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Dipeptidyl peptidase IV and quorum sensing signaling in biofilm-related virulence of Prevotella aurantiaca
Accepted Manuscript Dipeptidyl peptidase IV and quorum sensing signaling in biofilm-related virulence of Prevotella aurantiaca Dareen Fteita, Ahmed Ali Musrati, Eija Könönen, Xiaochu Ma, Mervi Gürsoy, Markus Peurla, Eva Söderling, Herman O. Sintim, Ulvi Kahraman Gürsoy PII:
S1075-9964(17)30168-3
DOI:
10.1016/j.anaerobe.2017.08.009
Reference:
YANAE 1789
To appear in:
Anaerobe
Received Date: 18 March 2017 Revised Date:
27 June 2017
Accepted Date: 14 August 2017
Please cite this article as: Fteita D, Musrati AA, Könönen E, Ma X, Gürsoy M, Peurla M, Söderling E, Sintim HO, Gürsoy UK, Dipeptidyl peptidase IV and quorum sensing signaling in biofilm-related virulence of Prevotella aurantiaca, Anaerobe (2017), doi: 10.1016/j.anaerobe.2017.08.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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DIPEPTIDYL PEPTIDASE IV AND QUORUM SENSING SIGNALING IN BIOFILM-
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RELATED VIRULENCE OF PREVOTELLA AURANTIACA
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Dareen Fteitaa,*, Ahmed Ali Musratia, Eija Könönena, b, Xiaochu Mac, Mervi Gürsoya, Markus
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Peurlad, Eva Söderlinga, Herman O. Sintimc, Ulvi Kahraman Gürsoya
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Finland
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b
Welfare Division, Oral Health Care, City of Turku, Turku, Finland
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c
Department of Chemistry and Purdue Institute for Drug Discovery and Purdue Institute of
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Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette,
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Indiana, USA
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Department of Periodontology, Institute of Dentistry, University of Turku, FI-20520, Turku,
Institute of Biomedicine, University of Turku, FI-20520, Turku, Finland
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*
Correspondence:
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Department of Periodontology, Institute of Dentistry, University of Turku,
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20520 Turku, Finland.
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Tel: +358 40 9650024
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E-mail addresses:
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[email protected] (D. Fteita), [email protected] (A. A. Musrati), [email protected] (E.
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Könönen), [email protected] (M. Gürsoy), [email protected] (M. Peurla),
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[email protected] (X. Ma), [email protected] (E. Söderling), [email protected] (H.
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O. Sintim), [email protected] (U.K. Gürsoy).
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Key words: autoinducer-2; biofilm; enzyme inhibitor; estradiol; pathogenicity; Prevotella
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ACCEPTED MANUSCRIPT ABSTRACT
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Biofilm formation and dipeptidyl peptidase IV (DPPIV) enzyme activity contribute to the
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virulence of oral bacteria, and these virulence factors are partly regulated by quorum sensing
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signaling system. We recently demonstrated that estradiol regulates growth properties and
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DPPIV activity of Prevotella intermedia, Prevotella nigrescens, and Prevotella pallens. Here,
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we examined the DPPIV dependency of biofilm formation of Prevotella aurantiaca. Three
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strains (two clinical strains AHN 37505 and 37552 and the type strain CCUG 57723) were
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incubated in three estradiol concentrations (30, 90, and 120 nmol/L). Regulation of DPPIV
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activity, biofilm and fimbria formation, and coaggregation of bacterial strains were analyzed
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after incubation with four concentrations (10 nM, 100 nM, 1 µM, 10 µM) of dihydroxy-2,3-
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pentaedione (DPD), the universal precursor of autoinducer -2 (AI-2), and analogs (ethyl-DPD,
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butyl-DPD, and isobutyl-DPD) for 24 hours. Estradiol enhanced the planktonic growth,
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coaggregation, and biofilm formation of P. aurantiaca strains. The whole cell extract of AHN
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37505 had the highest DPPIV activity, followed by CCUG 57723 and AHN 37552. Inhibition
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of DPPIV activity with di-isopropylfluorophosphate suppressed the effect of estradiol on
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biofilm formation. At 100 nM and 10 µM concentrations of DPD, butyl DPD, and isobutyl
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DPD, biofilm formation of P. aurantiaca was significantly inhibited. Fimbriae formation was
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enhanced up to concentrations of 100 nM and 1 µM followed by a significant inhibition at
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higher concentrations of DPD and all analogs. A slight but significant inhibitory effect of
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DPD and analogs on DPPIV activity was observed. Our results indicate that DPPIV plays a
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key role in the estradiol-regulated biofilm formation of P. aurantiaca. Quorum sensing
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autoinducer DPD and C1-alkyl analogs could inhibit biofilm-related virulence of P.
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aurantiaca.
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1. INTRODUCTION Prevotella aurantiaca is the newest member of the Prevotella intermedia group bacteria with
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a 16S rRNA gene sequence similarity of 96.4% and 96.1% to the type strains of P. intermedia
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and Prevotella pallens, respectively (Sakamoto et al., 2010). P. intermedia group organisms
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harbor distinct characteristics that may explain the observed difference in their association
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with the health status of the periodontium (Shah and Gharbia, 1992; Moore and Moore, 1994;
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Könönen et al., 1998; 2000). For instance, whereas P. intermedia is mainly associated with
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periodontal infections, P. nigrescens has been reported in both healthy and diseased
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periodontal subjects (Shah and Gharbia, 1992; Moore and Moore, 1994; Mättö et al., 1999;
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Könönen et al., 2000; Hashimoto et al., 2003). P. pallens seems to be mainly associated with
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the healthy periodontium (Könönen et al., 1998a; 1998b). Regarding P. aurantiaca, although
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the species were isolated from a periodontitis patient, a role for this organism in exacerbating
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periodontitis remains to be determined and thus far only scarce data are available on the
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involvement of P. aurantiaca in either periodontal or systemic diseases (Sakamoto et al.,
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2010; Fteita et al., 2015; Piccolo et al., 2015).
