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Hemagglutinin B Is Involved in the Adherence of Porphyromonas gingivalis to Human Coronary Artery Endothelial Cells

Hong Song,^1, Myriam Bélanger,^1, Joan Whitlock,¹ Emil Kozarov,² and Ann Progulske-Fox^1*

Department of Oral Biology, College of Dentistry, and Center for Molecular Microbiology, University of Florida, Gainesville,¹ College of Dental Medicine, Nova Southeastern University, Fort Lauderdale, Florida²

Received 26 April 2005/ Returned for modification 10 June 2005/ Accepted 7 August 2005

	ABSTRACT

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Porphyromonas gingivalis is a periodontopathogen that may play a role in cardiovascular diseases. Hemagglutinins may function as adhesins and are required for virulence of several bacterial pathogens. The aim of this study was to determine the role of hemagglutinin B (HagB) in adherence of P. gingivalis to human coronary artery endothelial (HCAE) cells. P. gingivalis strain 381, a P. gingivalis 381 HagB mutant, Escherichia coli JM109 expressing HagB (E. coli-HagB), and E. coli JM109 containing pUC9 (E. coli-pUC9) were tested for their ability to attach to HCAE cells. Inhibition assays were performed to determine the ability of purified recombinant HagB (rHagB) as well as antibodies to HagB, including the polyclonal antibody (PAb) A7985 and the monoclonal antibody (MAb) HL1858, to inhibit the attachment of P. gingivalis to HCAE cells. As expected, when the attachment of P. gingivalis and the HagB mutant were compared, no statistical significance was observed between the two groups (P = 0.331), likely due to the expression of the hagB homolog hagC. However, E. coli-HagB adhered significantly better to HCAE cells than did E. coli-pUC9, the control strain. In a competition assay, the presence of purified rHagB decreased bacterial adhesion of P. gingivalis or E. coli-HagB to HCAE cells. The presence of PAb A7985 or MAb HL1858 also significantly decreased attachment of P. gingivalis and E. coli-HagB to host cells. These results indicate that HagB is involved in the adherence of P. gingivalis to human primary endothelial cells.

	INTRODUCTION

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Porphyromonas gingivalis, a gram-negative anaerobic rod, is considered a major pathogen in the development of infectious periodontal diseases, such as periodontal abscesses, refractory periodontitis, generalized juvenile periodontitis, and rapidly progressing periodontitis (42, 56, 59). In addition, an association between periodontal disease and cardiovascular disease has been found in many epidemiological studies (3, 5, 6, 11, 44, 45). The accumulation of clinical and experimental evidence also suggests that periodontal infection may be a contributing risk factor for heart disease (27, 31, 34, 50, 51). For example, the occurrence of P. gingivalis in subgingival sites correlates with the detection of P. gingivalis in coronary artery plaque samples (25). A correlation between high levels of antibody to P. gingivalis and prevalence of coronary heart disease has also been observed (50, 51). Furthermore, the presence of P. gingivalis DNA has been detected within atherosclerotic plaques from vascular tissues (10, 21, 43, 54, 60). Recently, a potential role for this microorganism in atherosclerotic lesion formation has been suggested and evidence has been provided of a direct link between the presence of specific periodontal pathogens, including P. gingivalis, and subclincal atherosclerosis in humans (8, 11, 35, 52). Animal studies with apolipoprotein E-deficient mice, a model for atherosclerosis, provide additional evidence of a possible role for P. gingivalis in this disease, since oral infection with P. gingivalis accelerates early atherosclerosis (19, 37, 41). Rabbits which were experimentally induced with periodontitis showed more extensive accumulations of lipids in their aortas than control nonperiodontitis rabbits (26). Furthermore, intravenous injections of P. gingivalis lead to coronary and atherogenesis in pigs (7). In addition, we have isolated viable P. gingivalis from human atherosclerotic plaques (34). These results provide evidence that periodontitis and P. gingivalis are risk factors for and may contribute to the pathogenesis of atherosclerosis.

