Keywords
intracellular bacteria - immunoglobulin -
Porphyromonas gingivalis
Introduction
Porphyromonas gingivalis is a key stone pathogen of periodontitis.[1]
P. gingivalis is an anaerobic bacterium that expresses many virulent factors including fimbriae.
P. gingivalis fimbriae plays an important role in adherence to host oral cells, oral tissues, and
early bacterial colonizers, thus the bacterium can attach and colonize in the oral
cavity. P. gingivalis fimbriae are composed of major (FimA) and minor fimbriae, encoded by the fimA gene
and the mfa1 gene, respectively.[2]
[3]
[4] According to the genotype of FimA, P. gingivalis can be divided into six classes, FimA genotype I to V and Ib.[5] FimA genotype II and IV are associated with periodontitis, while type I is found
in healthy gingiva.[5]
[6]
After P. gingivalis use its fimbriae to adhere and colonize in the subgingival area, where the oxygen
is depleted and contains amino acids and hemin in the gingival crevicular fluid, they
proliferate and spread.[1]
[7]
[8]
P. gingivalis is able to tolerate and escape from host immune, and induce chronic inflammation
of the periodontal tissue.[1]
[8]
[9]
[10]
[11]
Planktonic or extracellular P. gingivalis growth can be inhibited by host immune system and antimicrobial substances.[12]
[13] However, P. gingivalis is able to stay alive and proliferate inside many types of host cells including epithelial
cells, periodontal ligament cells, and endothelial cells. Since it hides inside the
host cell, host immune system and extracellular antimicrobial substances cannot eliminate
the bacteria. Thus, host cells become a source of bacterial proliferation.[11]
[14]
[15] This causes recurrence of periodontitis after the periodontitis treatment.
In this study, we attempted to determine the effect of antibody specific to P. gingivalis FimA on intracellular P. gingivalis. Inside the cells, tripartite motif containing-21 (TRIM21) binds to immunoglobulin
(Ig) Fc and induces degradation of the protein that is captured by the Ig via proteasome
activation.[16]
[17]
[18] This mechanism was proposed to explain the ability of cells to eliminate intracellular
virus that was captured by the specific antibody. The internalized antibody might
also be able to inhibit or kill the intracellular bacteria.
Materials and Methods
P. gingivalis FimA Type I Immunization
Rabbit immunization with FimA Type I (FimI) was approved by the Naresuan University
Institutional Animal Care and Use Committee (project no. NU-AE600814). Histidine-tagged
FimI (His-FimI) was amplified from pcDNA3.FimA-PVXCP[19] and inserted into the pET28a plasmid. The recombinant plasmid was purified and transformed
into BL21DE3 Escherichia coli for His-FimI protein expression. His-FimI was purified using nickel column (Thermo
Scientific, United States) and histidine tag was removed by enterokinase enzyme (Bio
Basic Inc, Canada). FimI protein was administered intravenously to two female New
Zealand white rabbits (Nomura Siam International Co., Ltd., Thailand). FimI protein
was diluted to 200 μg in 0.5 mL sterile saline solution and then administered into
the marginal ear vein. FimI boosting was done on day 14, 28, and 42 with 100 μg FimI
protein diluted in 0.5 mL sterile saline solution. Rabbit blood samples (1 mL) were
collected from the marginal ear vein at day 0 as preimmune sera. And at day 49, seven
days after the last boost, the rabbit blood samples were collected for antibody titer
evaluation. Antibody titer was evaluated by enzyme-linked immunosorbent assay (ELISA).
Comparing to preimmune serum, the antibody titer was relatively high. Therefore, the
blood could be collected for further Ig purification. The final blood collection and
euthanasia were performed on day 55. The serum was prepared by allowing the blood
samples to clot at room temperature for 1 hour and spinning at 2,500 revolutions per
minute for 15 minutes at room temperature. The serum was transferred to new tubes
and kept at –20°C until use.
Rabbit Anti-FimI Ig Purification
Rabbit anti-FimI Ig was purified from rabbit serum using Protein A Sepharose column
(Abcam, United States) according to the manufacturer's instruction. Briefly, the serum
was mixed with phosphate buffer saline (PBS) and passed through the column. The column
was washed with PBS containing 0.5 M NaCl and the rabbit Ig was eluted from the column
using 0.1 M citric acid, pH 2.75. The eluent was neutralized by adding 0.1 volume
of 1 M Tris, pH 9.0. The rabbit Ig was concentrated and buffer exchanged to PBS using
Pall Macrosep Advance Centrifugal devices with Omega Membrane—10 kDa cutoff (Tisch
Scientific, United States). Rabbit anti-FimI Ig was aliquoted and kept at –20°C.
