- Email: [email protected]
Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multi-species communities
Accepted Manuscript Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multi-species communities
Jessica Neilands, Julia R. Davies, Floris J. Bikker, Gunnel Svensäter PII:
S1075-9964(18)30179-3
DOI:
10.1016/j.anaerobe.2018.10.007
Reference:
YANAE 1958
To appear in:
Anaerobe
Received Date:
09 August 2018
Accepted Date:
20 October 2018
Please cite this article as: Jessica Neilands, Julia R. Davies, Floris J. Bikker, Gunnel Svensäter, Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multispecies communities, Anaerobe (2018), doi: 10.1016/j.anaerobe.2018.10.007
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ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9
Parvimonas micra stimulates expression of gingipains from
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Porphyromonas gingivalis in multi-species communities
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Jessica Neilandsa*, Julia R. Daviesa, Floris J. Bikkerb and Gunnel Svensätera
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aDept
of Oral Biology, Faculty of Odontology, Malmö University, Malmö, Sweden
bDepartment
of Oral Biochemistry, Academic Centre for Dentistry Amsterdam,
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Free University and University of Amsterdam, Amsterdam, The Netherlands
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[email protected], [email protected], [email protected], [email protected]
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Corresponding author:
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Dr Jessica Neilands
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Dept. of Oral Biology, Faculty of Odontology, Malmö University, 205 06 Malmö, SWEDEN
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[email protected]
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+46-709-163515 1
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Abstract
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Dental biofilms are complex ecosystems containing many bacterial species that live in
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mutualistic relationships. These interactions can profoundly affect the virulence properties of
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the community. In this study we investigated how the production of gingipains, virulence
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factors from Porphyromonas gingivalis important in periodontal disease, was affected by
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other commonly found members of the sub-gingival microbiome.
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To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium
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nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus
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gordonii and Streptococcus cristatus, with or without Parvimonas micra) as well as dual
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species consortia (P. gingivalis with P. micra, S. oralis or F. nucleatum) were constructed and
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maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was
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determined by plating on Brucella agar and the gingipain specific fluorogenic substrate
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BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P.
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micra on gingipain activity was investigated by adding supernatants from P. micra to P.
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gingivalis cultures. The most prominent secreted proteins in the supernatant were identified
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using mass spectrometry.
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P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F.
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nucleatum. In contrast, with P. micra growth was significantly enhanced and this was
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associated with an increase in gingipain activity. In the multi-species consortia, the presence
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of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to
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supernatants from P. micra for 24 hours caused a 3-fold increase in gingipain activity. This
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effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel
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electrophoresis revealed that several of the most prominent proteins in the P. micra
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supernatant were glycolytic enzymes. The results from this study suggests that gingipains are
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produced in response to a P. micra derived signaling molecule that is most likely a protein.
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This is the first time it has been shown that P. micra can affect P. gingivalis virulence
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properties. This is likely to be of significance for the development of be of periodontitis since
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these two microorganisms are often found together in the subgingival biofilm.
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Key words: periodontitis, gingipain, Gram negative, bacterial interactions, Porphyromonas
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gingivalis
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Introduction
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It is estimated that more than 500 million people worldwide are affected by periodontitis [1].
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Periodontitis is a biofilm-induced inflammatory disease of the oral cavity that results in
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breakdown of the gingival tissues and supporting bone, which can be so extensive that it
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eventually leads to tooth loss. Disease is initiated when the inflammatory response of the host
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is stimulated by plaque accumulation at the gingival margin which results in an increased
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flow of nutrient-rich fluid from the gingival crevice (GCF). This environmental change
74
favours outgrowth and enrichment of various proteolytic bacteria within the biofilm while at
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the same time reducing the competitiveness of others. The change in composition,
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accompanied by an increase in proteolytic phenotypes within biofilms, is believed to be the
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driving force for progression of disease [2].
78
The oral cavity is a heterogeneous environment colonized by over 600 different bacterial
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species. Co-adhesion of species during biofilm formation promotes mutually beneficial
80
interactions by locating organisms in close proximity to appropriate partners [3,4]. The
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physical relationships facilitated by biofilm growth enable nutritional cooperation which is
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seen, for instance, in the expression of complementary enzymes by plaque bacteria for the
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degradation of complex salivary and serum glycoproteins [5, 6], and metabolic end-products
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of one species serving as primary energy source for others [7]. Thus, in health, the balance
85
between synergistic and antagonistic interactions within biofilms (biosis) supports a symbiotic
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relationship with the host, but when the biofilm is subjected to environmental pressure this
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balance can shift giving rise to a dysbiotic biofilm phenotype that may induce disease [8,9].
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A recent study by Abusleme et al, using 16S-rRNA sequencing to identify bacterial species
89
from the subgingival microbiome in healthy subjects and individuals with periodontitis,
90
revealed a core microbiome, found in the majority of subjects, which was present in equal
91
numbers in health and disease [10]. Species such as Actinomyces spp, Rothia spp and 4
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Streptococcus spp dominated in health whereas, for example, Treponema spp, TM7,
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Tannerella forsythia, Parvimonas micra, Porphyromonas gingivalis and
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Peptostreptococcacae appeared in the majority of subjects with periodontitis and had a higher
95
prevalence and abundance than in health [10]. These data support the earlier studies of
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Socransky et al. in which a complex of Treponema. denticola, P. gingivalis and T. forsythia
97
was found to be closely associated with periodontal disease [11]. P. gingivalis, has since been
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the focus of many studies due to its pro-inflammatory properties, for instance, activation of
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TLR2 receptors and impairment of leukocyte recruitment and function. In addition, P.
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gingivalis is capable of expressing a number of proteolytic enzymes including the
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extracellular endopeptidases, the arginine (RgpA & RgpB) and lysine (Kgp) gingipains,
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which are assumed to primarily be part of the proteolytic system for generation of nutrients
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[12]. However, these proteases can also modulate host defences through degradation of
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complement, immunoglobulins and cytokines [13]. Due to its ability to subvert immune
105
responses and support a dysbiotic biofilm phenotype, even when present at low levels, P.
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gingivalis is regarded as a keystone pathogen in periodontitis [14].
107
Gingipains are either outer membrane-associated or secreted [15].The activity of the
108
gingipains is enhanced under reducing conditions, for example in the presence of cysteine,
109
while the activity is diminished in the presence of oxygen [16,17]. An excess of hemin has
110
also been shown to enhance gingipain activity [18]. Despite the ability of P. gingivalis to
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produce these important virulence factors, animal studies have shown that it is not capable of
112
causing disease alone, but requires the presence of other bacterial species [19]. This is in
113
keeping with the concept that most infectious diseases are not the result of a single organism
114
but caused by the concerted actions of multi-species bacterial communities. One possible
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mechanism by which other bacteria could influence the ability of P. gingivalis to induce
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disease is by modulating the activity of virulence factors such as gingipains [20]. In this study
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we have therefore investigated how other members of the sub-gingival microbiome affected
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gingipain activity in P. gingivalis.
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Materials and methods
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Bacterial strains
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Clinical isolates recovered from the subgingival microbiome were used in this study. P.
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gingivalis (SUB1), Fusobacterium nucleatum (FMD), Actinomyces naeslundii (BJJ), P. micra
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(EME), Streptococcus oralis (2009-213A1) and Streptococcus gordonii (2010-203G) were
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isolated from patients with established periodontitis, each strain from a different patient
126
whereas Streptococcus mitis (CL) and Streptococcus cristatus (CL) were isolated from a
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healthy male donor. The bacteria were identified to species level as described previously
128
using a combination of colony morphology, appearance after Gram staining and biochemical
129
or molecular tests [21]. Isolates were stored in skimmed milk at -80ºC until use.
