 hpi. To harvest, three wells of a 24-well plate were scraped into 2SP, vortexed with 3-mm glass beads, and frozen at −80°C. Samples were titrated onto a fresh monolayer of HEp-2 cells with no treatment for enumeration. For assessment of cell viability, HEp-2 cells were plate in a 96-well dark-wall plate and were treated as described for IFU experiments. All wells were then mixed with PrestoBlue HS reagent (Invitrogen), incubated for 10 min, and measured for fluorescence using a Tecan plate reader. Values are given as percentages of the untreated sample.