Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

Bacterial strains and growth conditions.N. gonorrhoeae FA1090 (65) was cultured as specified in the text on gonococcal base solid medium (GCB; Difco) for 18 to 22 h at 37°C in the presence of a 5% atmospheric CO₂ or anaerobically as described previously (44) or in gonococcal base liquid medium (GCBL) supplemented with sodium bicarbonate at a final concentration of 0.042% and Kellogg's supplement I and II in ratios of 1:100 and 1:1,000, respectively (66). Piliated gonococci were used for DNA transformation, while nonpiliated variants were used in all other experiments. Escherichia coli strains either were grown on Luria-Bertani agar (Difco) or were cultured in Luria-Bertani broth (Difco) at 37°C.

Antibiotics were used on selected bacteria at the following concentrations: for N. gonorrhoeae, kanamycin was used at 40 μg/ml and erythromycin was used at 0.5 μg/ml, and for E. coli, kanamycin was used at 50 μg/ml and erythromycin was used at 250 μg/ml.

Genetic manipulations and site-directed mutagenesis.Oligonucleotides were designed on the basis of the genomic sequence of N. gonorrhoeae FA1090 (GenBank accession number NC_002946) using SnapGene software (version 2.8; GSL Biotech LLC) and synthesized by Integrated DNA Technologies. Genomic DNA was isolated with a Wizard genomic DNA purification kit (Promega). PCR products and plasmid DNA were purified using a QIAprep spin miniprep kit (Qiagen). PCRs were performed using chromosomal or plasmid DNA as the template, appropriate oligonucleotides, and Q5 high-fidelity DNA polymerase (NEB). E. coli MC1061 was used as the host during the molecular cloning and site-directed mutagenesis. The constructs obtained were verified by Sanger sequencing at the Center for Genomic Research and Biocomputing at Oregon State University. Transformation of N. gonorrhoeae was performed as described previously (67).

The clean deletion of aniA, resulting in the ΔaniA mutant, was constructed in N. gonorrhoeae FA1090 by in-frame replacement of aniA (ngo1276) in its native chromosomal locus with the nonpolar kanamycin resistance cassette using a strategy described by Zielke et al. (44). The constructs for the deletion of aniA were obtained using Gibson assembly (68) as described below. The 1-kb upstream DNA region was amplified using oligonucleotides aniA_up_fwd (5′-CCTTAATTAAGTCTAGAGTCGCCGGGACGGTTGGTCGA-3′) and aniA_up_rev (5′-CAGCCTACACGCGTTTCATAATGTTTTCCTTTTGTAAGAAAAGTAGGG-3′), and 1 kb downstream from aniA was amplified with primers aniA_down_fwd (5′-TAATTCCCATAGCGTTTATTAAATCGGATACCCGTCATTAGC-3′) and aniA_down_rev (5′-GCCTGCAGGTTTAAACAGTCGGCAAGGCGAGGCAACGC-3′). The kanamycin resistance cassette was amplified with oligonucleotides aniA_kan_fwd (5′-TATGAAACGCGTGTAGGCTGGAGCTGCT-3′) and aniA_kan_rev (5′-AATAAACGCTATGGGAATTAGCCATGGTCC-3′), using pKD4 as a template. The linearized pNEB193 was obtained by PCR amplification using primers pNEB193_fwd (5′-GACTGTTTAAACCTGCAG-3′) and pNEB193_rev (5′-GACTCTAGACTTAATTAAGGATCC-3′). All fragments were purified, mixed in equimolar proportions, and assembled using the Gibson assembly master mix. The plasmid obtained, pNEB193-ΔaniA, was linearized with HindIII and introduced into FA1090. Clones were selected on solid medium supplemented with kanamycin and verified by PCR with primers aniA_check_f (5′-CTGTCCCATTTTGAGAGCTCC-3′) and aniA_check_r (5′-CCTTGTGCGGCGCAATAG-3′) and immunoblotting analyses using polyclonal anti-AniA antiserum (67).

