https://orcid.org/0000-0002-7855-4949
Figures
Abstract
Porphyromonas gingivalis is a keystone pathogen for periodontal disease. The bacteria are black-pigmented and require heme for growth. P. gingivalis exhibit resistance to many antimicrobial peptides, which contributes to their success in the oral cavity. P. gingivalis W50 was resistant to the antimicrobial peptide LGL13K but susceptible to the all-D-amino acid stereoisomer, DGL13K. Upon prolonged exposure to DGL13K, a novel non-pigmented mutant was isolated. Exposure to the L-isomer, LGL13K, did not produce a non-pigmented mutant. The goal of this study was to characterize the genomic and cellular changes that led to the non-pigmented phenotype upon treatment with DGL13K. The non-pigmented mutant showed a low minimum inhibitory concentration and two-fold extended minimum duration for killing by DGL13K, consistent with tolerance to this peptide. The DGL13K-tolerant bacteria exhibited synonymous mutations in the hagA gene. The mutations did not prevent mRNA expression but were predicted to alter mRNA structure. The non-pigmented bacteria were deficient in hemagglutination and hemoglobin binding, suggesting that the HagA protein was not expressed. This was supported by whole cell enzyme-linked immunosorbent assay and gingipain activity assays, which suggested the absence of HagA but not of two closely related gingipains. In vivo virulence was similar for wild type and non-pigmented bacteria in the Galleria mellonella model. The results suggest that, unlike LGL13K, DGL13K can defeat multiple bacterial resistance mechanisms but bacteria can gain tolerance to DGL13K through mutations in the hagA gene.
Citation: Gorr S-U, Chen R, Abrahante JE, Joyce PBM (2024) The oral pathogen Porphyromonas gingivalis gains tolerance to the antimicrobial peptide DGL13K by synonymous mutations in hagA. PLoS ONE 19(10): e0312200. https://doi.org/10.1371/journal.pone.0312200
Editor: Maria Giulia Nosotti, University of Insubria, ITALY
Received: December 20, 2023; Accepted: October 2, 2024; Published: October 24, 2024
Copyright: © 2024 Gorr et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. Genome sequences have been uploaded to NCBI (https://www.ncbi.nlm.nih.gov/sra). SRA accession numbers: SRR28355461and SRR28355462.
Funding: SUG received funding from the University of Minnesota Academic Health Center seed grant program (No URL - this program is no longer available) The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: SUG holds three patents related to the research reported in this manuscript: Synthetic peptides and peptide mimetics. U.S. Patent number: US9914750. Date of Patent: March 13, 2018. Assignee: University of Louisville Research Foundation Inventor: Sven-Ulrik Gorr. Synthetic peptides and peptide mimetics. U.S. Patent number: US8569449, Date of Patent: October 29, 2013, Assignee: University of Louisville Research Foundation. Inventor: Sven-Ulrik Gorr. Peptides, hydrogel compositions and methods of use thereof. U.S. Patent number: US12037374. Date of Patent: July 16, 2024, Assignee: Regents of the University of Minnesota. Inventors: S.-U. Gorr, C. Aparicio and Z. Ye. SUG is Chief Scientific Officer and holds equity in Gavia BIO, LLC, which is developing the antimicrobial peptide DGL13K. These interests have been reviewed and managed by the University of Minnesota in accordance wit its Conflict of interest policies. The other authors declare no commercial funding, employment, consultancy, or commercial products in development related to these patents. This does not alter our adherence to PLoS ONE policies on sharing data. Mutant strains of P. gingivalis, developed in this research, are available from the University of Minnesota upon completion of a Materials Transfer Agreement
Abbreviations: AMP, antimicrobial peptide; CFU, colony-forming units; LPS, lipopolysaccharide; MDK, minimum duration for killing; MIC, minimum inhibitory concentration; OD, optical density; THM, Todd-Hewitt medium; WT, wild-type
Introduction
Periodontitis is one of the most common infectious diseases in middle-aged and older adults, with a prevalence of 50% [1–3]. Periodontitis has consistently been associated with a number of oral bacteria, including Porphyromonas gingivalis [4–6], which is considered a keystone pathogen that can initiate dysbiosis while remaining at relatively low frequency in the healthy oral microbiome [4, 6, 7]. P. gingivalis infections have been linked to several systemic diseases [8], including atherosclerosis [9, 10], rheumatoid arthritis [11], and Alzheimer’s disease [9, 12], although causation is still debated [13–16].