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Measurement of Arg gingipain activity using fluorescent substrate
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Arginine gingipain activity was assessed using the synthetic fluorogenic substrate BikKam-
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10, FITC–Arg–D-Glu–KDbc, (PepScan Presto B.V., Lelystad, The Netherlands). Fifty µL
134
bacterial suspension was added to 2 µL BikKam-10 in a 96-well plate (NUNC, ThermoFisher
135
Scientific, Roskilde, Denmark) (final concentration of 16mM) [17]. Fluorescence was
136
measured at one minute intervals in a BMG Fluostar Optima plate reader (BMG Labtech,
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Offenburg, Germany) (using excitation and emission wavelengths of 488 and 538 nm,
138
respectively) and expressed as change in fluorescence units over time (FU/min).
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P. gingivalis in dual-species consortia
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Bacterial suspensions of P. gingivalis, P. micra, S. oralis and F. nucleatum were created in
142
pre-reduced 10% heat-inactivated equine serum (Håtunalab, Bro, Sweden) to give an optical
143
density at wavelength 600 nm of 0.1. For P. gingivalis, this corresponded to approximately
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1.3x107 bacterial cells/mL, S. oralis 1.6x106 cells/mL, F. nucleatum 3.7x107 cells/mL and P.
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micra 7.2x106/mL. One mL of the P. gingivalis suspension were then mixed with an equal
146
volume of one of the other three bacterial suspensions to give dual-species consortia with a
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final concentration of 6.5x106 P. gingivalis cells/mL and incubated at 37° C anaerobically
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(10% H2, 5% CO2 in N2). The suspension with P. gingivalis only was incubated in the same
149
manner. After seven days the gingipain activity in the dual-species consortia was measured in
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50 µL cellsuspension and serial dilutions plated onto Brucella agar. The number of bacteria
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in each consortium was determined by counting the number of colonies of each species
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(easily distinguishable due to differences in colony morphology) after eight days incubation
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anaerobically at 37° C. In addition, the gingipain activity in the P. micra dual-species
154
consortium was measured after 24h and 72h.
155 156
Effect of P. micra on P. gingivalis in multi-species consortia
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Multi-species consortia were created by inoculating colonies (1µL loop) of the different
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bacteria (P. gingivalis, F. nucleatum, A. naeslundii, S. oralis, S. gordonii, S. mitis and S.
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cristatus) into four mL 10% pre-reduced heat-inactivated equine serum. The consortium was
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then split in half and a 1µL loop of P. micra added to only one of the parts. The consortia
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were incubated at 37° C under anaerobic conditions as described above. The gingipain
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activity in the consortia was measured after 24 hours and 7 days and the number of P.
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gingivalis determined after 7 days by plating on Brucella agar as described above. All
164
experiments where conducted in triplicate using independent bacterial cultures.
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Treatment of P. gingivalis with supernatants from P. micra cultures
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P. micra was grown overnight under anaerobic conditions in Brain Heart Infusion broth (BHI)
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and the culture medium filtered through a 0.45µm filter to create a cell-free supernatant. One
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mL of the supernatant was incubated at 80°C for 20 minutes to provide a heat-inactivated
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supernatant. To determine whether there was a direct stimulatory effect upon gingipain
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activity, 500 µL of the cell-free supernatant from P. micra and 500 µL of an overnight culture
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of P. gingivalis were mixed and the gingipain activity measured after 30 minutes as described
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above. Overnight cultures of P. gingivalis mixed with BHI in the same manner were used as a
174
control. In addition, overnight cultures of P. gingivalis were inoculated (1:10) into BHI and
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then mixed with an equal volume (1mL) of the P. micra cell-free supernatant (native or heat-
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inactivated). Control P. gingivalis cells were inoculated in the same manner in BHI only. The
177
cultures were then incubated anaerobically for 24 hours at 37° C and the gingipain activity
178
measured as described above.
179 180
Fluorescence in situ hybridization (FISH)
181
Multi-species consortia created as described above were added to Ibidi µ-slide 8-well slides
182
(Ibidi GmbH, Martinsried, Germany), and incubated anaerobically for 7 days. After removal
183
of the supernatant, the wells were washed with phosphate-buffered saline (PBS), and the
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bacteria fixed for 30 minutes with 4% paraformaldehyde in PBS. The 16S rRNA FISH
185
protocol was performed as described previously [28]. Briefly, bacteria were permeabilized
186
with a solution containing 10mg/mL lysozyme in 100mM Tris-HCl containing 5mM EDTA.
187
They were then washed with ultra-pure water and dehydrated with 50%, 80% and 99%
188
ethanol for 3 min each, after which 30 mL of hybridization buffer (0.9 M NaCl, 20 mM Tris-
189
HCl buffer, pH 7.5, with 0.01% sodium dodecyl sulfate and 25% formamide) containing
190
3.3µmol/L of each of the oligonucleotide probes [P. gingivalis: POGI-R
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(caatactcgtatcgcccgttattc) labelled with Atto565 [23] and P. micra: PAMIC1435
192
(tgcggttagatcggcggc) labelled with Atto390 [24] was added to the 8-well slides. Hybridization
193
was performed at 48ºC for 90 min in a humid chamber. After incubation the biofilms were
194
viewed in a Nikon Eclipse TE2000 inverted confocal scanning laser microscope. Confocal
195
illumination for the red fluorescence signal was provided by a G-HeNe laser (543 nm
196
excitation) and a UV laser was used for detection of blue fluorescence (390 nm excitation).
197
Images were recorded using Nikon NIS-Elements software.
198
2D gel electrophoresis and protein identification
199
The supernatant from an over-night culture of P. micra was examined using 2D gel
200
electrophoresis as previously described [25]. Briefly 23 µg protein was subjected to isoelectric
201
focusing on an 18 cm immobiline pH gradient (IPG) dry strip pH 4-7 using a Multiphor II
202
with cooling water (Amersham Pharmacia Biotech). After focusing the strips were stored at -
203
80 °C. Prior to running in the second dimension, the strips were equilibrated for 15 minutes in
204
50 mM Tris-HCl buffer (pH6.8), 2% (w/v) sodium dodecyl sulfate (SDS) and 26% (v/v)
205
glycerol containing 2.5% ditiotreitol (DTT) followed by a further 15 min in the same buffer
206
where the DTT was replaced with iodoacetamide (IAA) to alkylate any free DTT. The
207
equilibrated IPG strips were embedded on top of 14% polyacrylamide gradient gels using
208
0.5% (w/v) molten agarose. The SDS-PAGE was performed at constant current of 20 mA per
209
gel overnight at 10°C in a Protean II Cell (Bio-Rad). The following day the gels were stained
210
with colloidal Coomassie Brilliant Blue (Sigma Aldrich, USA) according to the
211
manufacturers instructions. The most prominent proteins were excised manually and tryptic
212
peptides subjected to liquid chromatography (LC), followed by tandem mass spectrometry
213
(MS/MS) as described previously [26]. Mass lists were used as the input for Mascot MS/MS
214
Ions searches of the NCBInr database using the Matrix Science web server
215
(www.matrixscience.com). 9
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Statistical analysis
218
The fluorescence measurements and the culture data were analyzed using paired one-sided
219
Student’s t-test or Mann-Whitney U-test using Prism 5 for MacOSX (Graphpad Inc, La Jolla,
220
CA, USA). All results were expressed as mean values with standard error of the mean from
221
three experiments.
222 223
Results
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Growth of P. gingivalis in single and dual-species consortia
225
To model the sub-gingival exudate-rich environment, P. gingivalis was initially grown in
226
serum alone (single-species culture). At baseline, the inoculum contained around 1 x 107 P.