For complementation studies, the wild-type aniA allele was amplified with primers aniA_pGCC4_f (5′-CTGTTAATTAAAAAAGGAAAACATTATCAAACGCC-3′) and aniA_pGCC4_r (5′-GCTAATGACGGGTATCCGAT-3′). Subsequently, the PCR product was digested with PacI and cloned into the PacI- and PmeI-digested pGCC4 vector under the control of the P_lac promoter, and the pGCC4 vector carrying the PCR product was introduced into the chromosome of FA1090 ΔaniA, creating the ΔaniA/P_lac::aniA strain.

To generate a construct for overexpression and purification of a mutated, recombinant version of AniA, AniA D137A H280A, site-directed mutagenesis was performed in consecutive reactions using as the template pET28-aniA (36), primer pair D137A-F (5′-CGCACAACGTCGCCTTCCACGCCGCAA-3′) and D137A-R (5′-TTGCGGCGTGGAAGGCGACGTTGTGCG-3′) and primer pair H280A-F (5′-GAACTTGGTGTCTTCCTTCGCCGTCATCGGCGAAATCTTC-3′) and H280A-R (5′-GAAGATTTCGCCGATGACGGCGAAGGAAGACACCAAGTTC-3′), and a Q5 site-directed mutagenesis kit (NEB), per the manufacturer's manual. The presence of mutated sites in the plasmid obtained, pET28-aniAD137A H280A, was confirmed by Sanger sequencing.

To generate pGCC4-aniAD137A H280A, the Gibson assembly method was used to replace wild-type aniA with a fragment containing mutated sites. The fragment containing the mutation was amplified using primers RAZ414 (5′-GAACCGCCGGCAGGCACGATGGTGCTTTGTACGTTTTCGTTAATCAG-3′) and RAZ415 (5′-CTACCGCCGAAACGCCTGCAGGCGAACTGCCCG-3′) and pET28-aniAD137A H280A as the template. Primers RAZ413 (5′-CGTGCCTGCCGGCGGTTC-3′) and RAZ416 (5′-GCGTTTCGGCGGTAGCTTGTG-3′) and plasmid pGCC4-aniA were used to amplify the remaining fragment. Both fragments were gel purified and mixed in equimolar proportions before the Gibson assembly master mix was added (NEB). pGCC4-aniAD137A H280A was introduced into the FA1090 ΔaniA chromosome.

Protein purification.Overproduction of recombinant variants of AniA, including a wild-type protein which lacks the N-terminal palmitoylation signal, sAniA (36), as well as mutated AniA, AniA D137A H280A, was performed in Escherichia coli BL21(DE3) by addition of IPTG to a final concentration of 0.5 mM when the cultures reached an optical density at 600 nm (OD₆₀₀) of 0.5. Following 3.5 h of incubation, the bacteria were pelleted by centrifugation and the pellets were resuspended in lysis buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 10 mM imidazole) supplemented with a Pierce protease inhibitor minitablet (Thermo Scientific). Cells were lysed by passaging them five times through a French pressure cell at 12,000 lb/in². Unbroken cells and cell debris were separated from the soluble protein fraction by centrifugation at 16,000 × g for 30 min at 4°C. The supernatants obtained were passed through a 0.45-μm-pore-size membrane filter (VWR International) and applied to a nickel affinity column (Profinity IMAC [immobilized metal affinity chromatography]; Bio-Rad). The columns were washed with 8 bed volumes of lysis buffer and elution buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 250 mM imidazole) at 97:3 (vol/vol), and proteins were eluted with 5 bed volumes of elution buffer. Eluates were subjected to dialysis against 20 mM HEPES, pH 7.5, supplemented with 0.1 mM cupric chloride dihydrate (EMD Chemicals Inc.) twice for 1 h each time and subsequently overnight at 4°C. The purified sAniA and AniA D137A H280A proteins were mixed with 10% glycerol and stored at −80°C.

