Importance of biofilm formation and dipeptidyl peptidase IV for the pathogenicity of clinical
Porphyromonas gingivalis isolates
For figures, tables and references we refer the reader to the original paper.
Introduction
Porphyromonas gingivalis is a natural member of the oral microbiome and regarded as a primary
etiological agent of periodontitis (Holt et al., 1999; Davey, 2006; Hayashi et al., 2010; Cugini et al.,
2013). This Gram-negative anaerobe is a key player in the shift from benign dental plaque into a
pathogenic microbial community (Cugini et al., 2013). Thanks to an arsenal of specialized virulence
factors, P. gingivalis can become highly destructive and trigger the progression of periodontal disease
(Cugini et al., 2013). Porphyromonas gingivalis is known to have proteolytic properties and digests
external sources of peptides to meet its nutritional requirements (Banbula et al., 2000; Kumagai et
al., 2000; Rea et al., 2004; Cugini et al., 2013). The serine protease dipeptidyl peptidase IV (DPPIV)
cleaves X-Pro or X-Ala dipeptides from the N-terminal end of polypeptide chains, leading to the
breakdown of periodontal tissues (Banbula et al., 2000; Kumagai et al., 2000). Moreover, DPPIV
promotes the activity of host-derived matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-8 and
MMP-9, which may augment tissue destruction (Kumagai et al., 2005). DPPIV also influences the
host's healing process, as it inhibits the adhesion of fibroblasts to fibronectin (Kumagai et al., 2005).
The ability of P. gingivalis to colonize the oral cavity and proliferate is influenced by its capacity to
participate in the oral biofilm (Davey, 2006). Biofilm formation may improve the adherence to the
subgingival tissue and provide protection against the immune system (Cos et al., 2010). Furthermore,
the presence in a microbial community may lead to environmental conditions and interactions that
are pivotal to elicit a virulent state and full pathogenicity of P. gingivalis (Cugini et al., 2013).
The aim of the study is to evaluate the importance of biofilm formation and DPPIV activity for the
virulence of clinical P. gingivalis isolates. Their biofilm-forming capacity and DPPIV activity were
assessed in relation to their in vivo pathogenic potential. Moreover, bacterial DPPIV activity was
compared between planktonic and sessile cells in biofilms to evaluate the influence of the mode of
growth on DPPIV.
Methods
Bacterial strains and culture
Clinical isolates of P. gingivalis with a different capsular type (K1–K5) were obtained from the oral
cavity of patients at the University Hospital Leuven (Belgium). The ethical committee of the
University Hospital approved the study (Code B32220071073) and the written informed consent was
obtained from all subjects. Capsular serotyping of the clinical isolates was performed by double
immunodiffusion and immunoelectrophoresis using polyclonal antisera, as described previously (van
Winkelhoff et al., 1993; Laine et al., 1996; Dierickx et al., 2003). All strains were grown on Wilkins–
Chalgren agar plates (Oxoid) supplemented with 5% sheep blood (Oxoid) and l-cysteine (500 mg L−1;
Sigma-Aldrich). The plates were incubated at 37 °C in an anaerobic atmosphere of 80% N2, 10% H2,
and 10% CO2 for 7–14 days.
Biofilm determination
A protocol using crystal violet for the quantification of biofilms was optimized for P. gingivalis (Tote
et al., 2010). Biofilms were grown in a 96-well plate using Wilkins–Chalgren broth (Oxoid)
supplemented with 500 mg L−1 l-cysteine (Sigma-Aldrich), 2 g L−1 maltose (Merck), and 2 g L−1
starch. The latter two compounds were added to improve the biofilm-forming capacity. After an
incubation period of 96 h on a horizontal shaking plate (Innova 4300; New Brunswick Scientific)
under anaerobic conditions, the medium was removed and the wells were washed with phosphatebuffered saline (Gibco) to remove nonadherent bacteria. The biofilm was methanol-fixed, stained
with 0.01% (w/v) crystal violet (Merck), and washed with running tap water to remove unbound dye.
The wells were air-dried, whereupon 33% (v/v) glacial acetic acid (Merck) was added to release the
dye from the biofilms. The optical density was measured at 570 nm using a microplate reader
(Multiskan microplate reader, Labsystems). P. gingivalis reference strains ATCC 33277, W50, and W83
were used as controls, as they are known for good, moderate, and weak biofilm formation,
respectively (Davey, 2006; Dashper et al., 2010; Biyikoglu et al., 2012). Based on these phenotypes,
three categories of biofilm formation were defined. An optical density under 0.2 was categorized as
no or weak biofilm formation, an optical density between 0.2 and 0.5 was considered as moderate
biofilm development and based on the reference strain ATCC 33277, an optical density higher than
0.5 was chosen as a cut-off for good biofilm formation. According to these arbitrary values, the
phenotype of the clinical isolates could be defined. For each strain, at least eight replicates were
taken and the experiments were repeated on three different days. The biofilm producing reference
strain ATCC 33277 was included as a positive control in each experiment and noninoculated Wilkins–
Chalgren broth as a blank.
