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Received: 2 July 2019    Revised: 13 August 2019    Accepted: 16 August 2019

DOI: 10.1111/omi.12268

ORIGINAL ARTICLE

Type IX secretion system is pivotal for expression of gingipain‐

associated virulence of Porphyromonas gingivalis

Malgorzata Benedyk1,2  | Agata Marczyk1 | Barbara Chruścicka2

1
Department of Microbiology, Faculty
of Biochemistry, Biophysics and Abstract
Biotechnology, Jagiellonian University, Porphyromonas gingivalis, a keystone pathogen in periodontitis, secretes an array
Krakow, Poland
2 of virulence factors including gingipains via the type IX secretion system (T9SS).
Malopolska Center of
Biotechnology, Jagiellonian University, Inactivation of any component of the T9SS leads to the accumulation of secreted pro‐
Krakow, Poland
teins in unprocessed and, in the case of progingipains, inactive forms in the periplasm.
Correspondence To cast light on the paradox that active gingipains are essential for P. gingivalis fitness
Malgorzata Benedyk, Department of
in vivo but a functional T9SS is not (Frontiers in Cellular and Infection Microbiology,
Microbiology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian 2017, 7:378), we have compared virulence of wild‐type P. gingivalis W83 and the
University, Krakow, Poland.
gingipain‐null strain with isogenic mutants deficient in individual T9SS components.
Email: malgorzata.benedyk@uj.edu.pl
Using an in vivo subcutaneous chamber mouse model of infection, gingipain‐null
Funding information
strain secretion mutants showed no virulence, but their pathogenic potential was
National Science Center Poland, Grant/
Award Number: 2017/01/X/NZ6/01953 reconstituted by coinfection with a low number of the parental strain. Apparently
The peer review history for this article is available the same mechanism compensated fitness of mutants lacking functional T9SS the
at https​://publo​ns.com/publo​n/10. 1111/
transposon library. In contrast to the parental strain, all mutants elicited significantly
omi.12268/​
lower but an effective inflammatory immune response, which cleared infection and
prevented systemic dissemination of P. gingivalis to organs. There were no significant
differences in immune responses to different secretion mutants, which were gener‐
ally more stimulatory than the gingipain‐null strain. Together, these results indicate
that functional T9SS is essential for P. gingivalis virulence apparently through delivery
of active gingipains to the bacterial surface. Therefore, T9SS is a legitimate target for
drug development to treat periodontitis.

KEYWORDS
fitness, gingipain, periodontitis, Porphyromonas gingivalis, protein secretion, T9SS, virulence

1 |  I NTRO D U C TI O N
Periodontitis is a chronic inflammatory disease of the tooth‐support‐
ing tissues. It is characterized by a progressive loss of attachment
caused by the destruction of the connective tissue and alveolar bone,
which can lead to tooth loss (Pihlstrom, Michalowicz, & Johnson,
2005). Globally, mild to moderate periodontitis affects nearly
half of the human population, with severe periodontitis affecting
about 11% of individuals (Kassebaum et al., 2014). Moreover, peri‐
odontitis is associated with various systemic conditions, including
atherosclerotic cardiovascular disease (Tonetti & Van Dyke, 2013),
diabetes (Chapple, Genco, Worksh, & Working Group 2 of the Joint
EFP/AAP Workshop, 2013), and rheumatoid arthritis (Potempa,
Mydel, & Koziel, 2017). The complex pathogenesis of periodontitis
implies interplay between the host response, cumulative effects
of various risk factors, and bacterial challenge posed by the dental
plaque (Hajishengallis & Korostoff, 2017).
Over the last two decades, extensive investigations of the oral
microbiome established that a wide variety of organisms are asso‐
ciated with periodontal pathogenesis. In a synergistic manner they

Mol Oral Microbiol. 2019;34:237–244. wileyonlinelibrary.com/journal/omi   © 2019 John Wiley & Sons A/S. |  237
Published by John Wiley & Sons Ltd
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238       BENEDYK et al.

