Abstract (English)

The Neisseria gonorrhoeae Type IV pilus is a dynamic fiber involved in host cell attachment, DNA transformation, twitching motility, and evading the innate immune system. We previously reported that pilus expression affects iron homeostasis and sensitivity to killing by oxidative (iron-dependent antibiotic streptonigrin and hydrogen peroxide and non-oxidative (antimicrobial peptide LL-37) agents. Here, we use in vitro evolution to identify genes involved in N. gonorrhoeae susceptibility to streptonigrin. We identified a mutation in the NGO0059 locus that encodes HpaC that results in a glycine to cysteine change in position 93. Although HpaC homologs are known as part of a two-component FAD-dependent monooxygenase system consisting of a hpaC reductase and a hpaB monooxygenase, Neisseria lack the monooxygenase. While HpaC increases streptonigrin sensitivity, HpaC also promotes hydrogen peroxide and LL-37 resistance. We tested whether the HpaC effect in streptonigrin, hydrogen peroxide and LL-37 sensitivity involved the Type IV pilus. We determined that HpaC affects streptonigrin independently of the pilus while hydrogen peroxide- and LL-37-mediated killing involves both HpaC and the pilus. We demonstrate that the Gly93Cys change conferred enhanced affinity for FAD and resulted in a loss-of-function phenotype in streptonigrin susceptibility. These data suggest that HpaC’s role in FAD oxidation and reduction impacts pilus-dependent and -independent resistance against neutrophil-mediated killing.

Methods (English)

Materials and Methods

Strains and Growth

          Gc and E. coli strains and plasmids used in the study are listed in Table 1. To control for T4p phase variation, the experiments performed here used an FA1090 strain designated N-1-60 which contains point mutations in the pilE G4 and is pilC2 phase-locked “on” [20]. Plasmids were introduced into Gc by spot transformation [56]. For solid medium, Gc was grown on gonococcal growth broth: 36.25 g/L GC Medium Base (Difco), 1.25 g/L agar, and Kellogg supplements I and II at 37 °C in 5% CO2. For liquid growth, 50 mM sodium bicarbonate and supplement I was added to Gc broth and cells were grown with aeration at 37 °C. Antibiotics and their concentrations used for selection in GCB were kanamycin (Kan) 50 µg/ml and erythromycin (Erm) 1 µg/ml. NEB 5-alpha competent E. coli (High Efficiency) cells used to propagate plasmids and NEB BL21 (DE3) Competent E. coli used to express protein were grown in Luria-Bertani (LB) solid medium containing 15 g/L agar or liquid media at 37 °C. The antibiotics and their concentrations used in LB were ampicillin (Amp) 100 µg/ml and Kan 50 µg/ml.

Mutant construction

hpaC::kan mutant

An approximately 650 bp fragment containing 270 bases upstream of NGO0059, the first 30 bases of NGO0059, a PacI restriction site, HA tag, NotI restriction site, the last 30 bp of NGO0059, and 270 bp downstream of NGO0059, was synthesized and cloned into pTwist-Amp-MC vector by Twist Biosciences.

A PacI and NotI flanked nptII fragment with a 12-mer DNA uptake sequence at the 3’ end of nptII from a previously generated plasmid [57] was introduced into the PacI- and NotI- digested plasmid from Twist Biosciences in between the upstream and downstream sequences of NGO0059. This plasmid pTwist-hpaC::kan was used to spot transform Gc strains to generate ΔhpaC::kan strains. Transformants were selected on GCB Kan and checked by diagnostic PCR and sequencing.

ΔhpaC::EcoR1-EcoRV-BamH1 polylinker mutant

We constructed the ΔhpaC::EcoR1-EcoRV-BamH1 mutant by replacing the kanamycin resistance cassette in ΔhpaC::kan with a EcoR1-EcoRV-BamHI (5’-GAATTCGATATCGGATCC-3’) polylinker sequence. To replace the kan cassette in pTwist-hpaC::kan, we ran site-directed mutagenesis on pTwist-hpaC::kan using primers NGO0059_ERB_F (5’-atcggatccAGGCCTTTAGACTGATATTC-3’) and NGO0059_ERB_R (5’-atcgaattcCTGCAAATCCGCCATTTTTC-3’) with Q5 polymerase according to manufacturer protocol (NEB E0554). The DNA mix was treated with a kinase, T4 ligase, and DpnI mix for 5 minutes at room temperature before transformation into NEB 5-alpha Competent E. coli (NEB C2987H). We confirmed the sequence of the fragment from ampicillin resistant clones by Sanger sequencing with primers hpaC_500F (5’-gatgacccaattcaggcctattct-3’) and hpaC_500R (5’- gtcgggcagcagggaaa-3’). The plasmid pTwist-hpaC::EcoR1-EcoRV-BamH1 was spot transformed into ΔhpaC::kan. We screened transformants with Kan sensitivity by PCR and Sanger sequencing to confirm the presence of the polylinker sequence in hpaC, generating the ΔhpaC::EcoR1-EcoRV-BamH1 mutant (N-8-57).