P. gingivalis can invade many cell types, including human oral epithelial cells (29, 46, 58), human gingival fibroblasts (2), human coronary artery smooth muscle cells, and human coronary artery endothelial (HCAE) cells (14, 38). Adherence to target cells is a required initial event for invasion of host cells (4). In order to avoid nonspecific host defenses, such as mechanical clearance, bacteria bind to host cells through adhesin molecules. Subsequent bacterial entry into host cells confers protection from the host immune system and may contribute to host tissue damage (4, 13, 15). Hemagglutinins can function as adhesins and are required for virulence of several bacterial pathogens (1, 9, 24, 35). Hemagglutinins are also considered important virulence factors, as they can be a mechanism to acquire hemin, necessary for bacterial growth, from erythrocytes (39). Several hag genes, encoding hemagglutinins of P. gingivalis, have been previously described and cloned (20, 40, 48, 49). However, the importance of the hemagglutinins in the colonization process of P. gingivalis remains to be determined. In this study, we investigated the role of hemagglutinin B of P. gingivalis in adhesion to and invasion of HCAE cells. Our results indicate that HagB promotes attachment of P. gingivalis to host cells but, alone, is not sufficient for internalization into host cells.

	MATERIALS AND METHODS

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Bacterial strains and cell culture conditions. P. gingivalis strain 381 was grown anaerobically on blood agar plates (Difco Laboratories, Detroit, MI) or in brain heart infusion broth (Difco), as described previously (39). Clindamycin was added to the media at 5 µg/ml to maintain the HagB mutant of P. gingivalis 381. Escherichia coli JM109 containing pUC9 with or without a 4.8-kb DNA fragment (ST7) containing hagB was grown aerobically on Luria-Bertani (LB) plates or in LB broth (Difco) with 100 µg/ml ampicillin, as described previously (49). For E. coli M15[pREP4]pQE-31 (QIAGEN Inc., Valencia, CA) and the HagB expression strain E. coli M15[pREP4]pQE-31-TX1, 100 µg/ml ampicillin and 5 µg/ml kanamycin were added to the media (33).

HCAE cells (Cambrex, Walkersville, MD) were cultured in endothelial cell basal medium-2 (EBM-2; Cambrex) supplemented with EGM-2-MV single-use aliquots (Single Quots; Cambrex) as described by the manufacturer. HCAE cells were maintained at 37°C with 5% CO₂ in a humidified atmosphere.

P. gingivalis HagB mutant construction. The BamHI/PstI fragment containing hagB from clone ST7 and was cloned into the BamHI/PstI site of pUC18 (Amersham Biosciences Corp., Piscataway, NJ) in E. coli JM109 (49). The ermF-ermAm cassette was cut out from plasmid pVA2198 (graciously provided by F. Macrina) using SacI and BamHI, and the ends were blunt ended using the Klenow enzyme. The 2.3-kb cassette was then ligated into the StuI site of the hagB fragment within the hagB-pUC18 construct. This plasmid was maintained in E. coli JM109 and designated pJW1. The purified plasmid was electroporated into P. gingivalis 381, the HagB mutant was obtained, and the mutation was confirmed by Southern hybridization (data not shown) as previously described (48). Sequencing was also performed to confirm the mutation. All restriction and modification enzymes were purchased from Promega Corporation (Madison, WI).

rHagB purification and analysis. The hagB gene of P. gingivalis (1.4 kb) was cloned into the vector pQE-31 (QIAGEN), and the construct was designated pQE-31-TX1 (32). The histidine-tagged HagB was purified on a nickel-nitrilotriacetic acid affinity column by fast protein liquid chromatography (FPLC) (Bio-Rad Laboratories, Hercules, CA) from E. coli M15[pREP4]pQE-31-TX1, as described previously (32). The eluted protein was dialyzed against 500 mM sodium chloride (NaCl) and 10 mM Tris, pH 7.4, and was concentrated using polyethylene glycol (PEG) 8000 (Fisher Scientific, Fair Lawn, NJ).

The purified recombinant HagB (rHagB) was run on a sodium dodecyl sulfate (SDS)-polyacrylamide gel, as described below. The 49-kDa band was excised from the gel and digested with trypsin, as previously described (22). Identification was confirmed by liquid chromatography-mass spectroscopy analysis performed at the Interdisciplinary Center for Biotechnology Research (ICBR) in the Protein Chemistry Core Laboratory of the University of Florida and by a SEQUEST database search.