P. gingivalis are black-pigmented bacteria that require heme for growth [17–19]. The bacteria produce multiple virulence factors that affect the host inflammatory response and contribute to the destruction of periodontal tissues supporting the teeth [20]. Among these virulence factors are three cysteine-proteases, Lys-gingipain (Kgp), Arg-gingipain A and B (RgpA and RgpB) [21], and a related hemagglutinin A (HagA) [20]. The latter shares adhesion domains with the gingipains Kgp and RgpA, but lacks proteolytic activity [22]. These proteins form a cell-surface complex [23] that is associated with heme-acquisition and colony pigmentation [18, 19].
A large number of non-pigmented mutants of P. gingivalis have been described [18, 24–33]. The mutations typically affect the gingipain-hemagglutinin complex [25, 28, 30, 32–34], other type IX secretion system (T9SS) components [24, 35], or LPS structure [26, 27, 29]. As a result, non-pigmented mutants may be less virulent than wild-type (WT) bacteria [31].
P. gingivalis thrives in the oral cavity, an environment that is rich in antimicrobial peptides (AMPs), both above and below the gum-line [36, 37]. The success of P. gingivalis in this environment can be attributed to the formation of biofilms [38], invasion of epithelial cells [39–41] and resistance to AMPs [36, 42–47]. The latter is likely mediated by modification of the lipopolysaccharide (LPS) structure in the outer membrane [42, 48, 49], which can generate a less negatively charged surface that does not attract cationic peptides [42]. In addition, gingipains can contribute to resistance [44, 48], although this may be peptide specific [43].
We have developed the AMP LGL13K (previously named GL13K) [45], which is derived from the sequence of the salivary protein BPIFA2 (BPI fold-containing family A member 2; former names: parotid secretory protein, PSP, SPLUNC2, C20orf70) [50–52]. An all-D-amino acid isomer of this peptide, DGL13K, was subsequently designed [53]. LGL13K, shows activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa [45] and biofilms of the latter [53] but LGL13K is degraded by bacterial proteases [53, 54] and the peptide is not active against P. gingivalis strains 55977, W50, and DPG3 [45]. To explore further the relative resistance of P. gingivalis to LGL13K, we compared the activity of LGL13K to that of the protease-resistant D-enantiomer, DGL13K [53, 54]. DGL13K, but not LGL13K, selected for a non-pigmented variant of P. gingivalis, which showed increased tolerance to DGL13K. The goal of this study was to characterize the genomic and cellular changes that led to the non-pigmented phenotype. The results suggest that, unlike LGL13K, DGL13K can defeat multiple bacterial resistance mechanisms but bacteria can gain tolerance to DGL13K through mutations in the hagA gene.
Materials and methods
Bacterial strains
WT Porphyromonas gingivalis W50 was kindly provided by Dr. Massimo Costalonga, University of Minnesota School of Dentistry. The P. gingivalis mutants KgpΔIg-B (Δkgp) [55] and Δ266 (ΔporN) [56] were created in a W83 background and kindly provided by Dr. Jan Potempa, University of Louisville School of Dentistry.
Unless stated otherwise for individual assays, P. gingivalis were routinely cultured in ‘Todd Hewitt Medium’ (THM) consisting of Todd Hewitt Broth (BD Bacto 249240) supplemented with 0.01% Hemin and 0.01% Menadione (both from Sigma-Aldrich, St. Louis, MO). Bacterial cultures were plated on THM-blood-agar consisting of 5% of sheep blood (Remel Lenexa, KS) in THM with 1.5% agar (Fisher Scientific). The bacteria were routinely cultured in an anaerobic chamber in an atmosphere consisting of 80% N2, 10% CO2 and 10% H2 at 37°C.
Peptides
LGL13K and DGL13K were purchased from AappTec (Louisville, KY) or Bachem (Torrance, CA) at >95% purity. Peptide identity and purity were confirmed by the supplier by mass spectrometry and RP-HPLC, respectively. Polymyxin B was purchased from Sigma (St. Louis, MO).