Crystallization, data collection, and structure solution.Crystallization trials were performed in the vapor diffusion hanging-drop format using a mosquito crystal robot (TTP Labtech). Crystallization solutions from JCSG core suites I to IV (Qiagen) were mixed with the protein solution at three different ratios (0.05 μl protein plus 0.15 μl crystallization solution, 0.1 μl protein plus 0.1 μl crystallization solution, 0.15 μl protein plus 0.05 μl crystallization solution). The initial crystals were harvested without optimization using suitable cryoprotection solutions and flash-cooled in liquid nitrogen. All data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. The data were processed and scaled using the XDS and XSCALE packages (69).

A 1.90-Å data set in space group P2₁2₁2₁ was collected from a single crystal grown in 0.1 M Tris-HCl, pH 8.5, 2.0 M ammonium dihydrogen phosphate (JCSG Core II-A11). The crystal was cryoprotected in crystallization solution supplemented with 20% glycerol. Initial phases were determined by molecular replacement using Phaser software (70) and the AniA structure (PDB accession number 1KBV) as a search model (27). The model was rebuilt using the Coot program (71) and refined using the Phenix program (72).

A 2.35-Å data set in space group I4₁22 was collected from a single crystal grown in 0.2 M potassium thiocyanate, 20% PEG 3350 (JCSG core I-C9) and cryoprotected in crystallization solution supplemented with 20% glycerol. The overall completeness of this data set was 84.8% due to the small crystal size and radiation damage. The structure was solved using Phaser software and the AniA structure in space group P2₁2₁2₁ (PDB accession number 5TB7) as a search model. The structure was corrected using the Coot program and refined using the Phenix program.

The quality of the structures was assessed using Coot and the MolProbity server (http://molprobity.biochem.duke.edu) (73). The structural superpositions were performed using the Dali server (http://ekhidna2.biocenter.helsinki.fi/dali/) (74). The structural figures were generated using the PyMOL molecular graphics system (version 1.8; Schrödinger, LLC).

Immunoblotting.Expression of AniA was assessed in a panel of geographically, temporally, and genetically diverse N. gonorrhoeae isolates, including commonly used laboratory strains FA1090 (65), MS11 (75), 1291 (76), and F62 (77); clinical isolates LGB1, LG2, LG14, LG20, and LGB26, which were collected from two public health clinics in Baltimore, MD, from 1991 to 1994 and differ in their porB variable region types and pulsed-field gel electrophoresis patterns (36, 79); 13 isolates from patients attending the Public Health-Seattle & King County, WA, Sexually Transmitted Disease clinic from 2011 to 2013 (the UW strains in Fig. 2) (44); and the 2016 WHO reference strains (46). All strains were cultured concurrently on solid medium collected from GCB plates for 20 h in 5% CO₂ at 37°C, and whole-cell lysates were prepared in SDS sample buffer in the presence of 50 mM dithiothreitol, normalized by the number of OD₆₀₀ units, and separated in 4 to 20% mini-Protean TGX precast gels (Bio-Rad). The immunoblotting analysis was performed using polyclonal rabbit antiserum against recombinant AniA (3).

Phage display.To identify peptide ligands interacting with AniA, two M13-based phage libraries, Ph.D.-C7C and Ph.D.-12 (New England BioLabs), were used in biopanning experiments following procedures described in a study reporting on the identification of peptide inhibitors targeting Clostridium difficile toxins A and B (80). Magnetic Ni-NTA bead-based affinity capture was used to immobilize sAniA. A preclearance step was included prior to each round of biopanning to remove Ni²⁺ and plastic binders from the phage pool (58, 80). The phages from each library (10¹⁰ PFU/ml) were incubated with magnetic Ni-NTA agarose beads (250 μg capacity) at room temperature for 1 h. The supernatants of this solution provided the precleared phage pool. For target immobilization, Ni-NTA beads (capacity, 50 μg) were coated with 100 μg of sAniA. Unbound sAniA was washed away, and the precleared phage pool was added. Following incubation, unbound phages were removed by washing 20 times with TBST (50 mM Tris-HCl, pH 8.6, 150 mM NaCl, 0.5% Tween 20). Three rounds of biopanning were performed, with increasing specificity being obtained by raising the Tween 20 concentration from 0.1% in the first biopanning experiment to 0.5% in the subsequent two rounds during the wash step (64, 81). Elution of phages was performed with 0.2 M glycine-HCl (pH 2.2). At the end of each round of selection, eluted phages were titrated and amplified in E. coli ER2738 (NEB) per the manufacturer's protocol. After the third round, 24 phage plaques from each library were randomly selected. Phage DNA was purified as recommended in the manufacturer's protocol (NEB) and sequenced using 96 gIII sequencing primer 5′-CCCTCATAGTTAGCGTAACG-3′.