227
gingivalis cells/mL but after 7 days, there was a 100-fold reduction in cell number (Fig 1)
228
indicating that P. gingivalis is unable to survive and grow alone in serum. To investigate
229
whether other members of the sub-gingival microbiome could influence its survival under
230
these conditions, P. gingivalis was co-cultured in dual-species consortia with one of the
231
following: P. micra, S. oralis or F. nucleatum. After seven days incubation, the number of S.
232
oralis, F. nucleatum and P. micra remained approximately the same (S. oralis 2.1x106
233
±7.0x105 CFU/mL, F. nucleatum 2.7x107 ±1.6x107 CFU/mL and P. micra 4.0x106 ±3.0x106
234
CFU/mL) while the number of P. gingivalis cells changed dramatically. In the presence of P.
235
micra, there was a 100-fold increase in the number of P. gingivalis cells, suggesting that P.
236
micra exerted a growth-stimulating effect on P. gingivalis. This effect was not seen for
237
consortia of P. gingivalis with either S. oralis or F. nucleatum (Fig 1).
238 239
Gingipain activity in dual-species consortia
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Since gingipain expression is known to be important for growth of P. gingivalis in serum, the
241
gingipain activity in the single- and dual-species cultures was investigated using the specific
242
substrate, BikKam-10. When P. gingivalis was cultured alone in serum for 7 days, BikKam-
243
10 revealed the gingipain activity to be low (107 ± 32 FU/min) whereas in the presence of P.
244
micra, the activity was increased more than 35-fold (3883 ± 979 FU/min) (p<0.05). In the
245
dual species consortia at 24 h, the gingpain activity was low but increased gradually over time
246
(Fig. 2). In the presence of the other species, gingipain activity was similar to that in the
247
single-species culture (S. oralis: 121 ±11 FU/min), although a small but not statistically
248
significant increase was seen for F. nucleatum (265 ± 92 FU/min). Thus the presence of P.
249
micra but not S. oralis or F. nucleatum significantly stimulated gingipain activity in P.
250
gingivalis.
251 252
Effect of P. micra on gingipain activity in a complex multispecies consortium
253
To more closely simulate conditions experienced by the sub-gingival microbiota, a seven-
254
species consortium (P. gingivalis, F. nucleatum, A. naeslundii, S. oralis, S. gordonii, S. mitis
255
and S. cristatus) was constructed and used to study the effect of P. micra on the activity of
256
gingipain from P. gingivalis in a more complex environment. Gingipain activity was thus
257
compared in the seven-species consortium under growth-restricted conditions with, and
258
without, the addition of P. micra. After 24 hours, the gingipain activity was low in both
259
consortia with (61±34 FU/min) and without (18±4 FU/min) P. micra (Fig 3). After seven
260
days however, the gingipain activity was significantly higher in the presence (3534±247
261
FU/min) than the absence (281±81 FU/min) of P. micra (p=0.01). Since the number of P.
262
gingivalis cells were the same in both test and control after seven days the difference in
263
activity cannot be attributed to a larger cell number. The data therefore show that P. micra
264
stimulates gingipain activity even in complex multi-species consortia. Inter-species 11
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interactions within the subgingival biofilm require that the bacteria are in close proximity to
266
each other. Analysis of the spatial arrangement of P. micra and P. gingivalis in the
267
multispecies consortium using FISH revealed that the two bacterial species were often present
268
in close association to each other (Fig. 4).
269 270
Effect of secreted products from P. micra on gingipain activity
271
To study the direct effect of P. micra products on gingipain activity, supernatant from P.
272
micra or an equivalent volume of growth medium alone was added to cultures of P. gingivalis
273
and the gingipain activity measured using BikKam-10 after 30 minutes. This revealed no
274
difference in the gingipain activity under the two conditions (56±11 FU/min as compared to
275
45±0.3 FU/min, p= 0,45), indicating that the increase in gingipain activity was not due to a
276
direct activation of the enzyme by products from P. micra. To further investigate the effect, P.
277
gingivalis was then cultured overnight in the presence or absence of cell-free spent medium
278
from P. micra and the gingipain activity assessed using the BikKam-10 substrate. This
279
revealed a 3-fold greater gingipain activity in the P. gingivalis culture exposed to P. micra
280
products (1714±242 FU/min) than in the absence of P. micra products (493±94 FU/min) (p
281
< 0.05) (Fig. 5). Again, the number of P. gingivalis cells in the two cultures was the same,
282
(3.0x108 ±1x108 CFU/mL in the presence and 3.5x108 ±0.9x108 CFU/mL in the absence of P.
283
micra products) and thus the difference in gingipain activity could not be attributed to a
284
difference in cell number. As expected, the P. micra products themselves gave no reactivity
285
with the gingipain specific BikKam-10 substrate (data not shown). Heat-treatment of the P.
286
micra products reduced the increase seen in gingipain activity after overnight culture by
287
approximately 60% (p= 0.008) suggesting that the stimulatory effect was mainly due to an
288
effect of one or more proteins produced by P. micra (Fig 5).
289 12
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Identification of secreted products from P. micra
291
The supernatant from P. micra was examined using 2D gel electrophoresis and ten of the
292
eleven most prominent proteins were successfully identified using mass spectrometry. Seven
293
proteins belonged to the glycolytic pathway; fructose-bisphosphate aldolase (EC 4.1.2.13),
294
phosphoglycerate kinase (EC 2.7.2.3), enolase, glyceraldehyde-3-phosphate dehydrogenase
295
(two isoforms), 2,3-bisphosphoglycerate-dependent phoshoglycerate mutase and
296
triosphosphate isomerase. The other three proteins were involved in amino acid metabolism,
297
transcription and translation and butyric acid metabolism (Fig 6). This shows that P. micra
298
secretes glycolytic enzymes into the external environment.
299 300
Discussion
301
The ability of the subgingival microbiome to successfully colonize the serum-rich
302
environment generated as a result of the inflammatory response in periodontitis depends in
303
part on their ability to exploit the exudate as a nutrient source [4]. We have shown that in
304
isolation P. gingivalis was unable to grow in serum, suggesting that it does not express the
305
range of glycosidases and proteases required to degrade complex serum glycoproteins. The
306
potential of other members of the sub-gingival biofilm to interact with P. gingivalis to
307
promote growth was therefore tested. This showed that P. micra, but not F. nucleatum or S.
308
oralis, was able to stimulate growth of P. gingivalis in serum, suggesting that some kind of
309
interaction occurred with P. micra that is beneficial for survival of P. gingivalis. Previously it
310
has been shown that when plaque is grown with human serum as a nutrient source, members
311
of the consortium cooperate to provide a battery of enzymes that can degrade the complex
312
glycoproteins found in this substrate [27]. Therefore, one potential mechanism underlying the
313
growth stimulation effect of P. micra on P. gingivalis could be the provision of
314
complementary enzymes for degradation of serum glycoproteins. P. micra is known to 13
ACCEPTED MANUSCRIPT 315
express a wide array of proteases and peptidases which could explain this effect [28]. A
316
similar synergistic interaction has been reported for another sub-gingival bacterium, T.
317
denticola, which was shown to be dependent on Eubacterium nodatum and Prevotella
318
intermedia for growth in serum [29]. However, F. nucleatum is also known to express a
319
number of peptidases but did not show any significant effect on growth of P. gingivalis in this
320
study, suggesting that the stimulation cannot be fully explained by nutritional cooperation.