Peptides were dissolved at 10 mg/ml in sterile 0.01% acetic acid and stored at 4°C until use, as described [53]. Peptide batches were tested for antimicrobial activity by MIC assays against P. aeruginosa Xen41 [57].
Timed-kill assay
P. gingivalis was cultured in THM for 72 h at 37°C followed by centrifugation at 6500 x g for 10 min at 4°C. The cell pellets were resuspended in 10 mM sodium phosphate, pH 7.4 at 107 CFU/ml. The bacterial suspension (495 μl) was mixed with 5 μl of peptide stock solution (10 mg/ml) and incubated in an anaerobic chamber at 37°C. Five μl aliquots were removed from the reaction mixture at the times indicated and plated on blood agar until brown colonies emerged.
Minimal Inhibitory Concentration (MIC)
P. gingivalis were cultured in THM until an approximate optical density at 600 nm (OD600) = 1 was reached. The bacteria were diluted in THM to 105 CFU/ml. MICs were determined as previously described [57, 58]. Briefly, 100 μl of bacterial stock (104 CFU/well) were mixed with 20 μl of a 2-fold serial peptide dilution (concentration range: 1025 μg/ml– 1 μg/ml; control samples 0 μg/ml) in 0.01% acetic acid. The samples were incubated in 96-well polypropylene plates in an anaerobic chamber at 37°C for 3 days. The OD630 was determined in a Synergy HT plate reader (BioTek, Winooski, VT) and plotted against peptide concentration. The MIC was read as the lowest peptide concentration that prevented any bacterial growth. The pigmentation status of the cultures was confirmed by plating aliquots of the MIC experiment on blood agar.
Genome sequencing
DNA samples for whole genome sequencing were prepared from colonies isolated from four independent experiments. Each experiment contained both untreated WT P. gingivalis W50 and W50 that had been treated with DGL13K for 60 min. Individual colonies were selected and subcultured in THM to stationary phase and genomic DNA prepared using the Masterpure Gram-positive DNA purification kit (Lucigen, Middleton, WI). The samples then were purified further using ZymoDNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA). DNA sample quality was verified by a fluorimetric Picogreen assay (>0.2 ng/μl).
Genomic library generation and MiSeq sequencing were performed by the University of Minnesota Genomics Center. Briefly, DNA libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA), according to the manufacturer’s specifications. Libraries were then sequenced using an Illumina MiSeq platform (2x300 bp) using Illumina’s SBS chemistry. The BBDuk tool for Geneious software version 168 9.1.8 was used to quality trim and filter Illumina adapters, artifacts, and PhiX from reads. Paired reads with quality scores averaging <6 before trimming or with a length <20 bp after trimming were discarded.
Remaining reads were mapped to the published W83 reference genome sequence (NCBI Reference Sequence: NC_002950.2) via BWA (0.7.17-r1188) to generate BAM files. Variant calling was done in parallel across all samples via Freebayes using a minimum variant frequency of 0.01 and minimum coverage of 34 reads. Polymorphism frequencies in each culture were determined and gated at >10% threshold.
Genome sequences have been uploaded to NCBI (https://www.ncbi.nlm.nih.gov/sra) with SRA accession numbers SRR28355461 for W50 hagA23/167 and SRR28355462 for W50 WT.
mRNA modeling
The mRNA secondary structure was modeled for a 493 nucleotide segment coding for the C-terminal Cleaved Adhesin Domain (PFAM: PF07675) in HagA. This domain was identified as the C-terminal orphan K3 domain [59] and contains the two synonymous SNPs identified in this study. Centroid structures [60] were calculated using the default parameters in the Vienna RNA web suite [61, 62], which is available from http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi
Reverse transcription-polymerase chain reaction (RT-PCR)
P. gingivalis were cultured for 48 h, as described above. Bacteria were centrifuged for 10 min at 10,000 x g (4°C) and the cells resuspended in 0.5 ml of TRIzol (Thermo Fisher Scientific). Direct-zol, RNA Miniprep Plus and RNA clean-up Quick-RNA Miniprep Kits (Zymo Research) were used to isolate and purify mRNA. The mRNA was converted to cDNA using the Invitrogen PhotoScript II First-Strand Synthesis System (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.