Phage ELISA.Individual wells in 96-well transparent plates (Greiner-Bio) were coated with 100 μl of 100 μg/ml of sAniA suspended in 0.1 M NaHCO₃, pH 8.6, and incubated overnight at 4°C in an airtight humidified box. After incubation, excess target solution was shaken out and the wells were filled with 300 μl of blocking buffer (0.1 M NaHCO₃, pH 8.6, 5 mg/ml bovine serum albumin). Following 2 h of incubation at 4°C, the blocking buffer was removed and the wells were washed six times with 200 μl of TBST (50 mM Tris-HCl, pH 8.6, 150 mM NaCl, 0.5% Tween 20). Isolated individual phage amplifications (10¹⁰ PFU) in 100 μl of TBST were incubated at room temperature for 1 h with rocking. All wells were then cleared by shaking and washed six times with 200 μl of TBST. A solution containing anti-M13 HRP-linked monoclonal antibodies (1:5,000 dilution in blocking buffer; NEB) was distributed at 200 μl per well, and the plate was incubated with rocking at room temperature for 1 h. The wells were washed six times with TBST and incubated by use of Turbo tetramethylbenzidine (TMB)-ELISA substrate for 30 min with rocking at room temperature. To stop the reaction, 100 μl of 1.78 M H₂SO₄ was added to each well. A Synergy HT plate reader (BioTek) measured the color intensity, and readings were compared to those in the control wells, which underwent a treatment that was identical to that described above but that lacked sAniA (designated the control), and to the signal from wild-type infectious virions that did not display peptides, wild-type phage M13KE (10¹⁰ PFU; designated the wild type), derived from the phage display cloning vector (NEB). Data from seven independent experiments are shown as means and standard errors of the means (SEMs).

Measurements of nitrite reductase activity with purified AniA variants.Enzymatic activity assessments with sAniA and AniA D137A H280A were performed on the basis of previous work (27, 41), but a highly sensitive fluorometric assay for nitrite measurements that relies on the reaction of nitrite with 2,3-diaminonaphthalene (DAN) to form the fluorescent product, 1-(H)-naphthotriazole, was used (42). All reactions were conducted under anoxic or microoxic conditions. Fluorescence was measured at an excitation wavelength of 360/40 nm and an emission wavelength of 460/40 nm at a gain of 50 using a Synergy HT plate reader (BioTek). The sAniA and AniA D137A H280A activities are expressed as the mean reaction rate (nanomoles of nitrite reduced per minute per microgram of protein) from at least 10 independent experiments over the course of a nitrite utilization assay (31).

Determination of peptide inhibitory concentrations.Synthetic peptides 12-5 (H-KHYYGGDTTTLWGGGS-NH₂), C7-3 (H-ACNYCRLNLWGGGS-NH₂), C7-3m1 (H-ACNYSRLNLWGGGS-NH₂), C7-3m2 (H-ACSYCRLNLWGGGS-NH₂), C7-3P (H-ACNFCRLNLWGGGS-NH₂), C7-3A (H-ACNACRLNLWGGGS-NH₂), and C7-3Bio, which is a version of C7-3 biotinylated at the N terminus (biotinAhx-ACNYCRLNLWGGGS-NH₂), were acquired from Pepmic Co., dissolved in double-distilled H₂O or dimethylformamide, and serially diluted before addition to sAniA (1 μM). Nitrite measurements with DAN were performed as described above after preincubation of samples for 1 h at room temperature. Control reactions consisted of sAniA alone and AniA D137A H280A, treated in the same manner as the experimental samples. Reactions were performed in at least 12 independent experiments. Percent inhibition was calculated using the formula 100 · {1 − [(x − y)/(z − y)]}, where x, y, and z are the concentrations of nitrite in samples containing sAniA incubated with synthetic peptides, AniA D137A H280A, and sAniA, respectively. The data were analyzed with a nonlinear log(inhibitor)-versus-response-variable slope (four-parameter) curve-fitting technique (GraphPad) to obtain IC₅₀s. All experiments were performed in at least 10 independent trials.