cause a shift in the microbial composition characterized by increased mouse model. To this end, we quantified numbers of bacteria in
levels of so‐called red‐complex bacteria, Porphyromonas gingivalis, chambers post‐inoculation, determined their ability to disseminate,
Treponema denticola, and Tannerella forsythia in the subgingival mi‐ and analyzed the local and systemic immune responses elicited by
croflora (Hajishengallis, Darveau, & Curtis, 2012; Hajishengallis & individual mutants.
Lamont, 2012; How, Song, & Chan, 2016). These anaerobic, Gram‐
negative microbes are uninvited and unwelcome guests in the very
2 | M ATE R I A L S A N D M E TH O DS
diverse community of microbial biofilm on the tooth surface and their
proliferation significantly changes the composition of the community
2.1 | Bacterial strains and cultures
(microbiota shift) (Hajishengallis, 2015) with all pathological conse‐
quences then following (Lamont & Hajishengallis, 2015). Among the The wild‐type strain of P. gingivalis (W83), its four T9SS mutants
red complex, P. gingivalis is the keystone pathogen with unsurpassed (∆Sov, ΔPorT, ΔPorZ, and ΔPorU; Table 1) and gingipains‐deletion
potential to initiate the transition of the commensal bacterial micro‐ null mutant (∆KRAB, Table 1) were grown under anaerobic condi‐
biota into a dysbiotic one that drives chronic inflammation in the tions (90% N2, 5% CO2, 5% H2) on blood (5% v/v sheep blood) agar
periodontium (Zenobia & Hajishengalliss, 2015). plates or in liquid tryptic soy broth (TSB; Sigma‐Aldrich) and yeast
Gram‐negative, anaerobic P. gingivalis bacterium expresses extract (5 mg/ml; BioShop) supplemented with hemin (5 μg/ml;
several well‐established virulence factors, including lipopolysac‐ Sigma‐Aldrich), l‐cysteine (250 μg/ml; Sigma‐Aldrich), and menadi‐
charide, phosphatase, fimbriae, hemagglutinins, and cysteine pro‐ one (0.5 μg/ml; Sigma‐Aldrich). The ∆Sov, ΔPorT, and ∆KRAB strains
teinases called gingipains (Bostanci & Belibasakis, 2012). The latter were cultured in medium supplemented with tetracycline (1 μg/ml;
are the most important P. gingivalis virulence factor with respect to Sigma‐Aldrich), and the ΔPorU and ΔPorZ strains were cultured in
pathogenicity and survival in vivo (Guo, Nguyen, & Potempa, 2010). medium supplemented with erythromycin (5 μg/ml; Sigma‐Aldrich).
Gingipains are encoded by three distinct genes: rgpA, rgpB, and kgp After overnight culture, bacteria were centrifuged (4,500 × g, 10 min)
(Potempa, Sroka, Imamura, & Travis, 2003). The Arg‐specific (RgpB to prepare inoculates. Subsequently, the bacterial pellet was washed
and RgpA) and Lys‐specific (Kgp) gingipains have been shown to three times with phosphate‐buffered saline (0.14 M NaCl, 0.0026 M
play a pivotal role in nutrient acquisition, colonization, penetration KCl, 0.01 M Na2HPO 4, 0.002 M KH2PO 4), pH 7.4 (PBS), and resus‐
into host tissue, deregulation of the immune response, and devel‐ pended in fresh PBS. Bacterial cell counts were standardized to an
opment of dysbiosis (Benedyk et al., 2016; Fleetwood et al., 2015; optical density of 1.0 at 600 nm (corresponding to 1 × 109 colony‐
Hajishengallis et al., 2012; Hajishengallis & Lamont, 2014). forming units [CFUs]/ml).
Gingipains are secreted to the bacterial cell surface and beyond
in a two‐step process (Fleetwood et al., 2015). First, preprogingipains
2.2 | Sov mutant construction
translocate the inner membrane using secretion pathways known as
the Sec system, and then progingipains across the outer membrane Using a similar strategy to that described previously to create the
using a protein secretion apparatus known as the type IX secretion ΔRgpA mutant (Nguyen et al., 2007), two 1.0 kb flanking regions,
system (T9SS) (Lasica, Ksiazek, Madej, & Potempa, 2017). The latter one 5′ and one 3′ to the sov gene (The Institute of Genomic Research
step is dependent on the presence of a conserved carboxy‐terminal [TIGR] accession numbers: PG0809 and PG0810), were amplified
domain, which tags them for secretion across the outer membrane by PCR using AccuPrime Pfx DNA Polymerase (Invitrogen Inc.)
by the T9SS (Nguyen, Travis, & Potempa, 2007). and inserted into the pUC19 plasmid (New England Biolabs Inc.) at
The T9SS is composed of several components localized in the
cytoplasmic membrane (PorL and PorM), in the periplasm (PorK and
PorN), in the outer membrane (β‐barrel proteins Sov, PorT, PorQ, TA B L E 1   Porphyromonas gingivalis strains used in this study
PorV, and PorP), and on the bacterial surface (PorU and PorZ) Strain Relevant genotype Source
(Lasica et al., 2017). Deletion of any component of that complex
P.g W83 WT Wild type Reference
system results in retention of the proteolytically inactive progin‐
strain
gipains in the mutant periplasm, together with 30 other proteins
P.g ∆PorU porU (NCBI: PG_RS00120; old Lasica et al.
transported by the T9SS (Nikolich, Shoemaker, & Salyers, 1992). locus PG0026) (Emr) (2016)
Interestingly, however, none of the T9SS components is essential P.g ∆PorT porT (NCBI: PG_RS03295; old Nguyen et al.
for the growth of P. gingivalis in epithelial cells and murine skin ab‐ locus PG0751) (Tcr) (2009)
scesses. This is a paradox considering that several proteins trans‐ P.g ∆PorZ porZ (NCBI: PGRS07070; old locus Lasica et al.
ported by this system, including gingipains, are absolutely essential PG1604 (Emr) (2016)
for P. gingivalis fitness in these models (Miller et al., 2017). Herein, P.g ∆Sov sov (NCBI: RC03550; old locus This study
we compared the propensity of P. gingivalis wild‐type (WT) strain PG0809/PG0810) (Tcr)
and individual secretory mutants (porU, ∆PorU; porZ, ∆PorZ; porT, P.g ∆KRAB kgpΔ598 rgpArgpBΔ410 Tcr Cmr Emr Rapala‐Kozik
∆PorT and sov, ∆Sov) to survive and proliferate in subcutaneous et al. (2011)