PCR reactions were conducted in a PCR Mastercycler (Eppendorf North America, Hauppauge, NY). A 20 μl reaction containing 1.0 μl of cDNA, 10 μM of each primer and 10 μl of OneTaq 2X Master Mix with Standard Buffer (New England Biolabs) was used for double-strand DNA synthesis. The RT-PCR reactions were carried out following the recommended thermal profile: 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 58°C for 45 s and 72°C for 2 min. The specificity of the amplicons was determined by electrophoresis of the PCR products on 1.5% agarose gels (BioExpress, Chicago, IL).
Primers for P. gingivalis 16S rRNA: 5’-TGTAGATGACTGATGGTGAAAACC-3’and 5’-ACGTCATCCCCACCTTCCTC-3’; Primers for hagA: 5’-GGAGGCTCACGATGTATGGG-3’ and 5’-ATCGGCATTGACCGGAACTT-3’.
Hemoglobin binding
P. gingivalis cultures were centrifuged at 10,000 x g for 10 min and the cell pellets resuspended in PBS to approximately OD600 = 1. Human red blood cells (1 ml) were centrifuged 10 min at 10,000 x g. The pellets were resuspended in an equal volume of dH2O and then frozen at -20°C. The cells were thawed at room temperature and centrifuged as before. The supernatant (RBC lysate) was used for hemoglobin binding. Aliquots of bacterial suspension (750 μl) were mixed with 250 μl of RBC lysate. One set of samples was immediately centrifuged for 10 min at 10,000 x g (0 h incubation). A second set of samples was incubated for 1 h at 37°C and then centrifuged (1 h incubation). The supernatants were collected and the OD450 read in a plate reader. Hemoglobin binding was calculated as (OD450@0h –OD450@1h) / OD450@0h.
Hemagglutination
This assay was based on published protocols [28, 63]. Human red blood cells were washed twice in PBS and resuspended in 10x their original volume. Cultures of P. gingivalis (2 days) were centrifuged at 10,000 x g for 10 min and the pellets resuspended in PBS to an approximate OD600 = 1. Aliquots of the bacterial culture (80 μl) were diluted 2-fold in 80 μl PBS in round bottom polyvinyl chloride 96-well plates. Diluted red blood cells (80 μl) were added to each well and the plates were incubated for 3–4 h at 37°C with rocking on a Bellydancer laboratory shaker (Stovall Life Science, Greensboro, NC). Hemagglutination was determined as the highest dilution of the bacterial culture that caused ‘matting’ of the red blood cells.
Whole-cell enzyme-linked immunosorbent assay (ELISA)
P. gingivalis was cultured on hydroxyapatite-coated pegs placed in a 96-well plate (MBEC biofilm incubator; Innovotech, Edmonton, AB, Canada) for 48h. The pegs were rinsed in 300 μl/well of PBS and transferred to a polypropylene 96-well plate containing 200 μl/well of PBS with 0.05% Tween 20 and 0.5% non-fat powdered milk and the monoclonal antibody 61BG1.3 diluted 1:10,000. This antibody was developed by R. Gmuer, Institute of Oral Biology, ZZMK, University of Zürich, Switzerland. It was obtained from the Developmental Studies Hybridoma Bank, which was created by the NICHD of the NIH and maintained at the University of Iowa, Department of Biology, Iowa City, IA.
The plates were incubated with the antibody for 2.5 h at 30°C with gentle shaking and then rinsed twice in PBS followed by a 20 min wash in 300 μl/well of PBS. Each well was then incubated for 90 min at 30°C with 200 μl PBS containing 0.05% Tween 20, 0.5% dry milk and horseradish peroxidase-conjugated rabbit anti-mouse IgG diluted 1:1,000. The pegs were washed in PBS as before and incubated with horseradish peroxidase substrate in a 96-well white wall polystyrene plate. Luminescence was measured in a Biotek Synergy HT platereader after 15 min incubation with ECL Western Blotting substrate (Thermo Pierce, Rockford, IL). For each strain, the average luminescence readings in four wells, which had been incubated without primary antibody, were subtracted from each antibody reading.
Hemoglobinase assay
Stationary phase cultures of P. gingivalis were centrifuged for 10 min at 10,000 x g. The supernatants (180 μl) were mixed with 20 μl of 5 mg/ml hemoglobin in PBS. The OD405 was read at T = 0 h and then the plates were incubated for 24 h at 37°C and OD405 determined. The net OD405 was determined by subtracting background values obtained in the absence of hemoglobin at each time point. Relative proteolysis was calculated as netOD405@0h – netOD405@24h.