321
P. gingivalis expresses gingipains which, as well as being significant contributors to
322
virulence, are important for the growth and survival of the organism in GCF. Mutant strains
323
lacking gingipains are unable to grow in defined medium supplemented with human serum
324
albumin [30]. In this study, no gingipain activity was detected even after 7 days, when P.
325
gingivalis was cultured in serum alone or in dual or multi-species consortia without P. micra.
326
However, in the presence of P. micra, activity was enhanced 10-30-fold. In order to test
327
whether the increased activity was due to activation of pre-synthesized gingipains, P.
328
gingivalis cultures were exposed for 30 minutes to cell-free spent medium from P. micra.
329
This revealed no increase in activity, suggesting that P. micra does not release factors or
330
modify the environment, for instance by providing a low redox potential, that resulted in
331
direct enzyme activation.
332
Another mechanism by which P. micra could influence overall gingipain activity in P.
333
gingivalis would be through increased expression of the enzymes. This was investigated in
334
multi-species consortia with or without P. micra, where the number of P. gingivalis cells was
335
the same. This revealed that, gingipain activity was enhanced more than 10-fold in the
336
presence of P. micra compared to consortia without P. micra, indicating that gingipain
337
expression was most likely upregulated within the cell population. This effect appeared to
338
occur after 3 days supporting the idea that gingpains are not constitutively expressed in serum
14
ACCEPTED MANUSCRIPT 339
but rather may be produced in response to a signal from an appropriate partner, such as P.
340
micra, in the subgingival microbiome.
341
To investigate the nature of a potential signal, P. gingivalis was cultured overnight in the
342
presence of the cell-free spent medium from P. micra. This gave rise to an increase in activity,
343
suggesting that gingipains are upregulated by soluble factors released from P. micra. The
344
effect was significantly reduced by heat-treatment, suggesting that the effect involves
345
proteins, although more than one molecule may be involved. The most prominent proteins in
346
the cell-free spent medium from P. micra were identified as glycolytic enzymes including
347
enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate
348
aldolase. To the best of our knowledge this is the first time it has been shown that glycolytic
349
enzymes from P. micra can be released into the external environment. It is generally accepted
350
that GAPDH can have a range of functions and can appear extracellularly in several different
351
species including streptococcal species such as Streptococcus pyogenes as well as Neisseria
352
meningitidis and Escherichia coli [31, 32, 33]. GAPDH is primarily cell-bound at lower pH
353
while at pH 7.5 90% is released into the extracellular environment [34]. Cell-bound GAPDH
354
on S. oralis has been shown to interact with FimA fimbriae on P. gingivalis leading to an
355
array of transcriptional and proteomic changes [35] including reduction in RgpB expression.
356
This corresponds well with the absence of gingipain activity seen in this study in the dual-
357
species culture of S. oralis and P. gingivalis. S. gordonii GAPDH as well as streptococcal
358
surface protein SspA/B has been shown to interact with P. gingivalis FimA and Mfa fimbriae
359
respectively [36]. The latter interaction has been shown to be important for the initiation the
360
Ltp1 signalling cascade which results in increased Rgp activity. Therefore, it is highly likely
361
that GAPDH or one of the other proteins from the P. micra supernatant has the ability to
362
interact with P. gingivalis and affect intracellular events, leading to increased gingipain
363
activity, although further studies are needed to confirm this. 15
ACCEPTED MANUSCRIPT 364 365
To the best of our knowledge this is the first time P. micra has been shown to affect virulence
366
properties of P. gingivalis. P. micra has been identified in greater amounts at sites affected by
367
periodontitis and, along with P. gingivalis, has been proposed to belong to the subgingival
368
core microbiome in this disease [10,37]. In the study of Socransky et al, P. micra was placed
369
in the orange complex along with Prevotella intermedia, Prevotella nigrescens and F.
370
nucleatum indicating that it is often found at periodontitis sites [11]. In addition, it is
371
associated with apical periodontitis lesions [38,39] and necrotic root canals [40]. Viable
372
bacteria have also been isolated from the bloodstream following dental procedures and
373
periodontal therapy and have been implicated in infections at many different sites in the body,
374
including chronic skin infections , destructive knee joint infections prosthetic hip joint
375
infections and soft tissue infections at remote sites [for a review see 41]. Studies have shown
376
that P. micra and P. gingivalis can coaggregate [42] and P. micra and P. gingivalis were
377
found in close proximity to each other in biofilms in this study - an interaction that may be
378
highly relevant in vivo.
379 380
Conclusion
381
The ability of P. gingivalis to act as a keystone species in periodontitis relies upon its ability
382
to manipulate the immune response via production of gingipains [17]. In this study, we show
383
that under conditions similar to those in the gingival pocket, virulence expression in the form
384
of gingipain activity is highly dependent on the presence of P. micra. Thus, interaction
385
between P. micra and P. gingivalis is likely to be of importance for the development of
386
periodontitis, although a contribution by other members of the subgingival biofilm cannot be
387
ruled out. 16
ACCEPTED MANUSCRIPT 388 389
Conflicts of interest
390
The authors declare no conflicts of interest
391 392
Acknowledgment
393
This study was supported by the Knowledge Foundation (#20150086) and the Foundation for
394
Dental Research in Malmö founded by Bertil Rohlin for Rohlin-Dentalen.
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42. Kremer BHA, van Steenbergen MTJ. (2000). Peptostreptococcus micros coaggregates
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Figure legends
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Figure 1. Growth of P. gingivalis in serum alone or in the presence of other species. The
507
number of P. gingivalis in the cultures was determined at baseline and after 7 days by
508
culturing on Brucella agar as described in the Materials and Methods.
509 510
Figure 2. Gingipain activity after incubation of P. gingivalis in serum for 7 days, alone or in
511
the presence of other species (a) and gingipain activity after 24h, 72h and 7 days in a dual-
512
species consortium with P. micra (b). Arginine gingipain activity measured using the specific
513
fluorescent substrate BikKam-10.
514 515
Figure 3. Gingipain activity in multispecies consortia after 24 h and 7 days. Arginine gingipain
516
activity was measured using the specific fluorescent substrate, BikKam-10, in a multi-species
517
consortium with or without P. micra.
518 519
Figure 4. FISH image showing the close proximity between P. micra (blue/violet) and P.
520
gingivalis (red) in the consortium.
521 522
Figure 5. Gingipain activity of P. gingivalis cultured in the presence of native or heat
523
inactivated (HI) secreted products from P. micra.
524 525
Figure 6. Secreted proteins from P. micra. Ten of the most prominent proteins were
526
successfully identified using mass spectrometry. pk = phosphoglycerate kinase, amt =
527
aminomethyltransferase, fba = fructose-bisphosphate aldolase, eno = enolase, gapdh =
528
glyceraldehyde-3-phosphate dehydrogenase, 2,3-bd = 2,3- butanediol dehydrogenase, pm =
23
ACCEPTED MANUSCRIPT 529
2,3-bisphosphoglycerate-dependent phoshoglycerate mutase, Ef = Elongation factor, tpi =
530
triosphosphate isomerase
24
ACCEPTED MANUSCRIPT
Parvimonas micra enhance growth of Porphyromonas gingivalis in 10% serum. P. micra significantly enhance gingipain activity in multispecies consortia. P. micra secretes glycolytic enzymes into the external environment. Gingipains are upregulated by soluble factors released from P. micra.