chambers or be eliminated by the immune system in an in vivo Abbreviations: P.g, Porphyromonas gingivalis; WT, wild‐type.
BENEDYK et al.
the SacI/SmaI and XbaI/SalI sites, respectively. All primers used in
this study are listed in Table 2. An intervening tetracycline resist‐
ance cassette (tetQ) from the pNFD13‐2 plasmid (Saiki & Konishi,
2007) was amplified and inserted between the flanking regions in
the modified pUC19 at the SmaI/XbaI site to create the final plas‐
mid construct, pSovAtB‐C. Correct placement and orientation of the
DNA segments were confirmed by sequencing. Purified plasmids
were electroporated into electro‐competent P. gingivalis W83 cells
as described previously (Nguyen et al., 2007) and integration of the
plasmid construct into the P. gingivalis genome by a double crossover
recombination event was selected for using 1 µg/ml tetracycline in
TSB agar. Deletion of the sov gene in the resulting mutants was con‐
firmed by PCR and DNA sequencing.
2.3 | Experimental animals and ethical approval
Specific pathogen‐free (SPF) female BALB/c mice, 8–10  weeks of
age, were purchased from Jackson Laboratory. Mice were housed in
individually ventilated cages and fed a standard laboratory diet and
water ad libitum in SPF conditions within the animal care facility at the
Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian
University, Krakow, Poland. Mice were maintained under a 60 ± 5%
relative humidity 12 hr‐light/dark cycle and at 22 ± 2°C. Control and
bacterially infected mice were housed in separate cages. All animal
experiments were reviewed and approved by the I Regional Ethics
Committee on Animal Experimentation, Krakow, Poland (approval
no: 131/2018).
2.4 | Subcutaneous chamber model procedure
To enable the best operative outcome, all mice used had a minimum
weight of 20 g. All mice were anesthetized by intraperitoneal in‐
jection of ketamine (22 mg/kg; VetaKetam, VetAgro) and xylazine
(2 mg/kg; Sedasin, Biowet), and the eyes were lubricated with oint‐
ment (Puralube Vet; Pharmaderm). Following general anesthesia,
using sterile instruments, the skin was incised on the right side of
the back and a sterile titanium‐coil chamber was implanted subcu‐
taneously. After 10 days, the mice were injected into the lumen of
the subcutaneous chamber with 1 × 109 CFUs of WT P. gingivalis
W83, ∆Sov, ΔPorT, ΔPorZ, ΔPorU, or ∆KRAB resuspended in a total
volume of 80 µl sterile PBS. Control mice were injected with 80 µl
sterile PBS into subcutaneous chambers (sham‐infection).
TA B L E 2   Primers used for SOV mutant construction
SovFrASacIF ctGAGCTCGCCTACAATCTCAAGCGTATGC
SovFrASmaIR ttaaCCCGGGCGTGATCCTTCTTTGTTTGAGA
SovFrBXbaIF acTCTAGATAGCCTAACGGCATTACCCAC
SovFrBSalIR attaGTCGACCTTGTATGCAGGAAGCAGGATAG
tetQSmaIF tataCCCGGGACAACGAATTATCTCCTTAACGT
tetQXbaIR cgcTCTAGATTTTATTGCCAAGTTCTAATGCT
|
      239
2.5 | CFU assay
After 24, 48, and 72  hr, 100  µl of the fluids were taken from the
chamber and then 10‐fold serially diluted chamber fluids were plated
on blood agar plates and cultured anaerobically for 14 days at 37°C.
Thereafter, the number of visible colonies was counted and the total
number of CFUs was calculated by multiplying this number by the
dilution factor.
2.6 | Myeloperoxidase assay
Polymorphonuclear neutrophil (PMN) influx into the subcutane‐
ous chambers was analyzed by measuring the enzymatic activity of
myeloperoxidase (MPO; as a marker of PMN accumulation). Briefly,
24 hr after bacterial injection into the chamber, 50 µl of 25‐fold di‐
luted chamber fluid was mixed with 100 µl o‐phenylenediamine dihy‐
drochloride (OPD) reagent (5 mg OPD, 5 µl 30% hydrogen peroxide,
5 ml buffer containing 0.05 M citric acid, and 0.05 M sodium phos‐
phate, pH 5) and incubated for 10 min at room temperature in the
dark. The reaction was terminated by addition of 50 µl 2.5 M H2SO4.
Then, absorbance was measured at 490 nm using a microplate reader
(SpectraMax Gemini; Molecular Devices) as direct accumulation
of MPO, which has been expressed as a percentage of the control
groups (chamber fluid from animals inoculated with PBS only).
2.7 | Gingipain activity
On day 1 (24 hr after bacterial injection into the chamber), the pro‐
teolytic activity of P. gingivalis gingipains was measured. Briefly,
5 µl chamber fluid was added to 95 µl TGC buffer (100 mM Tris
pH 7.6, 200  mM Gly‐Gly, 5  mM CaCl2, 100 µg/ml PEFABLOCK,
and 20 µg/ml aprotinin) supplemented with 10 mM l‐cysteine in
96‐well microtitre plates. After 5  min preincubation at 37°C, a
substrate of gingipain R (benzoyl‐arginine‐p‐nitroanilide [BAPNA];
Sigma‐Aldrich) was added to give a working concentration of 1 mM.