In vivo virulence
Stationary phase cultures of P. gingivalis were centrifuged for 10 min at 10,000 x g and the cells were resuspended in PBS to approximately 106 CFU/ml. Sixth instar larvae of Galleria mellonella (greater wax moth) were purchased locally and kept in sawdust at 4°C until use (typically 1–3 days). Groups of 9–10 larvae (270 ± 4 mg each) were injected with 10 μl (104 CFU) of P. gingivalis and incubated in a polystyrene Petri dish with oatmeal at 37°C in the dark. Dead larvae were identified by melanization and absence of movement when prodded [57, 64]. Surviving larvae were counted at the times indicated.
Results
We have previously reported that three strains of P. gingivalis (W50, ATCC 53977 and DPG3) are resistant to LGL13K, but not to the endogenous antimicrobial peptide LL-37 [45]. We have since designed an all-D amino acid version of this peptide (DGL13K) [53], which resists proteolytic degradation and kills both Gram negative and Gram positive bacteria [53, 54, 57, 65]. Timed-kill assays showed that P. gingivalis W50 were killed by DGL13K but not LGL13K or polymyxin B (Fig 1). In the course of DGL13K-treatment, a non-pigmented variant of W50 emerged and was dominant prior to complete killing of the bacteria (Fig 1). Thus, the median minimum duration for killing (MDK) [66] was about 60 min for pigmented colonies and 120 min for non-pigmented colonies.
P. gingivalis W50 were incubated for 2 hours with LGL13K, DGL13K or polymyxin B (PMX) (100 μg/ml) and aliquots plated on blood agar at the times indicated. The figure is a composite of separate culture plates from a single representative experiment.
Following DGL13K treatment, the non-pigmented colonies emerged on blood agar in the absence of the peptide (Fig 1), suggesting a stable mutant. These colonies were further expanded in culture medium in the absence of peptide. The minimum inhibitory concentrations (MICs) were determined for WT P. gingivalis and the non-pigmented W50 isolate. LGL13K (up to 1024 μg/ml) did not inhibit growth of any of the tested strains (Table 1), consistent with the timed-kill assays shown above. In contrast, DGL13K similarly inhibited the growth of WT and the non-pigmented isolate of W50.
Genome sequence and transcription
To investigate the molecular mechanism behind the lack of colony pigmentation and increased MDK for bacteria treated with DGL13K, non-pigmented colonies from four independent experiments were expanded and analyzed by whole-genome sequencing. The untreated W50 WT strain differed from the W83 reference genome sequence (NCBI Reference Sequence: NC_002950.2) in only three locations (61429, 1802991 and 1803013; W83 numbering system). In addition, sequence analyses revealed seven mutations that were present in at least 75% of sequence reads from DGL13K-treated samples but not in WT controls. Two of these mutations were found in two of four experiments (Table 2) and were selected for further analysis.
Both were synonymous SNPs located in the hagA gene. The corresponding non-pigmented mutant was named W50 hagA23/167 to denote the locations of the two SNPs. The two mutations did not prevent hagA transcription since the corresponding mRNA was readily identified by RT-PCR with no consistent differences between WT and non-pigmented bacteria (Fig 2).
Transcripts of 16S rRNA and hagA were amplified by RT-PCR and visualized by agarose gel electrophoresis. The images are representative of four (WT) and six (hagA23/167) independent samples. The raw gel image can be found in supporting data S1 Fig.
Codon usage can affect both transcription and translation [67]. Analysis of codon usage in hagA revealed that all six Ser codons and both Tyr codons were used. A T→C transition in the hagA23/167 sequence changed the more frequently used TAT Tyr codon (67 of the 100 Tyr codons in the WT sequence) to the less frequently used TAC Tyr codon (33 codons in the WT sequence) in hagA23/167. The G→A transition in the hagA23/167 sequence converted the low-use TCG Ser codon (8 of the 131 Ser codons in the WT sequence) to the 3-times more frequently used TCA Ser codon (23 codons in the WT sequence) (Table 3).