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Dipeptidyl peptidase IV (DPPIV) is a proline-specific serine protease that hydrolyzes
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the penultimate X-proline and X-alanine dipeptide residues from the N-terminus of the
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oligopeptide and some polypeptide substrates (Augustyns et al., 1999). In regards to bacterial
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DPPIV, the hydrolytic activity of its catalytic domain has a destructive effect on the
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periodontium (Abiko et al., 1985; Banbula et al., 1999; Kumagai et al., 2005). Furthermore,
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DPPIV of Porphyromonas gingivalis, a major periodontal pathogen, has been found to
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indirectly participate in periodontal tissue destruction through its promotive action on the
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superfamily of host-derived proteases, matrix metalloproteinases (MMPs), e.g. MMP-1,
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MMP-2, MMP-8, and MMP-9 (Kumagai et al., 2005). Among the genus Prevotella, P.
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intermedia and P. nigrescens (Gazi et al., 1997), and rumen Prevotella species (Depardon et
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al., 1996) are able to break down peptides through exhibiting DPP-like activities. Moreover,
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our recent in vitro fluorometric analysis revealed significant DPPIV activities within the P.
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intermedia group bacteria examined (Fteita et al., 2015).
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In biofilms, bacterial cells communicate through competitive interactions between genetically distinct species through a process called quorum sensing (QS) (Fuqua et al., 1994;
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Rasmussen and Givskov, 2006). QS is cell population density-dependent; after a threshold or
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quorum is reached, cellular processes are synchronized (Miller and Bassler, 2001) in order to
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enhance bacterial defense mechanism against the host and to modulate virulence expressions
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and diverse phenotypes (Sintim et al., 2010; Tateda et al., 2003; Shiner et al., 2006). One
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such universal communication signaling molecule is autoinducer-2 (AI-2), which is produced
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by both Gram positive and Gram-negative bacteria (Vendeville et al., 2005; Sintim and
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Gürsoy, 2016). Development of derivatives of natural QS molecules, which could compete
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with or inhibit native AI signaling pathways, has gained widespread interest worldwide
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(Lowery et al., 2009; Chung et al., 2011). Among the broad-spectrum anti-QS agents, 4,5-
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dihydroxy-2,3-pentanedione (DPD), which is the precursor of the universal AI-2, and its
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synthetic C1-alkyl analogs have been used to inhibit QS signalling and virulence factors
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produced by pathogenic bacteria (Roy et al., 2010; Guo et al., 2012). To date, such AI-2 QS
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antagonists have not been used to modulate oral bacteria.
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P. intermedia and P. nigrescens increase in subgingival sites and saliva during
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pregnancy-related gingivitis (Kornman and Loesche, 1980; Gürsoy et al., 2009; Carrillo-de-
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Albornoz et al., 2012). We recently demonstrated that estradiol regulates the growth
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properties and DPPIV activity of P. intermedia, P. nigrescens, and P. pallens in a species-
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and strain-dependent manner (Fteita et al., 2014; 2015), whereas data on DPPIV activity of P.
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aurantiaca are missing so far. Here, the aims were to examine the role of DPPIV in estradiol-
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regulated biofilm formation and to investigate the modulatory role of DPD and its analogs on
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biofilm formation, coaggregation, and fimbria morphology of P. aurantiaca.
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2. MATERIAL AND METHODS 2.1.
Bacterial strains and culture methods
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The type strain (CCUG 57723) and two clinical strains (AHN 37505 and AHN 37552) of P.
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aurantiaca were used in the experiments. The clinical strains had been collected from two
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periodontitis-free post-partum women and identified by using partial (ca. 550 bp) 16S rRNA
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gene sequencing (Estama et al., 2015).
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In all experiments, bacterial cells (revived from skimmed milk stocks kept in -70°C)
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were grown on Brucella blood agar plates supplemented with hemin (5 mg L-1) and vitamin
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K1 (10 mg L-1) were used for culturing the bacterial strains in an anaerobic chamber (Whitley
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A35 Anaerobic Workstation, Don Whitley Scientific Ltd., West Yorkshire, UK) with an
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atmosphere of 10% H2, 5% CO2, and 85% N2 at 37 ºC for 72 hours. To obtain pure cultures,
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clearly distinct colonies were passaged for another growth cycle on the same type of Brucella
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agar and with the same incubation conditions. After 72 hours, and to allow further growth,
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bacterial cells were collected and transferred to Todd-Hewitt broth (Becton, Difco™ and
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BBL™, USA) supplemented with 5 g L-1 yeast extracts, 750 mg L-1 cysteine, 5 mg L-1 hemin,
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and 5 mg L-1 menadione (Sigma Chemical Co., St. Louis, USA) for 24 hours in anaerobic
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condition.
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2.2.
Preparation of whole cell extracts
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Preparation of whole cell extract (WCE) was performed according to Itoh et al. (2009).
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Briefly, 72 hours old bacterial colonies from one full agar plate were collected using cotton
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swabs and suspended in 0.3% CHAPS detergent (Thermo Fisher Scientific, USA) and
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immediately incubated in an ice box for 30 minutes. Each bacterial suspension was then
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sonicated on ice for 20 seconds in order to avoid heating from sonication. The optical density
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(OD) of each bacterial suspension was adjusted to the lowest strain OD (2.3) at 490 nm.
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2.3.
Preparation of estradiol suspensions
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In each experiment, the estradiol concentrations of 30, 90, and 120 nmol L-1 were used,
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simulating the serum estradiol concentration equivalents during the first, second, and third
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trimester of pregnancy, respectively (O’Leary et al., 1991). In addition, an estradiol
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concentration of 0 nmol L-1 served as a control.
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2.4.
Synthesis and preparations of 4,5-dihydroxy-2,3-pentanedione (DPD) and its C1-alkyl
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analogs suspension
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DPD and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) were synthesized following
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the previously described protocol (Roy et al., 2010). Stock solutions (in dimethyl sulfoxide)
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were diluted with culture media in order to obtain the final molarity of 10 nM, 100 nM, 1 µM,
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and 10 µM. To avoid frequent thawing, each time the stock was taken out from -20°C to
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prepare a concentration, the rest was discarded.
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2.5.