hpaC(Gly93Cys) point mutant

A 1532 bp PCR product carrying hpaC(Gly93Cys) was generated by using primers NGO0059upF (5’- ATGCCGTCTGAAATACAGGCAAGGGAAGCC-3’) and NGO0059dnR (5’-TGAATGTCAGTCCGTTGCC-3’) from an evolved ΔpilE mutant carrying hpaC(Gly93Cys) and purified using a Qiagen PCR cleanup kit. The PCR product was sent for Sanger sequencing to confirm the presence of the Gly93Cys mutation (G277T nucleotide change) and spot transformed into CRISPRi-pilE (N-5-22, [27]). We swabbed the cells from the spot into liquid GCBL for SPN selection with IPTG and spread on GCB agar plates. We screened individual colonies for hpaC the G-to-T mutation by PCR and Sanger sequencing.

Complementation strains

We expressed hpaC from the transcriptionally silent lctP-aspC intergenic region [58]. PacI- and PmeI-flanked on the 5’ and 3’ ends of hpaC (NGO0059) was amplified by PCR using primers hpaC_PacI_F (5’-cctTTAATTAAatggcggatttgcagaaaact-3’) and hpaC_PmeI_R (5’-aggGTTTAAACtcagtctaaaggcctaaactgcc-3’) from Gc. After PCR cleanup, the PCR product was cloned into pCR2.1-TOPO vector following manufacturer’s protocol (Invitrogen K450002), resulting in pTOPO-hpaC. We sequenced hpaC using M13F and M13R (5'-CAGGAAACAGCTATGAC-3') primers. We amplified the hpaC from pTOPO-hpaC with KOD polymerase (Sigma 71085), gel extracted the products, digested the purified PCR product and pGCC4 [58] with PacI and PmeI in rCutsmart buffer (New England Biolabs) for 1 hour at 37 °C, heat inactivated the enzymes for 20 minutes at 65 °C, and ligated the digested PCR product and pGCC4 for 2 hours at room temperature. We transformed the ligation into NEB 5-alpha E. coli (NEB C2987H) or JM109 competent cells (Promega L2005) and selected for Kan (for pGCC4 and pTOPO) resistance and sensitivity to Amp (for pTOPO). We confirmed the inserted fragment DNA with Sanger sequencing using primers hpaC_PacI_F and aspCrev (5’-AGTGGAACGAAAACTCACGT-3’). We transformed pGCC4-hpaC plasmid into piliated hpaC null mutants and selected for erythromycin resistance, resulting in the construction of ΔhpaC/nics::hpaC (N-8-53). To construct ΔhpaC/nics::hpaC(Gly93Cys) (N-9-38), we used site-directed mutagenesis of pTOPO-hpaC, sequenced to confirm the presence of the mutation, cloned hpaC(Gly93Cys) into pGCC4, and spot transformed the plasmid into ΔhpaC (N-8-57). To construct ΔpilEΔhpaC/nics::hpaC (N-8-63), we deleted pilE by screening for nonpiliated colonies after backcrossing genomic DNA from FA1090 ΔpilE (N-1-69) and validating by PCR using primers pilRBS (5’-GGCTTTCCCCTTTCAATTAGGAG-3’) and SP3A (5’-CCGGAACGGACGACCCCG-3’) [20].

Construction of plasmids for HpaC and Gly-93 variant protein expression

The pET28a vector carrying FA1090 hpaC was synthesized (Twist Bioscience) and transformed into BL21 DE3 E. coli cells (Table 1, N-8-3). We performed site-directed mutagenesis (NEB E0554S) on pET28a-FA1090 hpaC to generate pET28a-FA1090 hpaC(Gly93Cys). For Gly93Cys, we used primers FA1090_hpaC_F (5’-CGGGCTGACCTGCCTGTCGCCCG-3’) and reverse primer (5’-GCAAAATGTTCGGCAACATCCTG-3’). We treated the reactions with a kit-provided kinase, T4 ligase, and DpnI mix for 5 minutes at room temperature before transforming 2.5 µl into NEB 5-alpha E. coli (NEB C2987H). After confirmation of the mutation with sequencing, we transformed BL21 DE3 cells and selected for Kan resistance.