The protease activity of gingipain R was represented by the release
of p‐nitroaniline from the substrate by determination of absorb‐
ance at 405 nm using a microplate reader (SpectraMax Gemini;
Molecular Devices).
2.8 | Cytokine and C‐reactive protein measurement
The level of IL‐6, TNFα, IL‐10, MCP‐1 in serum samples was deter‐
mined using a CBA Mouse Inflammatory Cytokine Kit (BD Bioscience)
according to the manufacturer's instructions and results were ana‐
lyzed using an FCAP Array v2.0.2 (BD Bioscience). Briefly, 24 hr after
bacterial injection into the chamber, mice were sacrificed by cervi‐
cal dislocation, and whole blood was immediately collected and al‐
lowed to clot at room temperature (60 min), and then centrifuged at
2,000 × g for 10 min to separate the serum. C‐reactive protein (CRP)
levels in the serum were measured using a commercially available
ELISA (Mouse CRP ELISA kit; Innovative Research), according to the
manufacturer's recommendations.
 |
240       BENEDYK et al.
2.9 | Statistical analysis
All the data were reported as the mean  ±  standard deviation (SD).
Significant differences between groups were examined using
Student's t test or two‐way repeated measures ANOVA (multi‐pa‐
rameter data) and one‐way ANOVA with the Bonferroni correction
or Dunnett's post‐test (multiple comparisons). Kaplan–Meier graphs
were analyzed using the log‐rank test with the Bonferroni correction
(post‐test) to correct for multiple comparisons. All statistical analyses
were performed using GraphPad Prism software (v5.0d GraphPad).
Values of p ≤ .05 were considered to be statistically significant.
3 |   R E S U LT S
3.1 | Inhibition of gingipain secretion prevents
fatality in the subcutaneous chamber model
A subcutaneous chamber infection mouse model was used to com‐
pare the virulence of the WT P. gingivalis W83 and the gingipain‐null
mutant ∆KRAB with secretion‐mutant strains ∆PorU, ∆PorZ, ∆PorT,
or ∆Sov. When 1 × 109 CFUs of bacteria were inoculated into the
chamber, only WT P. gingivalis W83 bacteria were lethal, with all
mice dying by 48  hr. By contrast, animals infected with the same
dose of the gingipain‐null mutant ∆KRAB strain or secretion mu‐
tants (∆PorU, ∆PorZ, ∆PorT, and ∆Sov) showed no signs of disease
throughout the course of the experiment (Figure 1a). Furthermore,
F I G U R E 1   Functional T9SS is necessary to maintain
Porphyromonas gingivalis virulence. BALB/c mice were challenged
with wild‐type (WT) P. gingivalis W83, four different P. gingivalis
T9SS‐secretion mutants (ΔPorU, ΔPorT, ΔSov, or ΔPorZ), and
gingipain‐null mutant (ΔKRAB), each at 1 × 109 CFUs (a) or with
P. gingivalis W83 (1 × 107 CFUs) alone or with ΔSov (1 × 109 CFUs)
(b). Mouse survival was monitored throughout the entire procedure
while subcutaneous chamber inocula of WT W83 at 107 CFUs and
the ∆Sov mutant at 109 CFUs alone each had no effect, when ad‐
ministered together they caused sickness and eventually killed the
mice (Figure 1b). This result suggests that the WT strain restored the
virulence of the secretory mutant.
Levels of gingipains in the chamber fluid were evaluated at the
end of the experiment and were found at very low (Rgp) or unde‐
tectable (Kgp) levels (Figure 2). This confirms that the lack of any
component of the T9SS impairs the secretion of gingipains (Figure 2)
and probably that of all other cargo proteins in vivo.
3.2 | Functional T9SS is necessary for P. gingivalis
survival in subcutaneous chambers
To establish the importance of T9SS components on P. gingivalis
survival/proliferation in vivo, we determined the number of bacteria
(CFUs) in the chamber fluid 24 hr after inoculation. We found that
while the number of CFUs of WT P. gingivalis W83 increased by 30‐
fold, CFUs of all strains deficient in the functional T9SS significantly
decreased in numbers (Figure 3). Although there were variations
in CFUs numbers among secretion mutants, with a tendency for
∆PorU > ∆PorZ > ∆PorT > ∆Sov, the differences between individual
strains were not significant. On the other hand, the gingipain‐null
mutant strain ∆KRAB was cleared more efficiently than secretion
F I G U R E 2   Gingipain activity is at a very low or undetectable
level in chamber fluid after infection with secretion mutants.
Arginine‐specific (a) and lysine‐specific (b) gingipain activity
was determined using l‐BAPNA and N‐(p‐Tosyl)‐Gly‐Pro‐Lys‐4‐
nitroanilide acetate salt as a substrate, respectively. Data are
presented as means ± SD of assays performed in triplicate and
were analyzed by one‐way ANOVA with the Bonferroni post‐test
correction (***p < .0001)
BENEDYK et al. |
      241