Planktonic growth and bacterial cell viability
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One-day-old bacterial suspensions were adjusted to 0.7 OD and measured at 490 nm with a
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spectrophotometer (Shimadzu Biotech, Tokyo, Japan). The adjusted OD of each strain
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corresponded to the logarithmic colony forming unit (log CFU mL-1) of 103.6x10-8 for P.
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aurantiaca AHN 37505, 95x10-8 for P. aurantiaca AHN 37552, and 120.3x10-8 for P.
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aurantiaca CCUG 57723. Optically adjusted bacterial suspensions were incubated with
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estradiol concentrations of 0, 30, 90, or 120 nmol L-1 in an anaerobic chamber for 24 hours.
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After incubation, each bacterial suspension was inoculated on Brucella agar plates and
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incubated in anaerobic conditions for 72 hours. Bacterial growth was counted as CFUs.
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performed. Each OD adjusted bacterial strain was incubated with 1 mM of the serine
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proteinase inhibitor di-isoprpylfluorophosphate (DPF) at 37 ℃ for 15 minutes. Afterwards, 10
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µl of each serially diluted bacterial suspension was spread on Brucella agar plates and
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incubated in an anaerobic atmosphere for 3-5 days until bacterial colonies were visible to be
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counted as CFUs.
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2.6.
Coaggregation assay
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In the coaggregation assay, the Kolenbrander’s standard method was used with slight
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modifications (Cisar et al., 1979; Kolenbrander, 1995). Briefly, P. aurantiaca strains were
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tested for their coaggregation abilities with Fusobacterium nucleatum ATCC 25586 in an
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anaerobic atmosphere. Overnight cultures of the test strains were harvested by centrifugation,
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washed, and centrifuged again for removing the supernatant, and cells were resuspended in
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the coaggregation buffer, Tris-HCl, pH 8.0 (Cisar et al., 1979). Equal amounts of each P.
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aurantiaca strain were incubated together with F. nucleatum, either in the presence of 0, 30,
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90, or 120 µg mL-1 estradiol, or, 10 nM, 100 nM, 1 µM, and 10 µM of DPD or its three
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analogs. Physical coaggregation was recorded at time 0 and after 30 minutes using a
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spectrophotometer (SHIMADZU UV-visible, BioSpec-mini, Kyoto, Japan) (Fteita et al.,
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2014). During the incubation period, the test cuvettes were kept covered and stored at 37°C.
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2.7.
Measurement of fluorometric DPPIV enzyme activity/inhibition
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The OD adjusted WCE from each P. aurantiaca strain was loaded in a 96-well plate
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containing Tris-HCl buffer (pH 8) with 0.05% Triton X-100. The buffer was added to
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enhance the enzyme solubility of the WCE. For detecting enzyme activities in the DPPIV
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fluorometric assay, 1 mM of the fluorogenic substrate H-Ala-Pro-7-amido-48
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buffer (pH 8), followed by an immediate enzyme activity measurement of all wells (Fteita et
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al., 2015). The experiment was performed in pentaplicate. To ensure the inhibition capacity of
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the serine protease inhibitor, di-isopropylfluorophosphate (DFP), against DPPIV, the above-
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mentioned protocol was applied using WCE to DPPIV inhibitor as 4:1 (Koreeda et al., 2001;
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Shibata et al., 2003). The DPPIV activity/inhibition measurements were recorded between 0-
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45 minutes and, to confirm the steadiness of the results, the final reading was done after 1
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hour after starting the reaction. All measurement steps of the fluorescence release by the
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substrate in both activity and inhibition conditions were performed kinetically (Fteita et al.,
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2015).
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2.8.
Analysis of DPPIV enzyme activity/inhibition by zymography
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Bacterial crude samples for zymography were prepared with fresh WCEs. The DPPIV
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fluorogenic activity and inhibition were examined with a DPPIV-specific zymography using
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8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels including a
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fluorogenic substrate, H-Ala-Pro-7-amido-4-trifluromethylcoumarin (Bachem, Bubendorf,
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Switzerland). A pre-stained low molecular weight SDS-PAGE standard with a range of 17-
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102 kDa (Bio-Rad, USA) was used as a reference. The gel was run at 110V for 1-1.5 hours.
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After electrophoresis, the gels were washed twice followed by an overnight incubation for
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enzyme activation. The enzyme activity was seen as dark bands in the UV-fluorescein device
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setting and as light bands in the stain-free gel setting of the imaging system used (Bio-Rad
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ChemiDoc™ MP Imaging System).
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2.9.
Determination of the role of DPPIV in biofilm formation
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ACCEPTED MANUSCRIPT The quantification of bacterial biofilms represented by protein levels was determined using
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the Bradford protein assay of biological samples (Hammond and Kruger, 1988) and
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incorporating the assay enhancement method using microwave (Akins and Tuan, 1995; Fteita
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et al., 2014). A saliva-coated 96-well plate was prepared as previously described (Fteita et al.
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2014), and each P. aurantiaca strain on the plate was incubated anaerobically with either
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estradiol (0, 30, 90, and 120 nmol L-1) or with estradiol and 1 mM of DPF (DPPIV inhibitor)
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in pentaplicates for 48 hours (Koreeda et al., 2001; Shibata et al., 2003). The wells were
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rinsed twice with PBS to eliminate unbounded cells and 0.2N of NaOH was loaded to each
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well. Sonication and microwave heating were performed, followed by the Bradford protein
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assay (Bio-Rad, USA) with the necessary incubation and shaking procedures (Fteita et al.,
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2015). A colorimetric detection of the amount of proteins in biofilms was performed at an
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absorbance of 595 nm using a micro-plate reader. To obtain actual protein levels, datasets
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were standardized using bovine serum albumin (Sigma-Aldrich, USA) concentrations (Fteita
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et al., 2014).
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2.10. TEM imaging
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After the incubation of the OD-adjusted P. aurantiaca strains with DPD and its analogs for 24
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hours, each suspension was centrifuged at 10 000 g for 5 minutes. During centrifugation, the
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centrifuge internal temperature was adjusted to 37°C simulating the anaerobic atmosphere.