MAb and PAb production. Mouse monoclonal antibodies (MAbs) against the purified rHagB were produced by standard protocols utilized by the ICBR Hybridoma Core Laboratory at the University of Florida (28, 29). Briefly, three 6- to 8-week-old female BALB/cByj mice were immunized subcutaneously with either 25 µg or 50 µg of the antigen, using the MPL + TDM adjuvant system (Sigma-Aldrich Company, Ltd., St. Louis, MO), or with 10 µg of the antigen mixed with the ImmunEasy adjuvant system (QIAGEN). Spleen cells from the immunized mice were fused with myeloma cells (sp₂/0) at a ratio of 7:1 by using 50% PEG (Hoffmann-LaRoche Inc., Nutley, NJ). Fused cells were grown in hypoxanthine, aminopterine, and thymidine selective medium. Hybridoma cells were evaluated by enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies that bound to the immunogen. Hybridomas from the ELISA-positive wells were transferred to 24-well plates and screened by ELISA a second time using anti-mouse whole-molecule- or heavy-chain (gamma or mu)-specific secondary antibodies (Sigma-Aldrich). Screening was also performed by Western blotting, as described below. Two positive hybridomas were selected and cloned using a single-cell-per-well procedure. The MAb HL1858 (immunoglobulin G1 [IgG1]) developed against HagB and an unrelated MAb, HL1830, were used in this study.

Specific rabbit polyclonal antibody (PAb) A7985 was raised against the purified rHagB protein (Strategic Biosolutions, Newark, DE). Preimmune rabbit serum was obtained prior to the first immunization and used as a negative control.

Antibody purification. The MAb HL1858 and the unrelated MAb HL1830 were purified by FPLC on a protein A cartridge (Bio-Rad), using ImmunoPure IgG binding and elution buffers as described by the manufacturer (Pierce, Rockford, IL). The purified MAbs were dialyzed in 10 mM sodium phosphate and 0.15 M NaCl, pH 7.5, and then concentrated using PEG 8000. Purity was determined by running them on gels, as described below. Concentrations of purified MAbs were determined using a bicinchoninic acid protein determination assay (Pierce).

Gel electrophoresis and Western immunoblot analysis. SDS-polyacrylamide gel electrophoresis was carried out by the method of Laemmli (36), using 4 to 20% Ready Gel Tris-HCl gels (Bio-Rad). Gels were stained with Coomassie brilliant blue R-250 or transferred onto polyvinylidene difluoride membranes (PerkinElmer Life Sciences, Inc., Boston, MA) (28). The specific protein HagB bands were subsequently detected by incubating with MAb HL1858 or PAb A7985 and then immunostaining with an anti-mouse or anti-rabbit IgG conjugated with alkaline phosphatase (ICN Biomedicals, Aurora, OH). The membranes were revealed using 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium (Sigma-Aldrich) as the substrate.

Immunoelectron microscopy. Immunoelectron microscopy was used to detect the presence of the HagB antigen on the surfaces of the wild-type and HagB mutant of P. gingivalis 381, E. coli JM109 expressing HagB (E. coli-HagB), and E. coli JM109 containing pUC9 (E. coli-pUC9). Bacteria were grown overnight, harvested, and incubated on 400-nm nickel hexagonal grids. Grids were blocked with 1% percent nonfat dry milk for 10 min, incubated with a 1/200 dilution of MAb HL1858 or unrelated MAb HL1831 for 1 h at room temperature, and then washed three times with phosphate-buffered saline (PBS). Anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) labeled with 18-nm-diameter gold particles was added, incubated for 1 h at room temperature, and then washed with PBS three times. The grids were examined using a Hitachi 7000 transmission electron microscope at the ICBR Electron Microscopy Core Laboratory of the University of Florida.

Adhesion and invasion assays. HCAE cells were seeded in 24-well tissue culture plates at 10⁵ cells per well and incubated for 18 h. Overnight broth cultures of P. gingivalis 381 (wild type and HagB mutant), E. coli-HagB, and E. coli-pUC9 were centrifuged and then resuspended in antibiotic-free EBM-2 medium to a concentration of 10⁷ CFU/ml, as determined with a spectrophotometer (Shimadzu, Kyoto, Japan). The HCAE cells were washed with PBS and infected with 1 ml per well of P. gingivalis 381 (wild type and HagB mutant), E. coli-HagB, or E. coli-pUC9. Three wells per bacterial strain were infected. The plates were incubated aerobically at 37°C for 90 min and then washed three times with PBS. Cells were lysed with sterile H₂O for 20 min at 37°C.