the ∆KRAB group and around sevenfold higher than in the groups
challenged with T9SS knockouts (∆PorU, ∆PorZ, ∆PorT, or ∆Sov;
Figure 4a). In contrast to the number of PMNs infiltrating chambers
being similar for all secretion mutants, the level of lymphocytes and
monocytes in the chamber fluid showed the following trends: ∆PorZ > 
∆PorT > ∆Sov > ∆PorU > ∆KRAB and ∆PorU > ∆PorT > ∆PorZ > ∆Sov 
> ∆KRAB, respectively (Figure 4b,c). The trend observed for monocyte
levels reflects the number of surviving bacteria in the chamber, which
was highest in ∆PorU and the lowest in ∆KRAB (Figure 3).
In addition to cytological evaluation, MPO activity was mea‐
sured in the chamber fluid to estimate PMN infiltration and ac‐
tivation. In line with cytological findings, the MPO activity was
F I G U R E 3   Functional T9SS is essential for Porphyromonas
gingivalis survival and proliferation in chambers. Mice were
challenged with 1 × 109 CFUs wild‐type (WT) P. gingivalis W83,
T9SS‐secretion mutants (ΔPorU, ΔPorT, ΔSov, or ΔPorZ) and
gingipain‐null strain ΔKRAB. Survival of bacteria in the chamber
was assessed by counting CFUs on agar plates inoculated with
chamber fluid at 24 hr post‐infection. Data are presented as
means ± SD and were analyzed by one‐way ANOVA (***p < .0001)