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Once centrifugation was done, the supernatants were discarded and the bacterial pellets were
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immediately collected and fixed with 5% glutaraldehyde in 0.16 mol 1-1 s-collidine–HCl
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buffer, pH 7.4 and sent to TEM facility for imaging, as described previously (Musrati et al.,
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2014).
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The TEM grid sections were visualized by a JEOL JEM-1400 Plus transmission electron
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microscope (JEOL, Tokyo, Japan) with an operation power of 80 kV acceleration voltage.
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section; the first magnification (x2,500) was to evaluate the section validity to be included in
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the study (bacterial contamination or artifacts), the second magnification (x12,000) was to
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visualize the fimbria formation changes among different bacterial cells, while the third
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magnification (x25,000) was mainly to run fimbria thickness calculations for obtaining
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quantitative data of the three P. aurantiaca strains under the effect of graded concentrations of
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DPD and its analogs. Bacterial fimbriae thickness was measured from five different sites of
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each bacterial cell using the software ImageJ 1.48v (Wayne Rasband National Institute of
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Health, USA) (Musrati et al., 2014).
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2.11. Statistical analysis
The data distribution from each experiment was checked for normality using the
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Kolmogorov-Smirnov and Shapiro-Wilk standard normality tests. Since all data were
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normally distributed, multiple comparisons between the estradiol concentration groups were
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performed by using the single factor “one-way ANOVA” with a two-tailed probability level
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of statistical significance. When applicable, each experiment was performed at least twice
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independently in five replicates. P-value below 0.05 was accepted as statistically significant.
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3. RESULTS 3.1.
Planktonic growth and coaggregation
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Elevated estradiol concentrations enhanced significantly not only the planktonic growth of the
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clinical P. aurantiaca strains (AHN 37505 and AHN 37552) (Fig. 1a) but also the
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coaggregation capability of AHN 37505 strain with F. nucleatum (Fig. 1b).
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3.2.
DPPIV activity of WCEs
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DPPIV activities of the three strains of P. aurantiaca showed a transitional increase over
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time. The strain AHN 37505 had the highest level of enzyme activity followed by the type
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strain CCUG 57723 and the other clinical strain (AHN 37552). A significant inhibition was
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observed in all three strains when 1 mM of DFP was added in each bacterial WCE (Fig. 2a).
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Based on CFU values, the 15-minute exposure of P. aurantiaca cells to DFP had no
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significant effect on their viability except for one strain, AHN 37505, where a significant
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decrease in the number of colonies was observed (Fig. 2b). In the zymogram, clear bands of
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DPPIV activity corresponded to the crude WCE of the three P. aurantiaca strains between the
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molecular weight markers of 102-79 kDa. In the wells containing the same WCE
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preincubated with 1 mM of DFP, no visible bands of DPPIV activity were observed in any of
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the tested strains (Fig. 2c).
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3.3.
Inhibition of DPPIV activity by DFP inhibited the biofilm formation
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Biofilm mass of all P. aurantiaca strains significantly increased with elevated estradiol
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concentrations of different peaks of protein production. This estradiol-regulated enhancement
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was totally depleted when the strains were incubated with 1 mM DFP; no significant change
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was seen in biofilm formation between different estradiol concentrations, except for P.
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aurantiaca AHN 37505, which exhibited a slight increase in its biofilm mass (Fig. 3).
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3.4.
Regulation of DPPIV enzyme activity by DPD and its analogs
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Fluorometric enzyme activity measurements showed significant differences in the DPPIV
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activity among the three P. aurantiaca strains incubated with graded concentrations of DPD
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and analogs in a strain- and dose-dependent response (Fig. 4). For all strains, the peak of
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inhibition in the enzyme activity was reached among DPD and its analogs at the
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concentrations of 100 nM and 1 µM.
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3.5.
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analogs
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Biofilm formation (Fig. 5) and coaggregation capabilities (Fig. 6) of the three strains of P.
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aurantiaca were slightly but significantly decreased when exposed to DPD, butyl DPD, and
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isobutyl DPD but not to ethyl DPD (P<0.05). Fimbriae thickness showed a transient and
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significant increase with increasing the DPD and its analogs concentrations followed by a
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significant decrease in the two highest concentrations (1 µM and 10 µM) in all strains (Fig. 7
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and Fig. 8). Although not considered quantitatively, TEM micrographs revealed clear
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changes in the number and color intensity of intracellular inclusions and granules of all
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strains with a clear visibility in the type strain P. aurantiaca CCUG 57723 (Fig. 8a, b, and c).
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Regulation of biofilm mass, coaggregation, and fimbriae thickness by DPD and its
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4. DISCUSSION
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In these in vitro experiments, biofilm formation of P. aurantiaca (which was treated with
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estradiol) was found to be dependent on DPPIV activity. To our knowledge, the present study
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is the first to demonstrate such a modulatory effect on this novel species within the P.
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intermedia group. Moreover, we present evidence that a disruption in QS signaling with C1-
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alkyl analogs of DPD may modify the biofilm-related virulence factors of P. aurantiaca.
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DPPIV activity of the P. aurantiaca strains was confirmed by zymography where clear bands of activities corresponded to a known molecular weight on the standard ladder.
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The observed enzyme activity bands were considered to be solely obtained from DPPIV with
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a high specificity based on previous reports where DFP totally inhibited the DPPIV activity as
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seen in the disappearance of the corresponding bands (Koreeda et al., 2001; Shibata et al.,
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2003). The 100% inhibition rate of DPPIV with no cytotoxic effect on the P. aurantiaca cells
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was performed using the gold standard method, i.e., counting the bacterial growth as CFUs.
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Only one clinical strain of P. aurantiaca (AHN 37505) showed a significant decrease in its
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proliferation when incubated with DFP.