Jessica Neilands, Julia R. Davies, Floris J. Bikker, Gunnel Svensäter PII:
S1075-9964(18)30179-3
DOI:
10.1016/j.anaerobe.2018.10.007
Reference:
YANAE 1958
To appear in:
Anaerobe
Received Date:
09 August 2018
Accepted Date:
20 October 2018
Please cite this article as: Jessica Neilands, Julia R. Davies, Floris J. Bikker, Gunnel Svensäter, Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multispecies communities, Anaerobe (2018), doi: 10.1016/j.anaerobe.2018.10.007
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ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9
Parvimonas micra stimulates expression of gingipains from
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Porphyromonas gingivalis in multi-species communities
11 12 13
Jessica Neilandsa*, Julia R. Daviesa, Floris J. Bikkerb and Gunnel Svensätera
14 15 16
aDept
of Oral Biology, Faculty of Odontology, Malmö University, Malmö, Sweden
bDepartment
of Oral Biochemistry, Academic Centre for Dentistry Amsterdam,
17
Free University and University of Amsterdam, Amsterdam, The Netherlands
18
[email protected], [email protected], [email protected], [email protected]
19 20 21 22 23 24 25 26 27 28
Corresponding author:
29
Dr Jessica Neilands
30
Dept. of Oral Biology, Faculty of Odontology, Malmö University, 205 06 Malmö, SWEDEN
31
[email protected]
32
+46-709-163515 1
ACCEPTED MANUSCRIPT 33
Abstract
34
Dental biofilms are complex ecosystems containing many bacterial species that live in
35
mutualistic relationships. These interactions can profoundly affect the virulence properties of
36
the community. In this study we investigated how the production of gingipains, virulence
37
factors from Porphyromonas gingivalis important in periodontal disease, was affected by
38
other commonly found members of the sub-gingival microbiome.
39
To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium
40
nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus
41
gordonii and Streptococcus cristatus, with or without Parvimonas micra) as well as dual
42
species consortia (P. gingivalis with P. micra, S. oralis or F. nucleatum) were constructed and
43
maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was
44
determined by plating on Brucella agar and the gingipain specific fluorogenic substrate
45
BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P.
46
micra on gingipain activity was investigated by adding supernatants from P. micra to P.
47
gingivalis cultures. The most prominent secreted proteins in the supernatant were identified
48
using mass spectrometry.
49
P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F.
50
nucleatum. In contrast, with P. micra growth was significantly enhanced and this was
51
associated with an increase in gingipain activity. In the multi-species consortia, the presence
52
of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to
53
supernatants from P. micra for 24 hours caused a 3-fold increase in gingipain activity. This
54
effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel
55
electrophoresis revealed that several of the most prominent proteins in the P. micra
56
supernatant were glycolytic enzymes. The results from this study suggests that gingipains are
57
produced in response to a P. micra derived signaling molecule that is most likely a protein.
2
ACCEPTED MANUSCRIPT 58
This is the first time it has been shown that P. micra can affect P. gingivalis virulence
59
properties. This is likely to be of significance for the development of be of periodontitis since
60
these two microorganisms are often found together in the subgingival biofilm.
61 62 63
Key words: periodontitis, gingipain, Gram negative, bacterial interactions, Porphyromonas
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gingivalis
65
3
ACCEPTED MANUSCRIPT 67
Introduction
68
It is estimated that more than 500 million people worldwide are affected by periodontitis [1].
69
Periodontitis is a biofilm-induced inflammatory disease of the oral cavity that results in
70
breakdown of the gingival tissues and supporting bone, which can be so extensive that it
71
eventually leads to tooth loss. Disease is initiated when the inflammatory response of the host
72
is stimulated by plaque accumulation at the gingival margin which results in an increased
73
flow of nutrient-rich fluid from the gingival crevice (GCF). This environmental change
74
favours outgrowth and enrichment of various proteolytic bacteria within the biofilm while at
75
the same time reducing the competitiveness of others. The change in composition,
76
accompanied by an increase in proteolytic phenotypes within biofilms, is believed to be the
77
driving force for progression of disease [2].
78
The oral cavity is a heterogeneous environment colonized by over 600 different bacterial
79
species. Co-adhesion of species during biofilm formation promotes mutually beneficial
80
interactions by locating organisms in close proximity to appropriate partners [3,4]. The
81
physical relationships facilitated by biofilm growth enable nutritional cooperation which is
82
seen, for instance, in the expression of complementary enzymes by plaque bacteria for the
83
degradation of complex salivary and serum glycoproteins [5, 6], and metabolic end-products
84
of one species serving as primary energy source for others [7]. Thus, in health, the balance
85
between synergistic and antagonistic interactions within biofilms (biosis) supports a symbiotic
86
relationship with the host, but when the biofilm is subjected to environmental pressure this
87
balance can shift giving rise to a dysbiotic biofilm phenotype that may induce disease [8,9].
88
A recent study by Abusleme et al, using 16S-rRNA sequencing to identify bacterial species
89
from the subgingival microbiome in healthy subjects and individuals with periodontitis,
90
revealed a core microbiome, found in the majority of subjects, which was present in equal
91
numbers in health and disease [10]. Species such as Actinomyces spp, Rothia spp and 4
ACCEPTED MANUSCRIPT 92
Streptococcus spp dominated in health whereas, for example, Treponema spp, TM7,
93
Tannerella forsythia, Parvimonas micra, Porphyromonas gingivalis and
94
Peptostreptococcacae appeared in the majority of subjects with periodontitis and had a higher
95
prevalence and abundance than in health [10]. These data support the earlier studies of
96
Socransky et al. in which a complex of Treponema. denticola, P. gingivalis and T. forsythia
97
was found to be closely associated with periodontal disease [11]. P. gingivalis, has since been
98
the focus of many studies due to its pro-inflammatory properties, for instance, activation of
99
TLR2 receptors and impairment of leukocyte recruitment and function. In addition, P.
100
gingivalis is capable of expressing a number of proteolytic enzymes including the
101
extracellular endopeptidases, the arginine (RgpA & RgpB) and lysine (Kgp) gingipains,
102
which are assumed to primarily be part of the proteolytic system for generation of nutrients
103
[12]. However, these proteases can also modulate host defences through degradation of
104
complement, immunoglobulins and cytokines [13]. Due to its ability to subvert immune
105
responses and support a dysbiotic biofilm phenotype, even when present at low levels, P.
106
gingivalis is regarded as a keystone pathogen in periodontitis [14].
107
Gingipains are either outer membrane-associated or secreted [15].The activity of the
108
gingipains is enhanced under reducing conditions, for example in the presence of cysteine,
109
while the activity is diminished in the presence of oxygen [16,17]. An excess of hemin has
110
also been shown to enhance gingipain activity [18]. Despite the ability of P. gingivalis to
111
produce these important virulence factors, animal studies have shown that it is not capable of
112
causing disease alone, but requires the presence of other bacterial species [19]. This is in
113
keeping with the concept that most infectious diseases are not the result of a single organism
114
but caused by the concerted actions of multi-species bacterial communities. One possible
115
mechanism by which other bacteria could influence the ability of P. gingivalis to induce
116
disease is by modulating the activity of virulence factors such as gingipains [20]. In this study
5
ACCEPTED MANUSCRIPT 117
we have therefore investigated how other members of the sub-gingival microbiome affected
118
gingipain activity in P. gingivalis.
119 120
Materials and methods
121
Bacterial strains
122
Clinical isolates recovered from the subgingival microbiome were used in this study. P.
123
gingivalis (SUB1), Fusobacterium nucleatum (FMD), Actinomyces naeslundii (BJJ), P. micra
124
(EME), Streptococcus oralis (2009-213A1) and Streptococcus gordonii (2010-203G) were
125
isolated from patients with established periodontitis, each strain from a different patient
126
whereas Streptococcus mitis (CL) and Streptococcus cristatus (CL) were isolated from a
127
healthy male donor. The bacteria were identified to species level as described previously
128
using a combination of colony morphology, appearance after Gram staining and biochemical
129
or molecular tests [21]. Isolates were stored in skimmed milk at -80ºC until use.