mutants, suggesting that gingipains are of pivotal importance for

P. gingivalis survival under attack by the immune system. In keeping
with WT P. gingivalis W83 having the exclusive ability to evade the
host immune system and proliferate inside chambers, only this strain
escaped from the chamber and systematically disseminated to vari‐
ous organs, including the lungs, liver, and kidneys as shown by plating
homogenates from these tissues prepared at 24 and 48 hr post‐in‐
oculation (Table 3). This confirms the importance of the functional
T9SS for the virulence of P. gingivalis in vivo and reaffirms the validity
of the T9SS as a drug target for the treatment of periodontitis.

3.3 | Functional T9SS is pivotal for robust host

inflammatory response
Cytological analysis of the chamber fluid showed the presence of
PMNs, lymphocytes, and monocytes regardless of the inoculated
strain. However, the number of each inflammatory cell type in the
chamber fluid determined 24 hr post‐challenge was far higher in ani‐
mals infected with WT P. gingivalis W83 than with any other strain.
The number of PMNs was around eightfold higher as compared with

TA B L E 3   Presence of bacteria in organs at 24 hr post‐infection

  Lungs Liver Kidney Spleen

P.g W83 WT + + + + F I G U R E 4   Operational T9SS is necessary for the recruitment

of inflammatory cells to the site of inflammation. Numbers of
P.g ∆PorU − − − −
polymorphonuclear neutrophils (PMNs) (a), lymphocytes (b), and
P.g ∆PorT − − − − monocytes (c) in chamber fluid after challenge (24 hr) with wild‐
P.g ∆PorZ − − − − type (WT) Porphyromonas gingivalis W83, T9SS‐secretion mutants
P.g ∆Sov − − − − (∆PorU, ∆PorT, ∆PorZ, or ∆Sov), and gingipain‐null mutant (ΔKRAB)
were counted in smears stained using Wright's stain. Cells were
P.g ∆KRAB − − − −
identified by morphology. Data are presented as a means ± SD
Abbreviations: P.g, Porphyromonas gingivalis; WT, wild‐type. (***p < .0001)
 |
242       BENEDYK et al.
F I G U R E 5   Functional T9SS is important for polymorphonuclear
neutrophil activation in Porphyromonas gingivalis‐infected
chambers. Subcutaneous chambers in BALB/c mice were inoculated
with 1 × 109 CFUs wild‐type (WT) P. gingivalis W83, T9SS‐secretion
mutants (∆PorU, ∆PorT, ∆PorZ, or ∆Sov), and the gingipain‐null
strain (∆KRAB). Then, 24 hr post‐infection myeloperoxidase (MPO)
activity in chamber fluid was determined. The MPO activity was
expressed as a % of control activity (control mice challenged with
PBS)
around fourfold higher in the chamber fluid from P. gingivalis W83
infected animals compared with ∆PorU‐, ∆PorZ‐, ∆PorT‐, ∆Sov‐,
or ∆KRAB‐infected animals (Figure 5). In this case, no clear trend
in the level of PMN activation was observed among the secretion
mutants.
3.4 | Functional T9SS is crucial for the
development of systemic inflammatory responses
during subcutaneous chamber infection with
P. gingivalis
Cytokine and chemokine production are an important part of the
host's defense system and play a critical role in regulation of the
inflammatory response against bacterial infections. To assess the
role of the T9SS in P. gingivalis‐stimulated systemic inflammation,
the concentration of TNFα, MCP‐1, IL‐6, and IL‐10 in the peripheral
blood was assessed 24 hr post‐inoculation. As expected, in the blood
of animals infected with WT P. gingivalis W83, a strong, statistically
significant increase of all cytokines tested was observed. Infection
with ∆PorU, ∆PorZ, ∆PorT, ∆Sov, or ∆KRAB resulted in only a small