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Several studies have addressed the impact of eukaryotic DPPIV in various biological,
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physiological, and pathological interactions, including the activation of immune cells, cancer
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pathogenesis, and metabolic disorders (Augustyns et al., 1999; Beckenkamp et al., 2016;
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Bellé et al., 2011). However, limited knowledge exists about the involvement of bacterial
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DPPIV in such systemic interactions in general, and in the oral cavity in particular. Among
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oral Gram-negative anaerobes, P. gingivalis and P. intermedia are known as significant
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producers of DPPIV (Gazi et al., 1997; Banbula et al., 2000; Clais et al., 2014; Fteita et al.,
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2015). Biofilm formation of P. gingivalis, a major pathogen in periodontal disease
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pathogenesis, was recently found to increase the bacterial DPPIV activity (Clais et al., 2014).
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Being a novel species isolated from the oral cavity and with scarce data available in the
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literature, P. aurantiaca gained our interest to investigate its potential pathogenicity under the
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effect of estradiol. In our previous studies dealing with members of the P. intermedia group, it
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was demonstrated that estradiol regulates various virulence characteristics potentially being
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involved in pregnancy-associated gingivitis (Fteita et al., 2014; 2015). In our previous study,
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of the other P. intermedia group organisms) in the presence and absence of estradiol (Fteita et
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al, 2015). The present results confirm our previous finding in which P. aurantiaca exhibited a
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significant DPPIV enzyme activity, which seems to play a key role in its estradiol-regulated
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capability to form biofilms. The latter observation was confirmed when the DPPIV inhibition
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by DFP (DPPIV inhibitor) abolished the significant differences between biofilm mass of P.
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aurantiaca grown in different estradiol concentrations.
In addition to host-derived molecules, such as estradiol, bacteria are exposed to
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bacterially-derived QS molecules. QS is a crucial process where bacteria can communicate
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with each other and respond to autoinducers in order to modify their gene expression and
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virulence properties (Fuqua et al., 1994). P. intermedia is one of the periodontal pathogens
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tested for their ability to produce extracellular autoinducer-like activities (Frias et al., 2001).
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Although complementation experiments were not achievable due to limited gene sequencing
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data in the database, Frias et al. (2001) was able to demonstrate the production of AI-2 by
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several P. intermedia strains in a glucose- and growth phase-independent manners. Disruption
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of QS plays a key role in modifying bacterial motility, adhesion, and biofilm-related
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virulence. Development of anti-QS agents through a pro-drug approach, targeting chemical
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structure alterations of analogs and bacterial membrane permeability, resulted in the
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production of next-generation non-bactericidal antimicrobial agents, the ester derivatives of
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DPD analogs (Kamaraju et al., 2011; Guo et al., 2012). Up to date, data on the modulatory
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effect of DPD and its analogs on bacterial cells are scarce; nevertheless, Guo et al. (2012)
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found that DPD and isobutyl DPD inhibit QS in Escherichia coli. In the present study, it is
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shown for the first time that DPD and its analogs have a dose-dependent inhibitory effect on
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fimbria thickness and a slight but significant inhibition of DPPIV enzyme activity,
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coaggregation, and biofilm formation.
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ACCEPTED MANUSCRIPT In conclusion, the results of our in vitro experiments indicate that estradiol regulates
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the proliferation and biofilm formation of P. aurantiaca and these events significantly depend
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on DPPIV activity. Biofilm and DPPIV enzyme activity-related virulence of P. aurantiaca
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may be inhibited, at least partly, by the disruption of QS signaling with C1-alkyl analogs of
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DPD.
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Funding
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This work was supported by the Finnish Doctoral Program in Oral Sciences (FINDOS) (D.F.),
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Turku University Foundation (D.F. and U.K.G.), and Finnish Dental Society Apollonia
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(U.K.G.). XCM thanks China Scholarship Council (No.201406140119) for financial support.
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Conflict of interests
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The authors declare no conflict of interest.
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ACCEPTED MANUSCRIPT References Abiko Y, Hayakawa M, Murai S, Takiguchi H (1985). Glycylprolyl dipeptidylaminopeptidase from Bacteroides gingivalis. J Dent Res 64:106-111. Akins RE & Tuan RS (1995) Ultrafast protein determinations using microwave enhancement.
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Kolenbrander PE (1995) Coaggregations among oral bacteria. Methods Enzymol 253:385397. Kornman KS, Loesche WJ (1980). The subgingival microbial flora during pregnancy. J Periodontal Res 15:111-122. 19
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Könönen E, Wolf J, Mättö J, Frandsen EV, Poulsen K, Jousimies-Somer H, et al. (2000). The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status. J Periodontal Res 35:329-334.
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Koreeda Y, Hayakawa M, Ikemi T, & Abiko Y (2001) Isolation and characterisation of
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Kumagai Y, Yagishita H, Yajima A, Okamoto T, & Konishi K (2005) Molecular mechanism for connective tissue destruction by dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis. Infect Immun 73:2655-2664.
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Lowery CA, Abe T, Park J, Eubanks LM, Sawada D, Kaufmann GF, et al. (2009) Revisiting AI-2 quorum sensing inhibitors: direct comparison of alkyl-DPD analogues and a
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natural product fimbrolide. J Am Chem Soc 131:15584-15585. Miller MB, Bassler BL. (2001) Quorum sensing in bacteria. Annu Rev Microbiol 55: 165-199.
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Moore WEC & Moore LVH (1994) The bacteria of periodontal diseases. Periodontol 2000 5: 66-77.
Musrati AA, Fteita D, Paranko J, Könönen E, Gürsoy UK (2016) Morphological and functional adaptations of Fusobacterium nucleatum exposed to human neutrophil peptide-1. Anaerobe 39:31-38. O'Leary P, Boyne P, Flett P, Beilby J, & James I (1991) Longitudinal assessment of changes in reproductive hormones during normal pregnancy. Clin Chem 37:667-672. 20
ACCEPTED MANUSCRIPT Piccolo M, De Angelis M, Lauriero G, et al. (2015) Salivary microbiota associated with immunoglobulin A nephropathy. Microb Ecol 70:557-565. Rasmussen TB, Givskov M (2006) Quorum-sensing inhibitors as anti-pathogenic drugs. Int J
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Med Microbiol 296: 149-161. Roy V, Smith JA, Wang J, Stewart JE, Bentley WE, Sintim HO (2010) Synthetic analogs tailor native AI-2 signaling across bacterial species. J Am Chem Soc 132: 11141-
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Sakamoto M, Suzuki N, & Okamoto M (2010) Prevotella aurantiaca sp. nov., isolated from
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the human oral cavity. Int J Syst Evol Microbiol 60:500-503.