Whole-cell nitrite utilization studies.N. gonorrhoeae FA1090 ΔaniA/P_lac::aniA and ΔaniA/P_lac::D137A H280A were grown to an OD₆₀₀ of ∼1.0, gently spun down, decanted, and resuspended in GCBL. Cell suspensions were incubated in the absence or presence of synthetic peptides 12-5, C7-3, C7-3m1, and C7-3m2 (0.15, 0.3, and 0.6 mM) at 37°C for 30 min. Subsequently, 0.1 mM sodium nitrite was added and the samples were transferred into an anaerobic chamber. After 1 h, nitrite consumption was measured using the Griess reagent (Biotium) as previously described for an N. gonorrhoeae nitrite reductase whole-cell assay (31). Absorbance (OD₅₄₅) values were measured using a Synergy HT plate reader (BioTek), and the nitrite concentration was assessed against a nitrite standard prepared in GCBL. Percent inhibition was calculated using the formula 100 · {1 − [(x − y)/(z − y)]}, where x, y, and z are the concentrations of nitrite in samples containing FA1090 ΔaniA/P_lac::aniA incubated with synthetic peptides, ΔaniA/P_lac::aniA D137A H280A, and ΔaniA/P_lac::aniA, respectively. Experiments were performed on at least three separate occasions, and means with SEMs are reported.

Determination of MIC₅₀.N. gonorrhoeae 1291 (82) cells were grown on GCB for 18 to 22 h at 37°C in the presence of 5% atmospheric CO₂. Bacteria were inoculated into GCBL supplemented with 0.042% sodium bicarbonate and incubated with shaking at 37°C for 3 h. Then, the culture was diluted to a concentration of approximately 5 × 10⁵ CFU/ml in brain heart infusion (BHI) broth supplemented with 10% (vol/vol) Levinthal's base, 1% (vol/vol) IsoVitaleX, and 2 mM sodium nitrite (31) containing serially diluted synthetic peptides C7-3, C7-3m1, and C7-3m2. Cultures were incubated anaerobically at 37°C for 18 h, serially diluted, and spotted onto GCB for CFU enumeration. The MIC₅₀ was defined as the minimum concentration of the peptide that reduced the mean number of CFU by 50% in comparison to that for the vehicle (dimethyl sulfoxide)-treated culture. Experiments were performed in biological triplicate.

Open state of AniA homotrimer structure.Normal mode analysis (NMA) is a method for characterizing the motions of macromolecules based on basis vectors (normal modes), which describes the flexibility of the molecule (83–85). The visual inspection of the 20 lowest-frequency normal modes computed by the elNémo server showed that the first four lowest frequencies of the normal mode of vibrations corresponded to the largest-amplitude motions of the extended and collapsed conformations of the AniA structure (86, 87). Therefore, these modes were used for generating alternative conformational transitions of the protein. To generate alternate conformations of AniA, we started from the structure of AniA in the P2₁2₁2₁ space group (PDB accession number 5TB7) and displaced the protein along the subspace defined by the first low-frequency mode. Such movements were made to generate a more extended state transition (conformation) of the whole structure. Next, the resulting protein conformation defined by the amplitude of variation was energy minimized. The resulting structure was then submitted to the elNémo server, the normal modes were computed, and the protein was displaced again using the second-lowest new normal mode. This process was repeated until the protein structure was opened using the fourth-lowest normal mode.