For the invasion assay, the wells were treated with 300 µg/ml gentamicin and 200 µg/ml metronidazole for 60 min at 37°C to kill all extracellular bacteria prior to lysing the cells with sterile H₂O. Decimal serial dilutions were plated, and CFU were enumerated. Each assay was performed in triplicate. The adhesion or invasion ratios were then calculated.

Competitive inhibition assays. (i) Competition using rHagB protein. Purified rHagB protein was used in the assay as a competitive inhibitor. A lysate of E. coli M15[pREP4]pQE-31 was run through a Ni-nitrilotriacetic acid column, and the eluted proteins were used as the negative control preparation. The E. coli M15[pREP4]pQE-31 eluted proteins, the purified rHagB protein, or bovine serum albumin (BSA) (Sigma-Aldrich) was added to HCAE cells in increasing concentrations of 50, 150, and 450 µg/ml. The HCAE cells were preincubated with the rHagB protein, the E. coli control proteins, or BSA (control) for 1 h at 37°C before addition of wild-type P. gingivalis or E. coli-HagB. Assays were then performed as described in the previous section.

(ii) Competition using antibodies. In the antibody inhibition assay, MAb HL1858 against HagB and an unrelated MAb, HL1830 (control), were diluted in threefold-increasing concentrations of 5.6 to 450 µg/ml. Polyclonal antibody A7985 or preimmune rabbit serum (control) was diluted in fivefold-increasing concentrations of 1/1250, 1/250, 1/50, and 1/10 and then mixed with bacteria. Bacteria without preincubation with serum were used as positive controls. After a 1-h preincubation period at 37°C, bacteria were centrifuged at 5,000 rpm for 15 min, washed three times with PBS, vortexed for 40 s, and then used in the adhesion assays, as described above.

Statistical analysis. Differences between groups were determined by analysis of variance. Normality and equal variance of the data were confirmed in preliminary analysis. When indicated, multiple pairwise comparisons were done using the Student-Newman-Keuls test (SigmaStat 3.0; SPSS Inc, Chicago, IL). For all comparisons, P values of <0.05 were considered significant.

	RESULTS

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rHagB purification. The recombinant hemagglutinin B protein of P. gingivalis was expressed in E. coli M15[pREP4]pQE-31 and purified by FPLC. The purified protein ran at approximately 49 kDa on SDS-polyacrylamide gels. No bands of this molecular mass were observed in the FPLC-purified lysate of E. coli M15[pREP4]pQE-31 control proteins (data not shown). A 56.3% coverage was obtained when liquid chromatography-mass spectrometry was performed on the excised 49-kDa band. Analysis with the SEQUEST software confirmed the identity of the purified protein as being hemagglutinin B of P. gingivalis.

Antibody purification and analysis. After FPLC purification, monoclonal antibodies were confirmed to be pure by SDS-polyacrylamide gel electrophoresis (data not shown). Immunoblotting was performed to confirm the reactivity of PAb A7985 or MAb HL1858 against HagB. Both antibodies strongly recognized the rHagB protein. Neither the PAb nor the MAb reacted with bands of similar molecular weight in the FPLC-purified lysate of E. coli M15[pREP4]pQE-31 control proteins (data not shown).

Adhesion and invasion assay. To determine whether HagB is involved in adhesion to and/or invasion of host cells, P. gingivalis 381 (wild type and HagB mutant), E. coli-HagB, and E. coli-pUC9 (negative control) were used to infect HCAE cells. The adhesion value for P. gingivalis 381 was normalized to 100% and was used as the control to derive the other adhesion values. The attachment and invasion of P. gingivalis 381 and of the HagB mutant to the HCAE cells were compared. No statistically significant difference (P = 0.331) was observed between the two groups. However, we observed a statistically significant difference (P < 0.001) between the E. coli control (6.6% ± 1.6%) and E. coli expressing the HagB protein (34.1% ± 8.5%). E. coli expressing the HagB protein attached approximately 5 times more to the HCAE cells than did the control E. coli. A statistically significant difference was also observed between P. gingivalis 381 (100% ± 27.8%) and both E. coli strains (see results above). Because no bacteria were recovered intracellularly from cells exposed to the E. coli strains but bacteria were recovered from strain 381 (control) incubated cells, it was concluded that E. coli-HagB and E. coli-pUC9 did not invade the HCAE cells.