Enzyme-Linked Immunosorbent Assay with P. gingivalis
P. gingivalis ATCC 33277 and ATCC 53978 (W50) strains were grown anaerobically at 37°C in a Thermo
Scientific anaerobic chamber (Thermo Scientific). Culture media was tryptic soy broth
(Becton, Dickinson and Company, United States) supplemented with 5.0 mg/mL of yeast
extract (Becton, Dickinson and Company), 0.5 mg/mL of L-cysteine hydrochloride (HiMedia
Laboratories Pvt. Ltd, India), 1.0 μg/mL of vitamin K1 (Sigma Chemical Co., St. Louis,
Missouri, United States), and 5 μg/mL of hemin (Sigma-Aldrich, United States).
ELISA was performed using formalin-fixed P. gingivalis. The bacteria were grown as described above and harvested by centrifugation. The
cell pellet was washed once with PBS and the washed cells were incubated with 0.5%
formalin in 0.9% NaCl overnight. The formalin-fixed cells were centrifuged and resuspended
at 1010 cells/mL in PBS before use.
For ELISA, 100 μL of formalin-fixed P. gingivalis was aliquoted into each well of 96-well ELISA plate and the plate was incubated overnight
at 4°C. After washing in PBST buffer (PBS with 0.05% (v/v) Tween-20), the plate was
blocked with 2% (w/v) bovine serum albumin in PBS for 1 hour. After washing, 100 μL
of rabbit anti-FimI Ig (1 μg/mL) in PBST buffer was added and the plate was incubated
at 37°C for 1 hour. After washing, the bound Ig was detected by incubation with peroxidase-conjugated
anti-rabbit Ig (Cell Signaling Technology, Massachusetts, United States) at 37°C for
1 hour. After washing, tetramethylbenzidine substrate was added and incubated in dark
for 30 minutes. Note that 1 N HCl was added and the OD450 was recorded using a microplate
reader.
P. gingivalis Labeling with PKH67 Green Fluorescent
Live P. gingivalis was labeled with PKH67 Green Fluorescent (Sigma-Aldrich) following the method provided
by the manufacturer with some modifications. One hundred microliters of PKH67 solution
were mixed with 1 × 109
P. gingivalis cells and incubated for 15 minutes at room temperature with protection from the light.
The labeling reaction was stopped by addition of fetal bovine serum (FBS). The labeled
bacteria were collected by centrifugation and resuspended in H357 cell growth media
for further use for invasion into H357 cells.
Invasion of Labeled P. Gingivalis into H357 Cells
H357 is a human tongue squamous cell carcinoma that has been used as a host cell model
in P. gingivalis host-cell invasion assays.[20] H357 cells were cultured in 1:1 mixture of DMEM (Dulbecco's Modified Eagle Medium)
and Ham's F12 media (Gibco, Thermo Fisher Scientific) supplemented with 2 mM glutamine
(Gibco), 10% FBS (Hyclone Laboratories Inc, United States), and 0.5 μg/mL sodium hydrocortisone
succinate (Sigma-Aldrich). The labeled bacterial suspension then replaced the medium
of H357 cells (1.4 × 106 cells) that were previously cultured for 24 hours in 60 mm culture dish. H357 cells
were incubated with the labeled bacteria for 4 hours before the culture medium containing
free bacteria was removed and replaced by complete culture media. Cells were cultured
for a period of time desired for each experiment.
Rabbit Anti-FimI Ig Labeling with Cy3
Rabbit anti-FimI Ig was linked to Cy3 using ReadiLink Rapid Cy3 Antibody Labeling
Kit (AAT Bioquest, United States). Fifty microliters of 1 mg/mL of rabbit anti-FimI
Ig was mixed with 5 μL of reaction buffer. The mixture was added into a vial containing
Cy3 dye and mixed by pipetting. The conjugation reaction mixture was incubated at
room temperature for 1 hour. The conjugation reaction was stopped by addition of 5
μL of TQ-Dyed Quench Buffer. The reaction was incubated at room temperature for 10 minutes
and the labeling Ig was stored at –20°C.