Molecular modeling of peptide binding with AniA.The multiple conformational states of the peptides were generated using OMEGA software (Open Eye Scientific Software) and molecular dynamics (MD) simulations (88). The docking poses of the multiconformations of peptide structures (ligands) were performed using 4Dshape+ software (Chem Design Solutions LLC) (89, 90). The docking strategy exhaustively docked/scored all possible positions of each ligand (each peptide conformation) in the AniA binding site. The rigid docking roughly consisted of two steps: shape fitting and application of optimization filters. During the shape fitting, the ligand (peptide structure) was placed into a 0.5-Å-resolution grid box encompassing all active-site atoms (including hydrogen atoms) using the smooth Gaussian potential. Two optimization filters were subsequently processed: rigid-body optimization and optimization of the ligand pose in the dihedral angle space. The pose ensemble was filtered to reject poses that did not have sufficient shape complementarity with the active site of the protein. In separate docking runs, the binding poses of the ligand structure were refined by MD simulations followed by free energy calculations using the Sander module from the Amber12 package (91) as previously described (92–95). The AniA-peptide binding complex was neutralized by adding appropriate counterions and was solvated in a rectangular box of TIP3P (transferable intermolecular potential with 3-points model) water molecules with a minimum solute wall distance of 10 Å (96). The solvated systems were energy minimized and carefully equilibrated. These systems were gradually heated from a temperature of 10 K to one of 298.15 K in 50 ps before an MD simulation was run. The MD simulations were performed with a periodic boundary condition in the NPT ensemble (ensemble in which number of atoms, pressure, and temperature are constant) at a temperature of 298.15 K with Berendsen temperature coupling and a constant pressure (pressure, 1 atm) with isotropic molecule-based scaling. A time step of 2.0 fs was used, with a cutoff of 12 Å being used for the nonbonded interactions, and the SHAKE algorithm was employed to keep all bonds involving hydrogen atoms rigid (97). Long-range interactions were handled using the particle mesh Ewald (PME) algorithm (98). During the energy minimization and MD simulations, only the ligand (peptide) and residue side chains in the binding pocket were permitted to move. We used this constraint to prevent any changes in the AniA structure due to the presence of residues in the loops on the top of the protein active site. A residue-based cutoff of 12 Å was utilized for noncovalent interactions. MD simulations were then carried out for ∼10.0 ns. During the simulations, the coordinates of the system were collected every 1 ps. The last 20 snapshots of the simulated structure of the MD trajectory were used to perform the binding free energy calculations.

Binding free energy calculation.The stable MD trajectory obtained for each AniA-peptide complex was used to estimate the binding free energy (ΔG_bind) by using the Sietraj program (99). The program calculates the solvated interactions energies (SIE) using five terms and three parameters that were fitted to reproduce the binding free energies of a data set of 99 ligand protein complexes by Naim et al. (99). Sietraj is a substitute for the molecular mechanism/Poisson-Boltzmann surface area (MM/PBSA) method (100).

Biolayer interferometry.The binding affinity of C7-3Bio, a biotinylated C7-3 peptide (biotinAhx-ACNYCRLNLWGGGS-NH₂), to sAniA was assessed by biolayer interferometry on an OctetRed 96 system (ForteBio, Menlo Park, CA). C7-3 was first dissolved in dimethylformamide to a final concentration of 1.2 mM and finally in kinetic buffer (ForteBio) to a final concentration of 20 μg/ml. Streptavidin (SA) biosensors (ForteBio) were loaded with C7-3Bio peptides for 10 min. Unloaded tips were used as a control. The sAniA samples were prepared in kinetic buffer at concentrations of 50, 100, 500, and 1,000 nM. The baseline was established for 240 s, and the association and dissociation steps were performed for 500 s. Experiments were performed in three biological replicates with curve fitting using a 2:1 (heterogeneous ligand) model, and K_D value calculations were completed using Octet data analysis software (version 9).

Statistical analyses.Statistical analyses were performed using one-way analysis of variance followed by Dunnett's multiple-comparison test with GraphPad Prism software (version 6). Differences were considered significant when P was <0.05.

Accession number(s).The PDB accession numbers of the newly solved P2₁2₁2₁ and I4₁22 crystals are 5TB7 and 5UE6, respectively.