rHagB competitive inhibition assay. Various concentrations of purified rHagB protein were preincubated with the HCAE cells before they were infected with the microorganisms. In the competitive assay with P. gingivalis 381, no inhibition was observed when the cells were preincubated with either 50 or 150 µg/ml of rHagB (Fig. 1A). However, a statistically significant difference in the attachment was observed between P. gingivalis 381 preincubated with 450 µg/ml of rHagB and all the other groups (0, 50, and 150 µg/ml of inhibitor). The addition of BSA (data not shown) or of control proteins from E. coli M15[pREP4]pQE-31 did not inhibit P. gingivalis 381 from attaching to the HCAE cells (P = 0.781) (Fig. 1A). Statistically significant differences were also observed in the attachment of E. coli-HagB to the HCAE cells when preincubations were performed in presence of 150 or 450 µg/ml rHagB (P < 0.001) (Fig. 1B). BSA (data not shown) or control proteins from E. coli M15[pREP4]pQE-31 at concentrations ranging from 50 to 450 µg/ml did not inhibit E. coli-HagB from attaching to HCAE cells (P = 0.666) (Fig. 1B).

View larger version (27K): [in this window] [in a new window]   FIG. 1. Inhibition of the attachment of (A) P. gingivalis or (B) E. coli-HagB to HCAE cells by preincubation with various concentrations of rHagB protein (stippled bars) or with control proteins from E. coli M15[pREP4]pQE-31 (solid bars) are reported as the mean (± standard error of the mean) of the bacterial attachment to the host cells. Different letters indicate a statistically significant difference (P < 0.05) between groups. The adhesion value for P. gingivalis 381 or E. coli-HagB was normalized to 100% and was used as the control to derive the other adhesion values.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Inhibition of the attachment of (A) P. gingivalis or (B) E. coli-HagB to HCAE cells by preincubation with various concentrations of rabbit PAb A7985 raised against HagB (stippled bars) or with control preimmune serum (solid bars). Results are reported as the mean (± standard error of the mean) of the bacterial attachment to the host cells. Different letters indicate a statistically significant difference (P < 0.05) between groups. The adhesion value for P. gingivalis 381 or E. coli-HagB was normalized to 100% and was used as the control to derive the other adhesion values.

View larger version (30K): [in this window] [in a new window]   FIG. 3. Inhibition of the attachment of (A) P. gingivalis or (B) E. coli-HagB to HCAE cells by preincubation with various concentrations of the control unrelated MAb HL1830 (solid bars) or with MAb HL1858 directed against HagB (stippled bars). Results are reported as mean (± standard error of the mean) of the bacterial attachment to the host cells. Different letters indicate a statistically significant difference (P < 0.05) between groups. The adhesion value for P. gingivalis 381 or E. coli-HagB was normalized to 100% and was used as the control to derive the other adhesion values.

View larger version (125K): [in this window] [in a new window]   FIG. 4. Transmission electron micrographs of P. gingivalis 381 (A), the P. gingivalis 381 HagB mutant (B), E. coli-HagB (C), and E. coli-pUC9 (D) cells that were first reacted with MAb HL1858 and then labeled with gold particles conjugated with a mouse IgG antibody.

	DISCUSSION

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Since multiple attempts to create a hagB hagC double mutant were unsuccessful, E. coli-HagB was used to test the involvement of HagB in adhesion to and invasion of HCAE cells. E. coli-HagB was found to have significantly increased attachment to HCAE cells compared to the parental E. coli strain. However, E. coli-HagB was not able to invade the host cells. This suggests that HagB is involved in P. gingivalis attachment to human cells but is not sufficient for invasion. Since multiple bacterial cell components and factors are involved in the complex process of entry into host cells (23, 46, 49), these results were expected. The role that HagB may play in host cell entry and trafficking within the cell thus cannot be deduced from this study.