mRNA structure prediction
In addition to codon usage, mRNA structure can affect translation efficiency [68]. To determine if the synonymous SNPs affected predicted mRNA structure, the WT and hagA23/167 sequences were modeled in a 493 nucleotide domain representing the C-terminal cleaved adhesion domain (orphan K3 domain [59]), which contains both synonymous SNPs. The predicted structure of the hagA23/167 mRNA differed substantially from the WT mRNA (Fig 3A and 3D). Hypothetical mRNA structures, which contained only one of the two synonymous SNPs, were modeled (Fig 3B and 3C) to determine if both SNPs contributed to the predicted structure of hagA23/167 mRNA. Based on this analysis, it appears that alteration of the Ser codon at position 1936023 (Fig 3C) causes most of the structural change determined in the hagA23/167 mRNA. The same conclusion was reached when the secondary structures were modeled by predicting the minimum free energy (S2 Fig), which strengthens the reliability of the predicted structures [61].
A. WT; B. Model of a hypothetical hagA167 mutant, which was altered at the Tyr codon at position 1936167. C. Model of a hypothetical hagA23 mutant, which was altered at the Ser codon at position 1936023. D. Model of a hypothetical hagA23/167 double mutant. An alternate structure prediction obtained by modeling the secondary structures by predicting the minimum free energy (MFE) is shown in the supplemental data, S2 Fig.
HagA expression and function
Heme-binding and hemagglutination are associated with proteins that contain the Cleaved Adhesin Domain (CAD) [22], including Lys-gingipain (Kgp), Arg-gingipain A (RgpA) and hemagglutinin A (HagA). The non-pigmented phenotype of the non-pigmented hagA23/167 mutant suggested that it could be deficient in heme binding and uptake, which was confirmed experimentally (Fig 4). Thus, the hagA23/167 mutant showed deficient heme-binding (Fig 4A) and hemagglutination (Fig 4B), consistent with the non-pigmented phenotype.
A. Hemoglobin-binding of P gingivalis. W50 (WT) and non-pigmented W50 (hagA23/167) cells were incubated for 60 min with hemoglobin and the bound fraction determined. Data from three independent experiments were analyzed by Student’s t-test, N = 7–11. Line indicates group mean. B. Hemagglutination by P. gingivalis. Two-fold serial dilutions of W50 (WT) and non-pigmented W50 (hagA23/167) were incubated with red blood cells and hemagglutination determined by the matting of red blood cells. The maximal bacterial dilution that resulted in hemagglutination was determined in five independent experiments and analyzed by Student’s t-test. Data are shown as mean ± SEM, N = 8–9.
To determine if the Cleaved Adhesin Domain proteins were expressed at the cell surface, the monoclonal antibody 61BG1.3, which recognizes a hemagglutinating epitope in these proteins [22], was used for whole-cell ELISA (Fig 5). WT P. gingivalis resulted in a strong antibody signal, consistent with the expression of all three proteins containing this epitope, while the hagA23/167 mutant showed reduced binding, consistent with the absence of one or more CAD proteins.
Biofilms of P. gingivalis W50 (WT), hagA23/167, Δkgp or ΔporN were cultured on hydroxyapatite-coated pegs. The surface expression of Kgp, RgpA and HagA was determined by immunolabeling and quantitated by chemiluminescence (RLU). Statistical outliers were removed and the data from two independent experiments were expressed as mean ± SEM. The data for hagA23/167 were compared to control groups by one-way ANOVA with Dunnett’s multiple comparison post-test, N = 16.
For comparison, the binding of 61BG1.3 was also tested with two mutants of the closely related P. gingivalis strain W83. As expected, the secretion mutant ΔporN, did not express the three proteins at the cell surface. The mutant Δkgp¸, which does not express the Lys-gingipain, exhibited intermediate antigen expression levels, similar to that observed for hagA23/167 mutant (Fig 5). These data suggest that the hagA23/167 mutant lacks surface expression of one of the CAD proteins.
Gingipain activity
To distinguish between HagA and Lys-gingipain expression, we tested if the mutant cells possessed hemoglobinase activity, a measure of the Lys-gingipain Kgp [25]. Fig 6 shows that the hemoglobinase activity in the hagA23/167 mutant did not differ from WT cells, suggesting that the latter mutation does not affect Lys-gingipain expression. This interpretation was confirmed with the Lys-gingipain deficient strain Δkgp, which showed a significant reduction of hemoglobinase activity (Fig 6). The secretion mutant ΔporN [56] was used as a negative control and showed very low background hemoglobinase activity in this assay.