130 131
Measurement of Arg gingipain activity using fluorescent substrate
132
Arginine gingipain activity was assessed using the synthetic fluorogenic substrate BikKam-
133
10, FITC–Arg–D-Glu–KDbc, (PepScan Presto B.V., Lelystad, The Netherlands). Fifty µL
134
bacterial suspension was added to 2 µL BikKam-10 in a 96-well plate (NUNC, ThermoFisher
135
Scientific, Roskilde, Denmark) (final concentration of 16mM) [17]. Fluorescence was
136
measured at one minute intervals in a BMG Fluostar Optima plate reader (BMG Labtech,
137
Offenburg, Germany) (using excitation and emission wavelengths of 488 and 538 nm,
138
respectively) and expressed as change in fluorescence units over time (FU/min).
139 140
P. gingivalis in dual-species consortia
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Bacterial suspensions of P. gingivalis, P. micra, S. oralis and F. nucleatum were created in
142
pre-reduced 10% heat-inactivated equine serum (Håtunalab, Bro, Sweden) to give an optical
143
density at wavelength 600 nm of 0.1. For P. gingivalis, this corresponded to approximately
144
1.3x107 bacterial cells/mL, S. oralis 1.6x106 cells/mL, F. nucleatum 3.7x107 cells/mL and P.
145
micra 7.2x106/mL. One mL of the P. gingivalis suspension were then mixed with an equal
146
volume of one of the other three bacterial suspensions to give dual-species consortia with a
147
final concentration of 6.5x106 P. gingivalis cells/mL and incubated at 37° C anaerobically
148
(10% H2, 5% CO2 in N2). The suspension with P. gingivalis only was incubated in the same
149
manner. After seven days the gingipain activity in the dual-species consortia was measured in
150
50 µL cellsuspension and serial dilutions plated onto Brucella agar. The number of bacteria
151
in each consortium was determined by counting the number of colonies of each species
152
(easily distinguishable due to differences in colony morphology) after eight days incubation
153
anaerobically at 37° C. In addition, the gingipain activity in the P. micra dual-species
154
consortium was measured after 24h and 72h.
155 156
Effect of P. micra on P. gingivalis in multi-species consortia
157
Multi-species consortia were created by inoculating colonies (1µL loop) of the different
158
bacteria (P. gingivalis, F. nucleatum, A. naeslundii, S. oralis, S. gordonii, S. mitis and S.
159
cristatus) into four mL 10% pre-reduced heat-inactivated equine serum. The consortium was
160
then split in half and a 1µL loop of P. micra added to only one of the parts. The consortia
161
were incubated at 37° C under anaerobic conditions as described above. The gingipain
162
activity in the consortia was measured after 24 hours and 7 days and the number of P.
163
gingivalis determined after 7 days by plating on Brucella agar as described above. All
164
experiments where conducted in triplicate using independent bacterial cultures.
165
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Treatment of P. gingivalis with supernatants from P. micra cultures
167
P. micra was grown overnight under anaerobic conditions in Brain Heart Infusion broth (BHI)
168
and the culture medium filtered through a 0.45µm filter to create a cell-free supernatant. One
169
mL of the supernatant was incubated at 80°C for 20 minutes to provide a heat-inactivated
170
supernatant. To determine whether there was a direct stimulatory effect upon gingipain
171
activity, 500 µL of the cell-free supernatant from P. micra and 500 µL of an overnight culture
172
of P. gingivalis were mixed and the gingipain activity measured after 30 minutes as described
173
above. Overnight cultures of P. gingivalis mixed with BHI in the same manner were used as a
174
control. In addition, overnight cultures of P. gingivalis were inoculated (1:10) into BHI and
175
then mixed with an equal volume (1mL) of the P. micra cell-free supernatant (native or heat-
176
inactivated). Control P. gingivalis cells were inoculated in the same manner in BHI only. The
177
cultures were then incubated anaerobically for 24 hours at 37° C and the gingipain activity
178
measured as described above.
179 180
Fluorescence in situ hybridization (FISH)
181
Multi-species consortia created as described above were added to Ibidi µ-slide 8-well slides
182
(Ibidi GmbH, Martinsried, Germany), and incubated anaerobically for 7 days. After removal
183
of the supernatant, the wells were washed with phosphate-buffered saline (PBS), and the
184
bacteria fixed for 30 minutes with 4% paraformaldehyde in PBS. The 16S rRNA FISH
185
protocol was performed as described previously [28]. Briefly, bacteria were permeabilized
186
with a solution containing 10mg/mL lysozyme in 100mM Tris-HCl containing 5mM EDTA.
187
They were then washed with ultra-pure water and dehydrated with 50%, 80% and 99%
188
ethanol for 3 min each, after which 30 mL of hybridization buffer (0.9 M NaCl, 20 mM Tris-
189
HCl buffer, pH 7.5, with 0.01% sodium dodecyl sulfate and 25% formamide) containing
190
3.3µmol/L of each of the oligonucleotide probes [P. gingivalis: POGI-R
8
ACCEPTED MANUSCRIPT 191
(caatactcgtatcgcccgttattc) labelled with Atto565 [23] and P. micra: PAMIC1435
192
(tgcggttagatcggcggc) labelled with Atto390 [24] was added to the 8-well slides. Hybridization
193
was performed at 48ºC for 90 min in a humid chamber. After incubation the biofilms were
194
viewed in a Nikon Eclipse TE2000 inverted confocal scanning laser microscope. Confocal
195
illumination for the red fluorescence signal was provided by a G-HeNe laser (543 nm
196
excitation) and a UV laser was used for detection of blue fluorescence (390 nm excitation).
197
Images were recorded using Nikon NIS-Elements software.
198
2D gel electrophoresis and protein identification
199
The supernatant from an over-night culture of P. micra was examined using 2D gel
200
electrophoresis as previously described [25]. Briefly 23 µg protein was subjected to isoelectric
201
focusing on an 18 cm immobiline pH gradient (IPG) dry strip pH 4-7 using a Multiphor II
202
with cooling water (Amersham Pharmacia Biotech). After focusing the strips were stored at -
203
80 °C. Prior to running in the second dimension, the strips were equilibrated for 15 minutes in
204
50 mM Tris-HCl buffer (pH6.8), 2% (w/v) sodium dodecyl sulfate (SDS) and 26% (v/v)
205
glycerol containing 2.5% ditiotreitol (DTT) followed by a further 15 min in the same buffer
206
where the DTT was replaced with iodoacetamide (IAA) to alkylate any free DTT. The
207
equilibrated IPG strips were embedded on top of 14% polyacrylamide gradient gels using
208
0.5% (w/v) molten agarose. The SDS-PAGE was performed at constant current of 20 mA per
209
gel overnight at 10°C in a Protean II Cell (Bio-Rad). The following day the gels were stained
210
with colloidal Coomassie Brilliant Blue (Sigma Aldrich, USA) according to the
211
manufacturers instructions. The most prominent proteins were excised manually and tryptic
212
peptides subjected to liquid chromatography (LC), followed by tandem mass spectrometry
213
(MS/MS) as described previously [26]. Mass lists were used as the input for Mascot MS/MS
214
Ions searches of the NCBInr database using the Matrix Science web server
215
(www.matrixscience.com). 9
ACCEPTED MANUSCRIPT 216 217
Statistical analysis
218
The fluorescence measurements and the culture data were analyzed using paired one-sided
219
Student’s t-test or Mann-Whitney U-test using Prism 5 for MacOSX (Graphpad Inc, La Jolla,
220
CA, USA). All results were expressed as mean values with standard error of the mean from
221
three experiments.