In an attempt to further verify the role of HagB in the adhesion of P. gingivalis to host cells, rHagB protein was also purified and used in competitive assays. The purified rHagB migrated as a 49-kDa protein during electrophoresis, as described previously (49), even though the predicted molecular mass of P. gingivalis HagB is 39.4 kDa. This differential migration could be due to the high pI of the protein and thus aberrant migration during electrophoresis, as has been described for other similar proteins (32), or possibly to posttranslational modifications. Using serum from a rabbit immunized with a whole preparation of P. gingivalis 381, we observed a reaction in Western blotting with the purified His₆-rHagB protein, demonstrating that at least some epitopes of rHagB are in the same conformation as when present in whole P. gingivalis cells (M. Bélanger, unpublished data). A competitive assay was performed using the rHagB protein to prevent bacterial adhesion to HCAE cells. Inhibition of the attachment to host cells was dose dependent for E. coli-HagB. Also, the presence of 450 µg/ml of rHagB inhibited the attachment of P. gingivalis to HCAE cells. Both MAb and a specific PAb directed against HagB and used in competitive assays also significantly decreased the adherence of P. gingivalis and E. coli-HagB to host cells. Furthermore, we observed that inhibition of E. coli-HagB was dose dependent, strongly suggesting that HagB is necessary for P. gingivalis binding to HCAE cells.

HagB is expressed on P. gingivalis and E. coli-HagB cell surfaces, as the MAb designed against HagB labeled these bacterial cells as tested by immunoelectron microscopy. However, the HagB mutant was also labeled by MAb HL1858, a MAb directed against HagB. As the hagC gene of P. gingivalis is 98.6% homologous to hagB (47), it is most likely that MAb HL1858 recognizes a common epitope of HagB and HagC. No difference in attachment of the wild type and the HagB mutant of P. gingivalis to HCAE cells was observed. Because of the high homology of hagB and hagC, a mutant deficient in HagB may not have decreased attachment to host cells, as HagC may "complement" the mutation. Furthermore, preliminary data obtained in our laboratory suggest that HagA is also responsible, at least in part, for binding to host cells (H. Song, 83rd Gen. Session Exhibit. Int. Assoc. Dent. Res. 2004, abstr. 3637). Double and, if possible, triple mutants of these different hemagglutinin genes would be desirable for defining the relative roles of each hemagglutinin in adherence. However, as indicated above, such mutant constructions have not been achieved. It is possible that a hagB hagC double mutant is lethal for some unknown reason.

P. gingivalis has been reported to invade HCAE cells and coronary artery smooth muscle cells (16, 19). Invasion of the endothelial and smooth muscle cells of the arterial wall by P. gingivalis might contribute to the pathogenesis of cardiovascular disease (14, 16). Avoidance of nonspecific host defenses, such as mechanical clearance, and adherence to target cells are required initial events for bacterial invasion of host cells. Therefore, HagB might play a role in adhesion to the arterial wall, whereas other P. gingivalis molecules would be responsible for the invasion step. One objective of defining the virulence factors of P. gingivalis is to develop a potential vaccine and/or a new antimicrobial agent. We and others have shown that an induction of protection against P. gingivalis infection occurred after subcutaneous or intranasal immunization with HagB in mice (17, 32, 61, 63). Also, purified rHagB has been found to elicit a protective immune response in the rat bone loss model (30). Our study has demonstrated that HagB is involved in P. gingivalis adhesion to HCAE cells. Taken together, these results provide preliminary evidence for a rationale for investigation of a HagB-based vaccine for the prevention and treatment not only of periodontal disease but also of cardiovascular disease.

	ACKNOWLEDGMENTS

 
We thank Christopher West for helpful discussions. We are grateful to Martin Handfield for use of the FPLC and technical advice. We also acknowledge Fred Bennett, Scherwin Henry, Linda Green, and Nancy Denslow from the ICBR Core Laboratories of the University of Florida for technical assistance.

This study was supported by National Institutes of Health grant DE 07496.

	FOOTNOTES

 
* Corresponding author. Mailing address: University of Florida, Center for Molecular Microbiology, Department of Oral Biology, College of Dentistry, P.O. Box 100424, Gainesville, FL 32610-0424. Phone: (352) 846-0770. Fax: (352) 392-2361. E-mail: apfox{at}dental.ufl.edu.

Editor: J. B. Bliska

Hong Song and Myriam Bélanger contributed equally to the present study.

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