Secretion medium from WT, the hagA23/167 mutant, Lys-gingipain mutant (Δkgp), or a secretion mutant (ΔporN) was incubated with hemoglobin and the decrease in OD405 recorded after 24h, as a measure of hemoglobinase activity. Data from two independent experiments were adjusted for differences in initial culture density between strains and analyzed by one-way ANOVA with Dunnett’s post-test. N = 14. Lines indicate the mean of each group.
In vivo virulence
The above results suggested that hagA23/167 does not express HagA at the cell surface. To test if depletion of this virulence factor [69] affected bacterial virulence, WT and mutant strains were injected into Galleria mellonella, an in vivo model of bacterial infection [64, 70]. The in vivo virulence of hagA23/167 is similar to that of WT W50 (Fig 7). Both strains show significant virulence compared to larvae that were injected with PBS.
6th instar larvae were injected with PBS, W50 (WT) or hagA23/167 and incubated for up to 110 hours. Survival was determined at the times indicated. The data are from three independent experiments; N = 29. The curves were compared by Mantel-Cox logrank test. WT differs from PBS (P<0.0001) but not from hagA23-167 (P<0.25).
Discussion
P. gingivalis depends on heme for growth and virulence [17]. Thus, there has been great interest in non-pigmented mutants, which are defective in heme uptake [18, 30, 32]. As part of an effort to understand the activity of the AMP GL13K against P. gingivalis, we identified a novel non-pigmented mutant that showed a low MIC and two-fold extended MDK to DGL13K, consistent with tolerance to this peptide (Table 4) [66]. In contrast, pigmented, WT cells were susceptible to DGL13K but resistant to LGL13K, as previously reported [45] (Table 4).
DELETE:LGL13K exhibits a high MIC, suggesting that the bacteria are resistant to this peptide. DGL13K exhibits a low MIC and WT cells show a low MDK, suggesting that these bacteria are susceptible to DGL13K. A non-pigmented mutant survives about twice as long as WT cells, indicating that this mutant (hagA23/167) is tolerant to DGL13K.
The non-pigmented variant hagA23/167 shows mutations in the hagA gene, which is one of the pigmentation-related genes identified in a Tn-seq screen of P. gingivalis [29]. Other genes identified in this screen are coding for the gingipains Krp and RgpA, which share a CAD with HagA [71]. The adhesion domains of HagA, RgpA and Kgp are involved in cell-cell interaction and biofilm formation [72].
An antibody to the CAD [22], showed less binding to hagA23/167 than WT cells (Fig 5). Since the adhesion domain is involved in biofilm formation [72], we cannot formally exclude that this result shows reduced biofilm formation in the hagA23/167 mutant. We consider this unlikely since the antibody is not expected to penetrate the biofilm and biofilm thickness would not affect surface labeling. Indeed, the hemoglobin binding, hemagglutination and hemoglobinase assays support the interpretation that the adhesion domain protein HagA is not transported to the cell surface in this mutant. The finding that hagA23/167 was defective in hemoglobin binding and hemagglutination (Fig 4) but not in proteolysis (Fig 6) is consistent with the ablation of HagA activity while preserving gingipain activity.
Reduced in vitro hemagglutination has been associated with a lower copy number of hagA in P. gingivalis strains [73] but it is not clear how the two SNPs in the hagA sequence affect protein expression. Although the predicted mRNA structure for WT hagA differed from hagA23/167, the mRNA levels appeared to be similar in WT and mutant strains, suggesting that differences in translation, rather than transcription, were responsible for the change in HagA activity. Indeed, changes in codon usage and mRNA structure, caused by the synonymous SNPs, could cause a change in translational efficiency [68].
In Bacillus subtilis, biofilm formation has been linked to the utilization of specific Ser codons and reduced expression of the regulatory protein SinR [74]. Since our mRNA modeling suggested that the synonymous SNP at a Ser codon was responsible for the observed reduction of HagA function, we propose that a serine sensor may also regulate biofilm formation in P. gingivalis. According to this model, depletion of HagA may allow bacteria to escape a biofilm that is under attack by antimicrobial peptides.