222 223
Results
224
Growth of P. gingivalis in single and dual-species consortia
225
To model the sub-gingival exudate-rich environment, P. gingivalis was initially grown in
226
serum alone (single-species culture). At baseline, the inoculum contained around 1 x 107 P.
227
gingivalis cells/mL but after 7 days, there was a 100-fold reduction in cell number (Fig 1)
228
indicating that P. gingivalis is unable to survive and grow alone in serum. To investigate
229
whether other members of the sub-gingival microbiome could influence its survival under
230
these conditions, P. gingivalis was co-cultured in dual-species consortia with one of the
231
following: P. micra, S. oralis or F. nucleatum. After seven days incubation, the number of S.
232
oralis, F. nucleatum and P. micra remained approximately the same (S. oralis 2.1x106
233
±7.0x105 CFU/mL, F. nucleatum 2.7x107 ±1.6x107 CFU/mL and P. micra 4.0x106 ±3.0x106
234
CFU/mL) while the number of P. gingivalis cells changed dramatically. In the presence of P.
235
micra, there was a 100-fold increase in the number of P. gingivalis cells, suggesting that P.
236
micra exerted a growth-stimulating effect on P. gingivalis. This effect was not seen for
237
consortia of P. gingivalis with either S. oralis or F. nucleatum (Fig 1).
238 239
Gingipain activity in dual-species consortia
10
ACCEPTED MANUSCRIPT 240
Since gingipain expression is known to be important for growth of P. gingivalis in serum, the
241
gingipain activity in the single- and dual-species cultures was investigated using the specific
242
substrate, BikKam-10. When P. gingivalis was cultured alone in serum for 7 days, BikKam-
243
10 revealed the gingipain activity to be low (107 ± 32 FU/min) whereas in the presence of P.
244
micra, the activity was increased more than 35-fold (3883 ± 979 FU/min) (p<0.05). In the
245
dual species consortia at 24 h, the gingpain activity was low but increased gradually over time
246
(Fig. 2). In the presence of the other species, gingipain activity was similar to that in the
247
single-species culture (S. oralis: 121 ±11 FU/min), although a small but not statistically
248
significant increase was seen for F. nucleatum (265 ± 92 FU/min). Thus the presence of P.
249
micra but not S. oralis or F. nucleatum significantly stimulated gingipain activity in P.
250
gingivalis.
251 252
Effect of P. micra on gingipain activity in a complex multispecies consortium
253
To more closely simulate conditions experienced by the sub-gingival microbiota, a seven-
254
species consortium (P. gingivalis, F. nucleatum, A. naeslundii, S. oralis, S. gordonii, S. mitis
255
and S. cristatus) was constructed and used to study the effect of P. micra on the activity of
256
gingipain from P. gingivalis in a more complex environment. Gingipain activity was thus
257
compared in the seven-species consortium under growth-restricted conditions with, and
258
without, the addition of P. micra. After 24 hours, the gingipain activity was low in both
259
consortia with (61±34 FU/min) and without (18±4 FU/min) P. micra (Fig 3). After seven
260
days however, the gingipain activity was significantly higher in the presence (3534±247
261
FU/min) than the absence (281±81 FU/min) of P. micra (p=0.01). Since the number of P.
262
gingivalis cells were the same in both test and control after seven days the difference in
263
activity cannot be attributed to a larger cell number. The data therefore show that P. micra
264
stimulates gingipain activity even in complex multi-species consortia. Inter-species 11
ACCEPTED MANUSCRIPT 265
interactions within the subgingival biofilm require that the bacteria are in close proximity to
266
each other. Analysis of the spatial arrangement of P. micra and P. gingivalis in the
267
multispecies consortium using FISH revealed that the two bacterial species were often present
268
in close association to each other (Fig. 4).
269 270
Effect of secreted products from P. micra on gingipain activity
271
To study the direct effect of P. micra products on gingipain activity, supernatant from P.
272
micra or an equivalent volume of growth medium alone was added to cultures of P. gingivalis
273
and the gingipain activity measured using BikKam-10 after 30 minutes. This revealed no
274
difference in the gingipain activity under the two conditions (56±11 FU/min as compared to
275
45±0.3 FU/min, p= 0,45), indicating that the increase in gingipain activity was not due to a
276
direct activation of the enzyme by products from P. micra. To further investigate the effect, P.
277
gingivalis was then cultured overnight in the presence or absence of cell-free spent medium
278
from P. micra and the gingipain activity assessed using the BikKam-10 substrate. This
279
revealed a 3-fold greater gingipain activity in the P. gingivalis culture exposed to P. micra
280
products (1714±242 FU/min) than in the absence of P. micra products (493±94 FU/min) (p
281
< 0.05) (Fig. 5). Again, the number of P. gingivalis cells in the two cultures was the same,
282
(3.0x108 ±1x108 CFU/mL in the presence and 3.5x108 ±0.9x108 CFU/mL in the absence of P.
283
micra products) and thus the difference in gingipain activity could not be attributed to a
284
difference in cell number. As expected, the P. micra products themselves gave no reactivity
285
with the gingipain specific BikKam-10 substrate (data not shown). Heat-treatment of the P.
286
micra products reduced the increase seen in gingipain activity after overnight culture by
287
approximately 60% (p= 0.008) suggesting that the stimulatory effect was mainly due to an
288
effect of one or more proteins produced by P. micra (Fig 5).
289 12
ACCEPTED MANUSCRIPT 290
Identification of secreted products from P. micra
291
The supernatant from P. micra was examined using 2D gel electrophoresis and ten of the
292
eleven most prominent proteins were successfully identified using mass spectrometry. Seven
293
proteins belonged to the glycolytic pathway; fructose-bisphosphate aldolase (EC 4.1.2.13),
294
phosphoglycerate kinase (EC 2.7.2.3), enolase, glyceraldehyde-3-phosphate dehydrogenase
295
(two isoforms), 2,3-bisphosphoglycerate-dependent phoshoglycerate mutase and
296
triosphosphate isomerase. The other three proteins were involved in amino acid metabolism,
297
transcription and translation and butyric acid metabolism (Fig 6). This shows that P. micra
298
secretes glycolytic enzymes into the external environment.
299 300
Discussion
301
The ability of the subgingival microbiome to successfully colonize the serum-rich
302
environment generated as a result of the inflammatory response in periodontitis depends in
303
part on their ability to exploit the exudate as a nutrient source [4]. We have shown that in
304
isolation P. gingivalis was unable to grow in serum, suggesting that it does not express the
305
range of glycosidases and proteases required to degrade complex serum glycoproteins. The
306
potential of other members of the sub-gingival biofilm to interact with P. gingivalis to
307
promote growth was therefore tested. This showed that P. micra, but not F. nucleatum or S.
308
oralis, was able to stimulate growth of P. gingivalis in serum, suggesting that some kind of
309
interaction occurred with P. micra that is beneficial for survival of P. gingivalis. Previously it
310
has been shown that when plaque is grown with human serum as a nutrient source, members
311
of the consortium cooperate to provide a battery of enzymes that can degrade the complex
312
glycoproteins found in this substrate [27]. Therefore, one potential mechanism underlying the
313
growth stimulation effect of P. micra on P. gingivalis could be the provision of
314
complementary enzymes for degradation of serum glycoproteins. P. micra is known to 13
ACCEPTED MANUSCRIPT 315
express a wide array of proteases and peptidases which could explain this effect [28]. A
316
similar synergistic interaction has been reported for another sub-gingival bacterium, T.