Measurement of DPPIV activity in planktonic and in sessile P. gingivalis cells
All strains were grown anaerobically in chopped meat carbohydrate broth (BD) for 24 h. Prior to
fractionation, the number of colony-forming units (CFU) was determined by viable plate count.
Figure 1 shows the fractionation protocol for DPPIV that was based on Banbula et al. (2000). Briefly,
the cells were washed and membrane-bound enzymes were detached from the purified bacteria
with a buffer solution containing 0.05% Triton X-100. To obtain the intracellular enzymes, cells were
lysed with 1% Triton X-100 buffer solution. To explore the subcellular localization of DPPIV, all
fractions of the reference strain W50 were examined. The DPPIV activity was measured kinetically
during 20 min using 0.5 mM of the chromogenic substrate glycyl-prolyl- para-nitroanilide in 100 mM
Tris-HCl buffer pH 8.3 at 37 °C (Durinx et al., 2000; Matheeussen et al., 2012). The release of paranitroaniline was measured at 405 nm using a microplate reader (VERSAmax™ microplate reader,
Molecular Devices). One unit of activity was defined as the amount of enzyme, which cleaves 1 μmol
substrate min−1. The enzyme activity was then related to the number of CFU and further expressed
as units (U) per 1010 CFU. The commercially available DPPIV inhibitor vildagliptin was included as a
control for enzyme specificity. One-way anova indicated assay repeatability, as the analysis of DPPIV
activity of W50 in triplicate was the same on five independent days.
The DPPIV activity in biofilms vs. planktonic bacteria was analyzed for the P. gingivalis strains with
good biofilm formation. Bacteria were grown in a 24-well plate using supplemented Wilkins–
Chalgren broth during 96 h and in an anaerobic atmosphere under shaking conditions. Bacteria in
their biofilm state were obtained by scraping and planktonic bacteria could be harvested from the
supernatant in the same wells. The amount of bacteria was determined using viable plate count and
quantitative PCR (qPCR). DNA extraction and purification were performed with a QIAamp DNA Mini
Kit (Qiagen) according to the manufacturer's guidelines. Primers and probes were designed from the
species-specific region on 16S rRNA gene. The sequence of the forward primer was 5′GAGGAACCTTACCCGGGAT-3′; the sequence of the reverse primer was 5′ATGCAGCACCTACATAGAAGC-3′; and the sequence of the TaqMan probe was 5′TAGATGACTGATGGTGAAAACCGTCTTCC-3′ (TIB MOLBIOL). The probe was labeled with the
fluorescent dyes 6-FAM at the 5′ end and BBQ at the 3′ end. Quantification of DNA with qPCR was
executed with a StepOnePlus™ RT-PCR system (Applied Biosystems). Thermal cycling consisted of an
initial denaturation step of 50 °C for 2 min and 95 °C for 10 min, followed by 40 amplification cycles
at 95 °C for 15 sec (denaturation) and 60 °C for 1 min (annealing + elongation). All samples were
processed in triplicate.
Animals
Eleven-week-old female BALB/c mice (Janvier) were maintained at 20 °C, 50% humidity and at a
light/dark cycle of 12/12 h. All mice were provided with standard rodent food pellets (Carfil Quality)
and tap water ad libitum. All animal studies were approved by the ethical committee of the
University of Antwerp (Code 2011-18).
Assessment of pathogenicity in mouse abscess model
The pathogenic capacity of the different P. gingivalis strains was assessed in a mouse abscess model.
For each strain, four mice were infected with a dorsal subcutaneous injection of bacterial suspension
(0.1 mL, 109 CFU mL−1). Prior to infection, the bacterial load was adjusted by measurement of the
optical density. The exact number of bacteria in the inoculum was determined by viable plate count.
Exposure to the aerobic atmosphere was limited prior to infection to ensure bacterial viability.
General health and weight of the mice and the presence and location of abscesses were evaluated
daily. Ten days after infection, the animals were euthanized by cervical dislocation. A macroscopic
evaluation was performed and the spread of P. gingivalis from the lesions to distant places was
assessed by two independent experienced researchers.
Statistical analysis
All data were analyzed using spss software version 20.0 (SPSS, Chicago). The repeatability of the
enzyme activity measurements, the localization of DPPIV, and the differences in biofilm formation
and DPPIV activity between the strains were analyzed using one-way anova and Tukey's HSD post hoc
tests. All values were expressed as the mean ± SD. A P-value lower than 0.05 was considered as
statistically significant.