increase in cytokine levels (Figure 6a–d). Similarly, CRP, another im‐
portant marker of inflammation, showed the highest increase in the
blood of animals challenged with WT P. gingivalis W83, while chal‐
lenge with secretion mutants (∆PorU, ∆PorZ, ∆PorT, and ∆Sov) and
the gingipain‐null mutant ∆KRAB caused a moderate increase during
F I G U R E 6   Inactivation of T9SS
suppresses the immune response to
Porphyromonas gingivalis. Subcutaneous
chambers in BALB/c mice were
challenged with 1 × 109 CFUs wild‐type
(WT) P. gingivalis W83, T9SS‐secretion
mutants (∆PorU, ∆PorT, ∆PorZ, or ∆Sov)
or gingipain‐null strain (∆KRAB). Then,
24 hr post‐infection level of IL‐6 (a), TNFα
(b), MCP‐1 (c), IL‐10 (d), and CRP (e) was
determined in blood serum. Significance
of observed differences between
P. gingivalis W83 WT and P. gingivalis
∆PorU‐, ∆PorT‐, ∆PorZ‐, ∆Sov‐, and
∆KRAB‐challenged animals was verified
with Student's t test, *p < .001, **p <
.0013, ***p < .0001 (a–d) or with one‐way
ANOVA test, ***p < .0001 (e)
BENEDYK et al.
the infection (Figure 6e). Interestingly, in the case of IL‐6 and TNFα, a
trend similar to that observed for CFUs, lymphocyte, and monocyte
numbers in the chamber fluid was noticed (Figure 6a,b).
4 | D I S CU S S I O N
The protein secretion system known as the T9SS is present in many
species of the Bacteroidetes phylum. In P. gingivalis, the keystone per‐
iodontal pathogen T9SS is responsible for the secretion of about 30
different proteins, including pivotal virulence factors: the gingipains
(Abby et al., 2016; Nguyen et al., 2009). It is surprising that to date no
virulence analysis of T9SS‐secretion mutants has been carried out
using an in vivo model of infection necessary to confirm the impor‐
tance of T9SS for the pathogenicity of P. gingivalis.
In this study, we used the well‐established subcutaneous cham‐
ber model that allows monitoring of bacterial growth or eradication
of infection in vivo. This provides a reliable way to sample and eval‐
uate inflammatory exudates to study the host local and systemic
immune response. Results of our in vivo experiments conclusively
demonstrated that a functional T9SS is pivotal for P. gingivalis growth,
dissemination, and pathogenic effects in vivo, including induction of
a strong inflammatory reaction. Disabling the T9SS in P. gingivalis
(as in the ∆PorU, ∆PorZ, ∆PorT, and ∆Sov mutants) led to a dras‐
tic decrease in CFUs in the chamber, which is in stark contrast to
WT P. gingivalis, which proliferated. Despite this finding, none of the
components of T9SS disabled by gene deletion were found to con‐
tribute to P. gingivalis fitness in three different models, in contrast
to several cargo proteins (Miller et al., 2017). Apparently, inoculating
with the transposon library of mutants, the lack of functional T9SS
was compensated by the mutants with a functional secretion system.
This contention was confirmed in the present study with the find‐
ing that a relatively small contingent of WT P. gingivalis restored the
bacterium virulence in vivo. It can be speculated that T9SS is leaky
and transported proteins essential for fitness are released into the
outside environment where they are processed by extracellular com‐
ponents of T9SS from secretion‐competent mutants in the library.
Such a mechanism would be especially important in processing
and activating gingipains. Only infection with WT P. gingivalis W83
was able to trigger a local and systemic immune response as mani‐
fested by the mass influx of inflammatory cells into the chamber and