In vitro evolution of ΔpilE mutant and backcrossing into CRISPRi-pilE

The pilE clean deletion mutant was grown on GCB agar plates for 16 hours at 37 °C with 5% CO2. Cells were swabbed and inoculated into 15 ml conical tubes containing 1 ml GCBL with supplement I and sodium bicarbonate, shaking for 1.5 hours at 37 °C. The culture was diluted 1:5 with 5 ml GCBL with supplement I and sodium bicarbonate and grew for 1.5 hours at 37 °C. The culture was diluted to an OD550 0.2 and treated with 0.5 SPN or DMSO for 30 minutes at 37 °C. Cells were pelleted at 4000 rpm for 2 minutes and resuspended in fresh GCBL. Cells were spread onto GCB agar plates, grown overnight at 37 °C with 5% CO2, and stored at -80 °C in GCBL and 20% glycerol and swabbed to enter the next round of growth and selection. The process of growth and selection was repeated six times and relative survival compared to the unevolved ΔpilE mutant was performed three times during the experiment. Genomic DNA was isolated at the sixth round of selection using phenol chloroform extraction and submitted for Illumina sequencing with SeqCenter in Pittsburgh, Pennsylvania. We aligned the reads for each isolate to the reference sequence N-1-60 (Accession number CP115654.1) and called variants relative to the reference using Snippy v4.6.0, then filtered only for variants that were unique to the evolved isolates (Table S1).

For isolating the mutations, we spot transformed the gDNA into the CRISPRi-pilE strain (N-5-22) [27]. The patch of cells that were exposed to the gDNA mix was swabbed into GCBL, diluted 1:5 with GCBL with supplement I and sodium bicarbonate and grew for 1.5 hours at 37 °C, cultures were diluted to an OD550 0.05 and grew with and without 1 mM IPTG for 2 hours before treatment with 0.5 SPN or DMSO for 30 minutes at 37 °C. Cells were washed by pelleting at 4000 rpm for 2 minutes and resuspending in fresh GCBL. We performed 10-fold dilutions of the cultures onto GCB agar plates and overnight at 37 °C with 5% CO₂. Individual colonies were frozen in 100 µl of GCBL with glycerol in 96 well plates. Isolates were tested for SPN sensitivity compared to CRISPRi-pilE in the presence and absence of IPTG. Genomic DNA prepared with phenol chloroform were sent for whole genome sequencing and variant calling with SeqCenter. Sample libraries were prepared using the Illumina DNA Prep kit and IDT 10bp UDI indices, and sequenced on an Illumina NextSeq 2000, producing 2x151bp reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert (v3.9.3), a proprietary Illumina software for the conversion of bcl files to basecalls. Variant calling was carried out using Breseq under default settings [59].

Gc antimicrobial killing assays

Cells were grown on GCB agar plates for 16-18 hours at 37 °C with 5% CO2. Individual colonies representing biological replicates were picked and streaked onto GCB agar plates and incubated for 16-18 hours at 37 °C with 5% CO2. Cells were inoculated to an OD550 between 0.2-0.3 in 1 ml GCBL with supplement I and sodium bicarbonate and grew for 1.5 hours at 37 °C. Cultures were diluted 1:5 with 5 ml GCBL with supplement I and sodium bicarbonate and grew for 1.5 at 37 °C. Cultures were diluted to an OD550 0.05 and grown with and without 1 mM IPTG if the strain carries a CRISPR interference array for 1.5-2 hours. The conditions for hydrogen peroxide, LL-37, and SPN killing assays and the method to determine the relative survival then follow the protocol described in Hu and Stohl et al.

Complementation

For complementation, we grew Gc to mid-exponential phase, normalized the cultures to an OD550 ~0.1, and treated the cultures with or without 12.5 µM IPTG for 1 hour at 37 °C. We then treated piliated FA1090 and ΔhpaC/nics::hpaC (N-1-60 and N-8-53) with 5 µM SPN and nonpiliated ΔpilE and ΔpilE ΔhpaC/nics::hpaC (N-1-69 and N-8-63) 1 µM SPN for 20 minutes at 37 °C.