Results
Clinical isolates K4 and K5 demonstrated the strongest biofilm formation
To evaluate the in vitro biofilm formation of the clinical isolates, P. gingivalis reference strains ATCC
33277, W50, and W83 were used as controls. Based on these phenotypes, three categories of biofilm
formation were defined with good, moderate, and weak biofilm formation, respectively (Fig. 2).
According to these criteria, the clinical isolates K1 and K2 formed weak biofilms, whereas K3
demonstrated moderate biofilm formation. The isolates K4 and K5 showed good biofilm
development, just as the ATCC reference strain.
Clinical isolates K4 and K5 showed the highest DPPIV activity in planktonic state
Our fractionation protocol allowed us to examine the DPPIV activity of the P. gingivalis reference
strains and clinical isolates. Fraction 2 contained detached membrane-associated proteins and
showed 100 times more DPPIV activity compared to the intracellular fraction 3. This confirms the
membrane association of DPPIV (Table 1) (Banbula et al., 2000). Moreover, addition of the DPPIV
inhibitor vildagliptin (20 μM) to fraction 2 resulted in complete inhibition of enzyme activity. Figure 3
illustrates the DPPIV activity for all the P. gingivalis strains in planktonic state. The clinical isolates K4
and K5 demonstrated the strongest DPPIV activity, as compared to the other strains.
The DPPIV activity differs significantly between the membrane-associated protein fraction (fraction
2) and the intracellular fraction (fraction 3). Fraction 1, containing whole bacteria, showed similar
DPPIV activities as the membrane-associated protein fraction. ( n = 5, P < 0.05, one-way anova and
Tukey's HSD post hoc test).
Biofilm sessile cells show increased DPPIV activity compared to planktonic cells
For the biofilm-forming clinical isolates, the DPPIV activity was compared between planktonic cells
and sessile cells in a biofilm. The biofilm-associated bacteria demonstrated significantly increased
DPPIV activities compared to the free-floating planktonic cells (Fig. 4).
Clinical isolates K4 and K5 induce abscess formation in vivo
A murine abscess model was used to study the pathogenicity of clinical isolates in vivo. The virulent
reference strains W50 and W83 induced abscesses in only one out of four mice. In contrast, infection
with the clinical isolates K4 and K5 resulted in clear abscess formation. In case of K4, one open and
two closed abscesses were seen. Infection with clinical isolate K5 resulted in closed abscess
formation in three of four mice. Two mice infected with isolate K5 even had an abdominal wound,
indicating the bacterial spread from the abscess point to distant places. Viable plate counts of the
pus allowed to quantify the bacterial load and confirmed the uniformity of the recovered P. gingivalis
population based on morphological characteristics (data not shown). No abscess formation was seen
for the reference strain ATCC 33277, or for the clinical isolates K1, K2 and K3.
Discussion
Dental plaque is an oral biofilm of bacteria that may induce periodontitis. The onset of periodontal
disease is characterized by the shift of a health-associated biofilm of Gram-positive aerobes into a
more pathogenic community with Gram-negative anaerobes (Holt et al., 1999; Cugini et al., 2013). P.
gingivalis has been considered as a key player in this dysbiosis (Cugini et al., 2013) and possesses an
arsenal of virulence factors for initiation and progression of periodontitis (Davey, 2006). In the oral
biofilm, P. gingivalis deploys fimbriae, adhesins and a capsule to efficiently colonize the oral cavity
(Davey, 2006). Because proteins are scarce and difficult to obtain in dental plaque, a variety of
bacterial proteases are required to support the asaccharolytic growth of P. gingivalis. Their enzymatic
activities may lead to destruction of periodontal tissue and affect the immune system (Cugini et al.,
2013). Furthermore, extensive proteolysis alters the oral microenvironment and triggers the shift in
biofilm composition (Cugini et al., 2013). Disruption of the bacterial interactions and disturbance of
the complex interplay between the microbial community and its host form the basis of periodontal
disease progression (Cugini et al., 2013).
Given the central role of P. gingivalis in the development of periodontitis, profound understanding of
its virulence factors and pathogenic properties may provide new insights in the pathogenesis and
clues for novel therapeutic approaches. This study assessed the biofilm-forming capacity and DPPIV
activity of clinical P. gingivalis isolates in relation to their in vivo pathogenic potential. Moreover, the
bacterial DPPIV activity was investigated according to its mode of growth to evaluate whether P.
gingivalis biofilm formation influences the regulation of other virulence factors.