Internalization of Rabbit Anti-FimI Ig into H357 Cells by Electroporation
After H357 cells were invaded by PKH67-labeled P. gingivalis for 16 hours, cells were harvested by trypsinization and 1 × 106 cells were resuspended in 200 μL of Opti-Mem (Gibco). The cell suspension was mixed
with 8 μL of 0.5 mg/mL of rabbit anti-FimI Ig or rabbit anti-FimI Ig-Cy3. Cells were
electroporated with square wave pulse type at 160 V for 15 ms using Gene Pulser Xcell
(Bio-Rad Laboratories, Inc., United States). After electroporation, 0.5 mL of media
was added and the cells were transferred to a 35-mm culture dish or 8-well Laboratory-Tek
II Chamber Slide (Nunc, United States).
Cell Visualization Under Microscope
For observing P. gingivalis-PKH67 and anti-FimI Ig-Cy3 inside the cells, cells were visualized using Zeiss Observer.D1
AXIO with Zen2 Software (Zeiss, Germany). In addition, cells containing P. gingivalis-PKH67 and anti-FimI Ig-Cy3 cultured in 8-well Laboratory-Tek II Chamber Slide were
fixed with 0.36% formaldehyde in PBS for 10 minutes, permeabilized with 0.1% Triton
X-100 in PBS for 1 minute, and stained with 4′,6-Diamidino-2-Phenylindole, dihydrochloride
(1:1,000 in PBS containing 0.2% Tween-20) for 10 minutes. The slide was mounted with
mount solution before visualization with Nikon A1R confocal microscope and NIS-Element
AR software (Nikon, Japan).
Internalization of Cy3-Rabbit Anti-FimI Ig into H357 Cells by Coincubation
After H357 cells were invaded by PKH67-labeled P. gingivalis for 16 hours, culture medium was replaced with culture medium containing 2.5 μg/mL
of anti-FimI Ig-Cy3. Cells were cultured for further 16 hours and the medium containing
the Ig was removed and replaced with normal culture medium. Cells were observed under
Zeiss Observer.D1 AXIO with Zen2 Software.
Flow Cytometry Analysis of H357 Cells Containing P. gingivalis
After H357 cells containing P. gingivalis-PKH were electroporated with rabbit anti-FimI Ig, cells were cultured up to 6 days
before analysis with BD FACSAria III and FACSDiva 8.0.1 software (BD Biosciences,
United States). Cells were trypsinized and resuspended in PBS. Fifty thousand single
cells were analyzed and fluorescein isothiocyanate signals were counted as H357 cells
containing P. gingivalis-PKH.
Statistical Analysis
Normality of data distribution was tested by Shapiro–Wilk test. The data failed normality
test and were further analyzed by Kolmogorov–Smirnov test. All statistical analyses
were performed using GraphPad Prism software version 10.2.3.
Results
Specificity of Rabbit Anti-FimI Ig
Binding of rabbit anti-FimI Ig to P. gingivalis strain ATCC33277, harboring FimA type I, and strain ATCC53978 or W50 strain, harboring
FimA type IV, was assessed by ELISA. The antibody binding gave OD450 3.55 ± 0.08 when
the antigen tested was ATCC33277 strain. In contrast, the OD was only 1.34 ± 0.10
when the antigen tested was W50 strain. This data showed that rabbit anti-FimI Ig
was specific to FimI harboring strain and could bind at a certain level to FimA type
IV harboring strain.
Rabbit Anti-FimI Ig Colocalization with P. gingivalis Inside the Cells
To localize P. gingivalis and anti-FimI Ig inside the cells, H357 cells were infected with P. gingivalis labeling with PKH67 dye, and then anti-FimI Ig labeling with Cy3 was internalized
into the infected cells by electroporation. One day after electroporation, cells were
fixed and stained with DAPI, and visualized with confocal microscopy. In addition,
2 days after electroporation, cells were visualized with fluorescence microscope.
Both P. gingivalis and the Ig were found colocalized near the nucleus ([Figs. 1] and [2]). The same result was observed on day 3 but the fluorescence signal was weaker.
On day 4, it was hard to observe fluorescence signal inside the cells as the signal
was weak and the boundary of the cells was hard to locate after cell number was increased
by cell proliferation.
Fig. 1 Confocal image of cells containing Porphyromonas gingivalis and anti-FimI immunoglobulin (Ig). Rabbit anti-FimI Ig-Cy3 was internalized into
H357 cells that were infected with P. gingivalis-PKH67 by electroporation. One day after electroporation, cells were fixed and stained
with DAPI, and visualized with confocal microscopy.