Purification of FA1090 HpaC and HpaC(Gly93Cys)

A 2.5 ml overnight culture of BL21 DE3/pET28a-FA1090 hpaC or BL21 DE3/pET28a-FA1090 hpaC(Gly93Cys) mutant was diluted into 250 ml LB with kanamycin in a 1 L flask and grew for 2 hours shaking at 37°C. IPTG was added to a final concentration of 0.1 mM and grew overnight with shaking at 37 °C. Cells were pelleted at 4500 rpm at 4°C. The cell pellets were resuspended in 10 ml resuspension buffer (50 mM Tris-HCl, pH 8, 2 mM ethylenediaminetetraacetic acid). Cells were centrifuged and frozen at -80 °C overnight. To lyse the cells, the pellets were resuspended in 25 ml lysis buffer (50 mM NaH2PO4, pH 8, 0.5 M NaCl, 1% triton X-100, with 2 tablets of freshly added cOmplete Mini protease inhibitor, Roche 11836153001) and pulse sonicated for 5 minutes at 35% amplitude in 30 second intervals separated by 20 seconds on ice. Lysates were centrifuged at 4500 rpm at 4°C for 30 minutes. The supernatants were treated with DNase (300 units/25 ml lysate), incubated for 1 hour at room temperature, and passed through a 0.45 µm filter and stored at 4°C. We equilibrated 2.5 ml of cOmplete His-Tag Purification Resin (Roche 5893682001) with 20 column volumes (50 ml) of buffer A (50 mM NaH2PO4, pH 8, 300 mM NaCl). We added the equilibrated resin to the cell lysates and mixed gently overnight at 4°C. We packed the resin-lysate mix by gravity-assisted flow onto a column and the flow-through was collected. We washed the resin with buffer A with 20 mM imidazole and collected the washes in separate tubes for later analysis. We eluted the His-tagged protein with buffer A with 70 mM imidazole into separate fractions and stored the samples at 4°C. We ran the flow-through, washes and elutions on an SDS-PA gel to check the quality of the purification. The most concentrated elution fractions were pooled, diluted to 1 mg/ml, and dialyzed with protein storage buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 50% v/v glycerol) overnight at 4°C and stored at -80 °C. We determined the concentration of protein using a BCA kit. We purified 51 µM HpaC and 15 nM HpaC(Gly93Cys).

FAD assays

We used a kit that uses FAD as a cofactor for an oxidase enzyme mix, generating a product that a probe detects, resulting in either fluorescence or a change in color (Abcam ab204710). We used the kit according to manufacturer provided protocol except at room temperature and with half of the recommended OxiRed probe and oxidase enzyme to conserve reagents. Purified His6-tagged FA1090 HpaC or HpaC(Gly93Cys) was denatured by heating at 95 °C for 5 minutes or not, cooled on ice, and added to the FAD assay reactions in black polystyrene 96-well assay plates (costar 3915). Fluorescence was measured every two minutes with excitation and emission maxima of 535/587 nm.

Aligning FA1090 HpaC to B. cepacia TftC

We aligned the peptide sequence of FA1090 HpaC to TftC from Burkholderia cepacia (PDB 3K88). We used the “align” command in PyMOL with five refinement cycles for outlier rejection. The analysis yielded an alignment with a root mean square deviation (RMSD) 0.684 Å for the backbone C_α atoms.

Bioinformatic analysis of bacterial genomes containing hpaB and/or hpaC

We searched for the terms “hpaC” and “hpaB” on the National Center for Biotechnology Institute Gene database [50], accessed each genus listed in the taxonomic groups of Betaproteobacteria (26 genera) and Gammaproteobacteria (57 genera), confirmed the presence of HpaC and HpaB by protein sequence homology, and analyzed the genomic context surrounding either hpaC or hpaB (~10 genes up or downstream) to identify if the other gene was present. We manually curated the genera into categories of genomes having either hpaC, hpaB, or both. After ClustalW alignment of the 16S rRNA sequences, we generated neighbor-joining rooted phylogenetic trees in Jalview.

Acknowledgements (English)

The authors thank the current and past members of the Seifert lab for their insight and support. In particular, we thank Drs. Selma Metaane for her valuable feedback of the manuscript and Wendy Geslewitz for her thoughtful conversations on this work.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH). This manuscript is the result of funding in whole or in part by the NIH. It is subject to the NIH Public Access Policy. Through acceptance of this federal funding, NIH has been given a right to make this manuscript publicly available in PubMed Central upon the Official Date of Publication, as defined by NIH.