In our study population, bacteria with good biofilm-forming properties demonstrated a high DPPIV
activity compared to the weak or nonbiofilm formers. Interestingly, our study is the first to show that
DPPIV activity even increased during biofilm formation, which might suggest that biofilm
development promotes bacterial survival through the upregulation of enzyme activity. The molecular
basis of the high DPPIV activity upon biofilm formation should be further elucidated at the gene and
protein expression level. A recent gene expression study using DNA microarrays showed that several
genes involved in growth and metabolism are downregulated, while a number of virulence
determinants were highly upregulated when P. gingivalis grows as a biofilm (Lo et al., 2009). The
augmented DPPIV activity in our biofilms might be related to its importance for bacterial virulence in
the oral biofilm, as the proteolytic action of DPPIV contributes to the nutritional requirements of P.
gingivalis and the colonization of the oral cavity through tissue disruption. Kumagai et al. (2000) have
already established DPPIV as a P. gingivalis virulence factor using DPPIV-deficient mutants in a
murine abscess model. Infection with the wild-type strains caused more severe abscess formation
and increased mortality compared to DPPIV-deficient mutants (Kumagai et al., 2000).
Also in our study, a murine dorsal subcutaneous abscess model was used to compare the pathogenic
potential of the clinical isolates. Although this model does not reproduce all aspects of human
periodontitis, it provides a convenient approach to evaluate bacterial virulence. Only the good
biofilm formers with a high DPPIV activity induced abscesses, which may further indicate the
potential role of these bacterial properties for the pathogenicity of P. gingivalis.
Experimental conditions determine the absolute quantitative outcomes of in vitro assessments. In
our setup, different growth media resulted in different absolute DPPIV activities, as shown in Figs 3
and 4, indicating the importance of standardization in this type of experiments. However, the relative
differences in DPPIV activities between the clinical isolates were independent of the growth medium.
Also biofilm formation, which was quantified by absorbance measurement after crystal violet
staining, suffered from some variability due to minor differences in the experimental circumstances.
To handle this variability, P. gingivalis reference strains were used to allow relative assessment of
biofilm growth. Overall, the biofilm-forming capacity of the reference strains was in line with
literature, that is, ATCC 33277, W50, and W83 formed good, moderate, and weak biofilms,
respectively. ATCC 33277 proved to form biofilms, but showed only low DPPIV activity. In vivo, this
strain was nonpathogenic, which further supports the importance of DPPIV in the full package of
virulence factors for pathogenicity. The fact that ATCC 33277 forms biofilms despite its low DPPIV
activity is in line with the concept that DPPIV is upregulated in biofilms, but not directly contributes
to biofilm formation. However, we can hypothesize that membrane-associated DPPIV enhances
biofilm growth by acting as an adhesin, which has already been described for proteases such as
gingipains (Tokuda et al., 1996; Grenier et al., 2003). Many bacterial surface structures, such as
fimbriae, outer membrane proteins and lipopolysaccharides, influence bacterial hydrophobicity and
therefore surface attachment and biofilm formation (Dierickx et al., 2003; Grenier et al., 2003). The
difference in biofilm-forming capacity of the clinical isolates could also be related to their difference
in capsular type, as this significantly contributes to the bacterial surface properties (Dierickx et al.,
2003; Davey, 2006; Biyikoglu et al., 2012). Our straightforward method to assess biofilm growth in
vitro allows the evaluation of the biofilm-forming capacity of additional clinical isolates with different
capsular types to clarify the importance of the capsular type for biofilm growth (Davey, 2006).
Overall, our data obtained in clinical isolates are in accordance with literature data and strengthen
the hypothesis that biofilm formation and DPPIV activity are important tools for P. gingivalis
pathogenicity (Yagishita et al., 2001; Kumagai et al., 2005). As such, these bacterial factors could be
considered as potential therapeutic targets for virulence inhibition. Given the increasing number of
reports on resistance to current antibiotics and the lack of new drugs in the developmental pipeline,
alternative therapies to improve the eradication of bacterial infections are needed (Escaich, 2008;
Rasko & Sperandio, 2010).
In conclusion, biofilm formation, DPPIV activity, and in vivo pathogenicity strongly correlate in our
clinical P. gingivalis isolates. Moreover, our data suggest that the biofilm mode of growth may
improve the virulence potential of P. gingivalis by upregulation of other virulence factors such as
DPPIV. Considering the key roles of biofilm formation and bacterial proteases for oral colonization
and growth, these virulence factors could present interesting targets to tackle periodontitis.
Acknowledgements
This project was supported by the Agency for Innovation by Science and Technology in Flanders (IWT,
Belgium), the University of Antwerp (BOF-GOA, ID: 25624), and the Fund for Scientific Research
(FWO-Flanders, Belgium, Project code G.0173.09N). S.C. is a Ph.D. fellow of the IWT-Flanders. We
thank Pim-Bart Feijens for his support during the in vivo assays.