Shah HN & Gharbia SE (1992) Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov. Int J Syst Bacteriol 42: 542-546.
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Shibata Y, Miwa Y, Hirai K, & Fujimura S (2003) Purification and partial characterization of a dipeptidyl peptidase from Prevotella intermedia. Oral Microbiol Immunol 18:196-
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Shiner EK, Terentyev D, Bryan A, Sennoune S, Martinez-Zaguilan R, Li G, et al. (2006)
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Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling. Cell Microbiol 8: 1601-1610.
Sintim HO, Smith JA, Wang J, Nakayama S, Yan L (2010) Paradigm shift in discovering next-generation anti-infective agents: targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules. Future Med Chem 2:10051035.
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ACCEPTED MANUSCRIPT Sintim HO, Gürsoy UK (2016) Biofilms as "Connectors" for oral and systems medicine: A new opportunity for biomarkers, molecular targets, and bacterial eradication. OMICS 20:3-11. Tateda K, Ishii Y, Horikawa M, Matsumoto T, Miyairi S, Pechere JC, et al. (2003) The
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Pseudomonas aeruginosa autoinducer N-3-oxododecanoyl homoserine lactone
accelerates apoptosis in macrophages and neutrophils. Infect Immun 71: 5785-5793. Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR (2005) Making ‘sense’ of
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metabolism: autoinducer-2, LuxS and pathogenic bacteria. Nat Rev Microbiol 3: 383-
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ACCEPTED MANUSCRIPT Figure legends Fig. 1a) Planktonic growth properties of three Prevotella aurantiaca strains within gradually increased estradiol concentrations (0, 30, 90, and 120 nmol L-1). Data represent the colony forming unit (CFU) mL-1. 1b) Spectrophotometrically evaluated coaggregation between
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Fusobacterium nucleatum ATCC 25586 and the P. aurantiaca strains in different estradiol concentrations (0, 30, 90, and 120 nmol L-1). Data represent the optical density (OD) readings of 0.7 at 660 nm. Asterisks indicate significant differences with the control (*P < 0.05,
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**P<0.01).
Fig. 2) DPPIV activity, zymography, and proliferation rate of three P. aurantiaca strains
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without and with DPPIV inhibition by di-isopropylfluorophosphate (DFP-/+). a) For DPPIV activity, data are presented as fluorescence excitation and emission intensity values, and b) for proliferation measurements, data are represented as colony forming unit (CFU) mL-1. c) For zymography, the enzyme assay was performed twice, only one representative gel is
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presented. Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01). Fig. 3) Protein levels representing the biofilm mass of the P. aurantiaca strains in different estradiol concentrations (0, 30, 90, and 120 nmol L-1) without and with dipeptidyl peptidase
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IV (DPPIV) inhibition by di-isopropylfluorophosphate (DFP-/+). Data are presented as mg mL-1. Solid bars represent the groups with no DFP, while the dashed bars represent the groups
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with DFP. Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01). Fig. 4) DPPIV enzyme activity of the P. aurantiaca strains in concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM). Data are presented as excitation and emition OD values. Asterisks indicate significant differences with the control (*P<0.05, **P<0.01, and ***P<0.001).
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ACCEPTED MANUSCRIPT Fig. 5) Protein levels representing the biofilm mass of the P. aurantiaca strains in different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM). Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01). Fig. 6) Coaggregation between Fusobacterium nucleatum ATCC 25586 and the P. aurantiaca
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strains in different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM). Data represent the optical density (OD) readings of 0.7 at 660 nm. Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01).
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Fig. 7) Fimbriae thickness measurements of the three P. aurantiaca strains incubated in
different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100
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nM, 1 µM, and 10 µM). Data are presented in nanometers scale (nm). Measurements were taken from images of 25,000x magnification. Asterisks indicate significant differences with the control (*P<0.05, **P<0.01, and ***P<0.001).
Fig. 8a, b, and c) TEM micrographs reveal the difference in Fimbriae thickness and
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intracellular changes in bacterial cell morphology between the control and different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM) of the three study strains: (a) P. aurantiaca AHN 37505, (b) P. aurantiaca AHN
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37552, and (c) P. aurantiaca CCUG 57723. Scale bars indicate 200 nm.
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ACCEPTED MANUSCRIPT Highlights
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Quorum sensing (QS) and estradiol effect on P. aurantiaca virulence were evaluated. Biofilm mass and DPPIV enzyme activity were used for the evaluation. Estradiol enhanced the growth, coaggregation, and biofilm mass of P. aurantiaca. QS Signaling disruption may, partly, inhibit virulence of P. aurantiaca.
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S1075-9964(17)30168-3
DOI:
10.1016/j.anaerobe.2017.08.009
Reference:
YANAE 1789
To appear in:
Anaerobe
Received Date: 18 March 2017 Revised Date:
27 June 2017
Accepted Date: 14 August 2017
Please cite this article as: Fteita D, Musrati AA, Könönen E, Ma X, Gürsoy M, Peurla M, Söderling E, Sintim HO, Gürsoy UK, Dipeptidyl peptidase IV and quorum sensing signaling in biofilm-related virulence of Prevotella aurantiaca, Anaerobe (2017), doi: 10.1016/j.anaerobe.2017.08.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
DIPEPTIDYL PEPTIDASE IV AND QUORUM SENSING SIGNALING IN BIOFILM-
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RELATED VIRULENCE OF PREVOTELLA AURANTIACA
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Dareen Fteitaa,*, Ahmed Ali Musratia, Eija Könönena, b, Xiaochu Mac, Mervi Gürsoya, Markus
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Peurlad, Eva Söderlinga, Herman O. Sintimc, Ulvi Kahraman Gürsoya
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a
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Finland
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b
Welfare Division, Oral Health Care, City of Turku, Turku, Finland
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c
Department of Chemistry and Purdue Institute for Drug Discovery and Purdue Institute of
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Inflammation, Immunology and Infectious Disease, Purdue University, West Lafayette,
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Indiana, USA
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d
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Department of Periodontology, Institute of Dentistry, University of Turku, FI-20520, Turku,
Institute of Biomedicine, University of Turku, FI-20520, Turku, Finland
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*
Correspondence:
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Department of Periodontology, Institute of Dentistry, University of Turku,
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20520 Turku, Finland.