Black pigmentation and hemin binding previously have been linked to the presence of anionic LPS (A-LPS) in the outer membrane of P. gingivalis [27, 75]. The structure of P gingivalis LPS also plays a role in bacterial resistance to the AMP polymyxin B [42, 76]. Specifically, a lack of phosphate groups in lipid A reduces the electrostatic attraction between the cationic peptide and LPS [76]. Indeed, a phosphatase mutant that is defective in dephosphorylation of lipid A exhibits increased sensitivity to polymyxin B [42]. HagA contains a conserved LPS attachment site [77]. Thus, it is possible that reduced levels of HagA at the cell surface in the hagA23/167 variant also alter the cell surface complement of LPS and thereby affects bacterial sensitivity to AMPs and pigmentation.
It is not entirely clear how potential differences in cell surface charges, mediated by LPS, differentially affect the two GL13K enantiomers. However, it has been reported that the two peptides are not merely mirror images of one another [78]. Thus, the pKa values of the four ionizable groups on LGL13K are somewhat higher than the pKa values for those same groups on DGL13K and the latter peptide adopts its secondary structure more rapidly than the L-enantiomer [78]. This secondary structure is associated with nanofibril formation, which proceeds more rapidly for DGL13K than for LGL13K [78]. However, in the presence of LPS, only subtle differences in fibril formation were found for LGL13K and DGL13K, whereas a random sequence peptide, LGL13K-R, did not interact with LPS [79]. Others have reported that the D-enantiomer of an antimicrobial peptide showed stronger binding to LPS than the L-enantiomer [80]. It remains to be determined if these structural changes affect the interaction of the peptides with the bacterial outer membrane of P. gingivalis.
Colony pigmentation has been associated with bacterial virulence in P. gingivalis [31]. Lack of pigmentation has been mapped to the hagA and kgp genes [30] and this region has been linked to both low pigmentation and lack of virulence in spontaneous mutants of P. gingivalis W50 [81]. Given the role of LPS and HagA in bacterial virulence [20, 82], it is surprising that the hagA23/167 mutant bacteria were at least as virulent as the WT strain. It is possible that, despite the non-pigmented phenotype, these mutants compensate for the loss of a virulence factor by functional redundancy [83], e.g., as expressed by the gingipain/hemagglutinin protein family [22].
Conclusions
A non-pigmented mutant of P. gingivalis emerged in cultures treated with the antimicrobial peptide DGL13K. This mutant showed increased tolerance, but not resistance, to the peptide. The non-pigmented mutant does not express HagA at the cell surface, but this change does not affect in vivo virulence of the mutant bacteria. The observed inability of P. gingivalis to develop resistance to DGL13K suggests that this peptide could be a candidate for local adjunctive antibiotic therapy of periodontitis.
Supporting information
S1 Fig. Raw gel image for Fig 2.
The original gel contains three sets (A, B,C) of 10 lanes. Lanes marked with X are not included in Fig 2. Lane sets B and C are not included in Fig 2. Lane A1: Mr markers; Lane A2: WT - 16S RNA; Lane A3: WT–hagA; Lane A7: hagA23-176 – 16S RNA; Lane A8: hagA23-176 –hagA. The image was captured on a BioRad gel imaging system.
https://doi.org/10.1371/journal.pone.0312200.s001
(PDF)
S2 Fig. MFE predicted RNA structure.
Supplement to the predicted structures shown in Fig 3, obtained by modeling the secondary structures by predicting the minimum free energy (MFE). The predicted structures show a 493 nucleotide segment of the mRNA sequence coding for the C-terminal K3 cleaved adhesin domain of hagA.
https://doi.org/10.1371/journal.pone.0312200.s002
(PDF)
Acknowledgments
Dr. Helmut Hirt, University of Minnesota is acknowledged for the initial discovery of non-pigmented colonies upon DGL13K treatment. We thank Dr. Donald Demuth, University of Louisville for helpful suggestions and Dr. Pamela Hanic-Joyce, Concordia University, Montreal for helpful suggestions and careful reading of the manuscript. Dr. Massimo Costalonga, University of Minnesota School of Dentistry is thanked for providing P. gingivalis W50 and Dr. Jan Potempa, University of Louisville is thanked for making gingipain mutants available for this work.
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