the increase in proinflammatory cytokine production. Furthermore,
only in response to infection with WT P. gingivalis W83 we did ob‐
serve a rapid recruitment of a large number of lymphocytes, PMNs,
and monocytes to the site of infection, leading to increased produc‐
tion of IL‐6, TNFα, MCP‐1, and IL‐10 cytokines as well as an increase
in CRP. Apparently, the lack of gingipain activity in T9SS mutants
most significantly contributed to abolishment of P. gingivalis viru‐
lence, which corroborates the finding that inhibition of gingipains
completely eliminates P. gingivalis virulence in vivo (Bostanci &
Belibasakis, 2012; Guo et al., 2010; Kadowaki et al., 2007). Finally,
taking into account that Sov is the OM beta‐barrel secretion pore
essential for OM translocation while PorU is the surface sortase
|
      243
involved in cleavage of the CTD and attachment of T9SS cargo pro‐
teins, it is interesting to note a clear trend for the higher CFU and
monocyte accumulation in chambers inoculated with delta‐PorU
than delta‐Sov. This may be related to the “partial secretion” pheno‐
type of the former mutant in which significant amount of T9SS cargo
proteins were found on the cell surface (Glew et al., 2012).
Previous studies using the subcutaneous chamber model
have shown that challenge with bacteria possessing a properly
functioning T9SS, and therefore capable of secretion of pro‐
teins including gingipains, leads to exaggerated inflammation
and immune activation, and an increase in bacterial tissue bur‐
den with resultant bacteria dissemination, chamber exfoliation,
tissue injury, and ultimately death. The microbicidal mechanisms
of phagocytes with production of TNF and other cytokines seem
to be primarily responsible for the necrotic lesions of infection.
None of the above effects were observed after chamber inoc‐
ulation with secretion mutants (∆PorT, ∆PorU, ∆PorZ, or ∆Sov)
or the gingipain‐null mutant ∆KRAB, confirming the pivotal role
of gingipains as the primary virulence factor responsible for an
immediate and uncontrolled inflammatory response, bacteria dis‐
semination, and subsequently death of the animals (Guo et al.,
2010; Kadowaki et al., 2007; Lin, Huang, Lai, Huang, & Hu, 2005).
These results are in line with our previous studies using an animal
model of aspiration pneumonia, where infection with P. gingivalis
W83 induced fatal pneumonia, while the gingipain‐null mutant
∆KRAB induced only a transient and non‐threatening infection
(Benedyk et al., 2016).
In summary, we conclude that the complex T9SS and all of its
identified components play an important role at the site of infec‐
tion as the source of gingipain, the most important virulence fac‐
tor of P. gingivalis. More importantly, our data clearly confirm that
all components of T9SS are a viable target for the development
of novel treatment strategies for periodontitis as T9SS inhibition
leads to utter loss of bacterial virulence and quick clearance from
the tissue.
AC K N OW L E D G E M E N T S
This work was funded by the National Science Center, Poland
(2017/01/X/NZ6/01953).
C O N FL I C T O F I N T E R E S T
The authors have no conflicts of interest to declare.
ORCID
Malgorzata Benedyk  https://orcid.org/0000-0001-5432-8739
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How to cite this article: Benedyk M, Marczyk A, Chruścicka B.
Type IX secretion system is pivotal for expression of gingipain‐
associated virulence of Porphyromonas gingivalis. Mol Oral
Microbiol. 2019;34:237–244. https​://doi.org/10.1111/
omi.12268​