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Tel: +358 40 9650024
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E-mail addresses:
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[email protected] (D. Fteita), [email protected] (A. A. Musrati), [email protected] (E.
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Könönen), [email protected] (M. Gürsoy), [email protected] (M. Peurla),
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[email protected] (X. Ma), [email protected] (E. Söderling), [email protected] (H.
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O. Sintim), [email protected] (U.K. Gürsoy).
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Key words: autoinducer-2; biofilm; enzyme inhibitor; estradiol; pathogenicity; Prevotella
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ACCEPTED MANUSCRIPT ABSTRACT
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Biofilm formation and dipeptidyl peptidase IV (DPPIV) enzyme activity contribute to the
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virulence of oral bacteria, and these virulence factors are partly regulated by quorum sensing
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signaling system. We recently demonstrated that estradiol regulates growth properties and
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DPPIV activity of Prevotella intermedia, Prevotella nigrescens, and Prevotella pallens. Here,
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we examined the DPPIV dependency of biofilm formation of Prevotella aurantiaca. Three
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strains (two clinical strains AHN 37505 and 37552 and the type strain CCUG 57723) were
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incubated in three estradiol concentrations (30, 90, and 120 nmol/L). Regulation of DPPIV
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activity, biofilm and fimbria formation, and coaggregation of bacterial strains were analyzed
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after incubation with four concentrations (10 nM, 100 nM, 1 µM, 10 µM) of dihydroxy-2,3-
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pentaedione (DPD), the universal precursor of autoinducer -2 (AI-2), and analogs (ethyl-DPD,
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butyl-DPD, and isobutyl-DPD) for 24 hours. Estradiol enhanced the planktonic growth,
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coaggregation, and biofilm formation of P. aurantiaca strains. The whole cell extract of AHN
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37505 had the highest DPPIV activity, followed by CCUG 57723 and AHN 37552. Inhibition
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of DPPIV activity with di-isopropylfluorophosphate suppressed the effect of estradiol on
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biofilm formation. At 100 nM and 10 µM concentrations of DPD, butyl DPD, and isobutyl
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DPD, biofilm formation of P. aurantiaca was significantly inhibited. Fimbriae formation was
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enhanced up to concentrations of 100 nM and 1 µM followed by a significant inhibition at
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higher concentrations of DPD and all analogs. A slight but significant inhibitory effect of
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DPD and analogs on DPPIV activity was observed. Our results indicate that DPPIV plays a
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key role in the estradiol-regulated biofilm formation of P. aurantiaca. Quorum sensing
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autoinducer DPD and C1-alkyl analogs could inhibit biofilm-related virulence of P.
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aurantiaca.
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1. INTRODUCTION Prevotella aurantiaca is the newest member of the Prevotella intermedia group bacteria with
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a 16S rRNA gene sequence similarity of 96.4% and 96.1% to the type strains of P. intermedia
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and Prevotella pallens, respectively (Sakamoto et al., 2010). P. intermedia group organisms
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harbor distinct characteristics that may explain the observed difference in their association
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with the health status of the periodontium (Shah and Gharbia, 1992; Moore and Moore, 1994;
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Könönen et al., 1998; 2000). For instance, whereas P. intermedia is mainly associated with
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periodontal infections, P. nigrescens has been reported in both healthy and diseased
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periodontal subjects (Shah and Gharbia, 1992; Moore and Moore, 1994; Mättö et al., 1999;
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Könönen et al., 2000; Hashimoto et al., 2003). P. pallens seems to be mainly associated with
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the healthy periodontium (Könönen et al., 1998a; 1998b). Regarding P. aurantiaca, although
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the species were isolated from a periodontitis patient, a role for this organism in exacerbating
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periodontitis remains to be determined and thus far only scarce data are available on the
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involvement of P. aurantiaca in either periodontal or systemic diseases (Sakamoto et al.,
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2010; Fteita et al., 2015; Piccolo et al., 2015).
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Dipeptidyl peptidase IV (DPPIV) is a proline-specific serine protease that hydrolyzes
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the penultimate X-proline and X-alanine dipeptide residues from the N-terminus of the
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oligopeptide and some polypeptide substrates (Augustyns et al., 1999). In regards to bacterial
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DPPIV, the hydrolytic activity of its catalytic domain has a destructive effect on the
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periodontium (Abiko et al., 1985; Banbula et al., 1999; Kumagai et al., 2005). Furthermore,
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DPPIV of Porphyromonas gingivalis, a major periodontal pathogen, has been found to
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indirectly participate in periodontal tissue destruction through its promotive action on the
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superfamily of host-derived proteases, matrix metalloproteinases (MMPs), e.g. MMP-1,
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MMP-2, MMP-8, and MMP-9 (Kumagai et al., 2005). Among the genus Prevotella, P.
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intermedia and P. nigrescens (Gazi et al., 1997), and rumen Prevotella species (Depardon et
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al., 1996) are able to break down peptides through exhibiting DPP-like activities. Moreover,
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our recent in vitro fluorometric analysis revealed significant DPPIV activities within the P.
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intermedia group bacteria examined (Fteita et al., 2015).
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In biofilms, bacterial cells communicate through competitive interactions between genetically distinct species through a process called quorum sensing (QS) (Fuqua et al., 1994;
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Rasmussen and Givskov, 2006). QS is cell population density-dependent; after a threshold or
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quorum is reached, cellular processes are synchronized (Miller and Bassler, 2001) in order to
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enhance bacterial defense mechanism against the host and to modulate virulence expressions
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and diverse phenotypes (Sintim et al., 2010; Tateda et al., 2003; Shiner et al., 2006). One
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such universal communication signaling molecule is autoinducer-2 (AI-2), which is produced
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by both Gram positive and Gram-negative bacteria (Vendeville et al., 2005; Sintim and
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Gürsoy, 2016). Development of derivatives of natural QS molecules, which could compete
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with or inhibit native AI signaling pathways, has gained widespread interest worldwide
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(Lowery et al., 2009; Chung et al., 2011). Among the broad-spectrum anti-QS agents, 4,5-
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dihydroxy-2,3-pentanedione (DPD), which is the precursor of the universal AI-2, and its
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synthetic C1-alkyl analogs have been used to inhibit QS signalling and virulence factors
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produced by pathogenic bacteria (Roy et al., 2010; Guo et al., 2012). To date, such AI-2 QS
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antagonists have not been used to modulate oral bacteria.