Fig. 2 Localization of Porphyromonas gingivalis and anti-FimI immunoglobulin (Ig). Rabbit anti-FimI Ig-Cy3 was internalized into
H357 cells that were infected with P. gingivalis-PKH67 by electroporation. Two days after electroporation, cells were imaged by fluorescence
microscopy to visualize the cells in phase contrast (A), for Cy3 signal (B), and PKH67 signal (C) before merging (D).
Rabbit Anti-FimI Ig Entered Cells by Coincubation
After culturing H357 cells in culture medium containing rabbit anti-FimI Ig for 16 hours,
cells were observed with fluorescence microscope 1 day after the medium containing
Ig was replaced with normal medium. Similar to that observed in the previous section,
the Ig was found near the nucleus ([Fig. 3]).
Fig. 3 Localization of anti-FimI immunoglobulin (Ig) after coincubation with H357 cells.
H357 cells that were infected with Porphyromonas
gingivalis-PKH67 were cultured in the medium containing rabbit anti-FimI Ig-Cy3 for 16 hours.
One day after the medium containing Ig was replaced with normal medium, cells were
imaged by fluorescence microscopy to visualize the cells in phase contrast (A) and for Cy3 signal (B) before merging (C).
Proportion of P. gingivalis Infected Cells after Anti-FimI Ig Internalization
Rabbit anti-FimI Ig was internalized into H357 cells containing P. gingivalis-PKH67 by electroporation. Percentage of the infected cells was observed using flow
cytometry ([Table 1]). The observation was done up to 6 days after electroporation since the level of
fluorescence was low at day 6. Comparing with the control group with no electroporation,
the electroporated cells group showed lower level of infected cells. At day 2 and
3, the percentage of infected cells were similar between the mock electroporation
control group and the anti-FimI Ig electroporation group. At day 4, the percentage
of infected cells in the anti-FimI Ig electroporation group seemed to be lower than
that of the electroporation control group but the difference was not statistically
significant.
Table 1
Percentage of Porphyromonas gingivali
s infected cells after anti-FimI Ig internalization
|
Percentage of H357 cells containing P. gingivalis
|
|
Control
|
Electroporation control
|
Electroporation with anti-FimI Ig
|
|
Day 2
|
22.25 ± 1.34
|
19.10 ± 0.92
|
18.90 ± 0.14
|
|
Day 3
|
21.45 ± 0.07
|
16.15 ± 0.49
|
16.35 ± 0.07
|
|
Day 4
|
20.5 ± 0.00
|
13.43 ± 0.28
|
12.83 ± 0.25
|
|
Day 6
|
12.67 ± 0.42
|
9.27 ± 0.31
|
9.03 ± 0.32
|
Abbreviation: Ig, immunoglobulin.
Discussion
After H357 cells were infected with P. gingivalis, P. gingivalis cells were found near the nucleus, the location was speculated to be endoplasmic
reticulum (ER). This was also found by another work, which showed that intracellular
P. gingivalis localized to ER-perinuclear regions.[21] After electroporation or coincubation with rabbit anti-FimI Ig, the Ig was also
found near the nucleus. The colocalization of the Ig and P. gingivalis makes a chance that the antibodies can bind to and inhibit the bacterial proliferation
and survival.
The effect of the antibody to inhibition and killing of P. gingivalis is not clear in this study. Four days after the internalization of anti-FimI Ig into
the infected cells, the infected cells seemed to be lower. However, the difference
was not statistically significant. It might need to be investigated for a longer period
of time, and the other technique of P. gingivalis fluorescence labeling will be required since the signal of PKH67 faded at day 6.
Rabbit anti-FimI Ig could enter H357 cells by only coincubation. Internalization of
the antibody by this method might occur through the function of Ig receptor on H357
cell surface. However, there was no report of Ig receptor on H357 cells or normal
oral epithelial cells. There are evidences showing that FcRn is expressed in normal
human epidermal keratinocytes[22] and intestinal epithelial cells.[23] Fc gamma receptors also express in human nasal epithelial cells[24] and carcinoma cells.[25] It was shown that rabbit IgG could bind to human Fc gamma receptor.[26] If H357 cells have Ig Fc receptor on cell surface, anti-FimI Ig internalization
after coincubation might be modified by this receptor.
Conclusion
Rabbit anti-P. gingivalis FimI polyclonal antibody and P. gingivalis were colocalized near the nucleus. And the antibody was able to enter H357 cells
by the coincubation method.