317
denticola, which was shown to be dependent on Eubacterium nodatum and Prevotella
318
intermedia for growth in serum [29]. However, F. nucleatum is also known to express a
319
number of peptidases but did not show any significant effect on growth of P. gingivalis in this
320
study, suggesting that the stimulation cannot be fully explained by nutritional cooperation.
321
P. gingivalis expresses gingipains which, as well as being significant contributors to
322
virulence, are important for the growth and survival of the organism in GCF. Mutant strains
323
lacking gingipains are unable to grow in defined medium supplemented with human serum
324
albumin [30]. In this study, no gingipain activity was detected even after 7 days, when P.
325
gingivalis was cultured in serum alone or in dual or multi-species consortia without P. micra.
326
However, in the presence of P. micra, activity was enhanced 10-30-fold. In order to test
327
whether the increased activity was due to activation of pre-synthesized gingipains, P.
328
gingivalis cultures were exposed for 30 minutes to cell-free spent medium from P. micra.
329
This revealed no increase in activity, suggesting that P. micra does not release factors or
330
modify the environment, for instance by providing a low redox potential, that resulted in
331
direct enzyme activation.
332
Another mechanism by which P. micra could influence overall gingipain activity in P.
333
gingivalis would be through increased expression of the enzymes. This was investigated in
334
multi-species consortia with or without P. micra, where the number of P. gingivalis cells was
335
the same. This revealed that, gingipain activity was enhanced more than 10-fold in the
336
presence of P. micra compared to consortia without P. micra, indicating that gingipain
337
expression was most likely upregulated within the cell population. This effect appeared to
338
occur after 3 days supporting the idea that gingpains are not constitutively expressed in serum
14
ACCEPTED MANUSCRIPT 339
but rather may be produced in response to a signal from an appropriate partner, such as P.
340
micra, in the subgingival microbiome.
341
To investigate the nature of a potential signal, P. gingivalis was cultured overnight in the
342
presence of the cell-free spent medium from P. micra. This gave rise to an increase in activity,
343
suggesting that gingipains are upregulated by soluble factors released from P. micra. The
344
effect was significantly reduced by heat-treatment, suggesting that the effect involves
345
proteins, although more than one molecule may be involved. The most prominent proteins in
346
the cell-free spent medium from P. micra were identified as glycolytic enzymes including
347
enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose bisphosphate
348
aldolase. To the best of our knowledge this is the first time it has been shown that glycolytic
349
enzymes from P. micra can be released into the external environment. It is generally accepted
350
that GAPDH can have a range of functions and can appear extracellularly in several different
351
species including streptococcal species such as Streptococcus pyogenes as well as Neisseria
352
meningitidis and Escherichia coli [31, 32, 33]. GAPDH is primarily cell-bound at lower pH
353
while at pH 7.5 90% is released into the extracellular environment [34]. Cell-bound GAPDH
354
on S. oralis has been shown to interact with FimA fimbriae on P. gingivalis leading to an
355
array of transcriptional and proteomic changes [35] including reduction in RgpB expression.
356
This corresponds well with the absence of gingipain activity seen in this study in the dual-
357
species culture of S. oralis and P. gingivalis. S. gordonii GAPDH as well as streptococcal
358
surface protein SspA/B has been shown to interact with P. gingivalis FimA and Mfa fimbriae
359
respectively [36]. The latter interaction has been shown to be important for the initiation the
360
Ltp1 signalling cascade which results in increased Rgp activity. Therefore, it is highly likely
361
that GAPDH or one of the other proteins from the P. micra supernatant has the ability to
362
interact with P. gingivalis and affect intracellular events, leading to increased gingipain
363
activity, although further studies are needed to confirm this. 15
ACCEPTED MANUSCRIPT 364 365
To the best of our knowledge this is the first time P. micra has been shown to affect virulence
366
properties of P. gingivalis. P. micra has been identified in greater amounts at sites affected by
367
periodontitis and, along with P. gingivalis, has been proposed to belong to the subgingival
368
core microbiome in this disease [10,37]. In the study of Socransky et al, P. micra was placed
369
in the orange complex along with Prevotella intermedia, Prevotella nigrescens and F.
370
nucleatum indicating that it is often found at periodontitis sites [11]. In addition, it is
371
associated with apical periodontitis lesions [38,39] and necrotic root canals [40]. Viable
372
bacteria have also been isolated from the bloodstream following dental procedures and
373
periodontal therapy and have been implicated in infections at many different sites in the body,
374
including chronic skin infections , destructive knee joint infections prosthetic hip joint
375
infections and soft tissue infections at remote sites [for a review see 41]. Studies have shown
376
that P. micra and P. gingivalis can coaggregate [42] and P. micra and P. gingivalis were
377
found in close proximity to each other in biofilms in this study - an interaction that may be
378
highly relevant in vivo.
379 380
Conclusion
381
The ability of P. gingivalis to act as a keystone species in periodontitis relies upon its ability
382
to manipulate the immune response via production of gingipains [17]. In this study, we show
383
that under conditions similar to those in the gingival pocket, virulence expression in the form
384
of gingipain activity is highly dependent on the presence of P. micra. Thus, interaction
385
between P. micra and P. gingivalis is likely to be of importance for the development of
386
periodontitis, although a contribution by other members of the subgingival biofilm cannot be
387
ruled out. 16
ACCEPTED MANUSCRIPT 388 389
Conflicts of interest
390
The authors declare no conflicts of interest
391 392
Acknowledgment
393
This study was supported by the Knowledge Foundation (#20150086) and the Foundation for
394
Dental Research in Malmö founded by Bertil Rohlin for Rohlin-Dentalen.
17
ACCEPTED MANUSCRIPT 395
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with Fusobacterium nucleatum and non-encapsulated Porphyromonas gingivalis. FEMS
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Microbiol Lett 182:57-61
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Figure legends
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Figure 1. Growth of P. gingivalis in serum alone or in the presence of other species. The
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number of P. gingivalis in the cultures was determined at baseline and after 7 days by
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culturing on Brucella agar as described in the Materials and Methods.
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Figure 2. Gingipain activity after incubation of P. gingivalis in serum for 7 days, alone or in
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the presence of other species (a) and gingipain activity after 24h, 72h and 7 days in a dual-
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species consortium with P. micra (b). Arginine gingipain activity measured using the specific
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fluorescent substrate BikKam-10.
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Figure 3. Gingipain activity in multispecies consortia after 24 h and 7 days. Arginine gingipain
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activity was measured using the specific fluorescent substrate, BikKam-10, in a multi-species
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consortium with or without P. micra.
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Figure 4. FISH image showing the close proximity between P. micra (blue/violet) and P.
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gingivalis (red) in the consortium.
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Figure 5. Gingipain activity of P. gingivalis cultured in the presence of native or heat
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inactivated (HI) secreted products from P. micra.
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Figure 6. Secreted proteins from P. micra. Ten of the most prominent proteins were
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successfully identified using mass spectrometry. pk = phosphoglycerate kinase, amt =
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aminomethyltransferase, fba = fructose-bisphosphate aldolase, eno = enolase, gapdh =
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glyceraldehyde-3-phosphate dehydrogenase, 2,3-bd = 2,3- butanediol dehydrogenase, pm =
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2,3-bisphosphoglycerate-dependent phoshoglycerate mutase, Ef = Elongation factor, tpi =
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triosphosphate isomerase
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ACCEPTED MANUSCRIPT
Parvimonas micra enhance growth of Porphyromonas gingivalis in 10% serum. P. micra significantly enhance gingipain activity in multispecies consortia. P. micra secretes glycolytic enzymes into the external environment. Gingipains are upregulated by soluble factors released from P. micra.