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P. intermedia and P. nigrescens increase in subgingival sites and saliva during
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pregnancy-related gingivitis (Kornman and Loesche, 1980; Gürsoy et al., 2009; Carrillo-de-
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Albornoz et al., 2012). We recently demonstrated that estradiol regulates the growth
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properties and DPPIV activity of P. intermedia, P. nigrescens, and P. pallens in a species-
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and strain-dependent manner (Fteita et al., 2014; 2015), whereas data on DPPIV activity of P.
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aurantiaca are missing so far. Here, the aims were to examine the role of DPPIV in estradiol-
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regulated biofilm formation and to investigate the modulatory role of DPD and its analogs on
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biofilm formation, coaggregation, and fimbria morphology of P. aurantiaca.
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2. MATERIAL AND METHODS 2.1.
Bacterial strains and culture methods
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The type strain (CCUG 57723) and two clinical strains (AHN 37505 and AHN 37552) of P.
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aurantiaca were used in the experiments. The clinical strains had been collected from two
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periodontitis-free post-partum women and identified by using partial (ca. 550 bp) 16S rRNA
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gene sequencing (Estama et al., 2015).
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In all experiments, bacterial cells (revived from skimmed milk stocks kept in -70°C)
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were grown on Brucella blood agar plates supplemented with hemin (5 mg L-1) and vitamin
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K1 (10 mg L-1) were used for culturing the bacterial strains in an anaerobic chamber (Whitley
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A35 Anaerobic Workstation, Don Whitley Scientific Ltd., West Yorkshire, UK) with an
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atmosphere of 10% H2, 5% CO2, and 85% N2 at 37 ºC for 72 hours. To obtain pure cultures,
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clearly distinct colonies were passaged for another growth cycle on the same type of Brucella
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agar and with the same incubation conditions. After 72 hours, and to allow further growth,
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bacterial cells were collected and transferred to Todd-Hewitt broth (Becton, Difco™ and
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BBL™, USA) supplemented with 5 g L-1 yeast extracts, 750 mg L-1 cysteine, 5 mg L-1 hemin,
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and 5 mg L-1 menadione (Sigma Chemical Co., St. Louis, USA) for 24 hours in anaerobic
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condition.
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2.2.
Preparation of whole cell extracts
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Preparation of whole cell extract (WCE) was performed according to Itoh et al. (2009).
145
Briefly, 72 hours old bacterial colonies from one full agar plate were collected using cotton
146
swabs and suspended in 0.3% CHAPS detergent (Thermo Fisher Scientific, USA) and
147
immediately incubated in an ice box for 30 minutes. Each bacterial suspension was then
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sonicated on ice for 20 seconds in order to avoid heating from sonication. The optical density
149
(OD) of each bacterial suspension was adjusted to the lowest strain OD (2.3) at 490 nm.
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2.3.
Preparation of estradiol suspensions
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In each experiment, the estradiol concentrations of 30, 90, and 120 nmol L-1 were used,
153
simulating the serum estradiol concentration equivalents during the first, second, and third
154
trimester of pregnancy, respectively (O’Leary et al., 1991). In addition, an estradiol
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concentration of 0 nmol L-1 served as a control.
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2.4.
Synthesis and preparations of 4,5-dihydroxy-2,3-pentanedione (DPD) and its C1-alkyl
158
analogs suspension
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DPD and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) were synthesized following
160
the previously described protocol (Roy et al., 2010). Stock solutions (in dimethyl sulfoxide)
161
were diluted with culture media in order to obtain the final molarity of 10 nM, 100 nM, 1 µM,
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and 10 µM. To avoid frequent thawing, each time the stock was taken out from -20°C to
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prepare a concentration, the rest was discarded.
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2.5.
Planktonic growth and bacterial cell viability
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One-day-old bacterial suspensions were adjusted to 0.7 OD and measured at 490 nm with a
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spectrophotometer (Shimadzu Biotech, Tokyo, Japan). The adjusted OD of each strain
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corresponded to the logarithmic colony forming unit (log CFU mL-1) of 103.6x10-8 for P.
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aurantiaca AHN 37505, 95x10-8 for P. aurantiaca AHN 37552, and 120.3x10-8 for P.
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aurantiaca CCUG 57723. Optically adjusted bacterial suspensions were incubated with
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estradiol concentrations of 0, 30, 90, or 120 nmol L-1 in an anaerobic chamber for 24 hours.
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After incubation, each bacterial suspension was inoculated on Brucella agar plates and
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incubated in anaerobic conditions for 72 hours. Bacterial growth was counted as CFUs.
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performed. Each OD adjusted bacterial strain was incubated with 1 mM of the serine
176
proteinase inhibitor di-isoprpylfluorophosphate (DPF) at 37 ℃ for 15 minutes. Afterwards, 10
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µl of each serially diluted bacterial suspension was spread on Brucella agar plates and
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incubated in an anaerobic atmosphere for 3-5 days until bacterial colonies were visible to be
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counted as CFUs.
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2.6.
Coaggregation assay
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In the coaggregation assay, the Kolenbrander’s standard method was used with slight
183
modifications (Cisar et al., 1979; Kolenbrander, 1995). Briefly, P. aurantiaca strains were
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tested for their coaggregation abilities with Fusobacterium nucleatum ATCC 25586 in an
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anaerobic atmosphere. Overnight cultures of the test strains were harvested by centrifugation,
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washed, and centrifuged again for removing the supernatant, and cells were resuspended in
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the coaggregation buffer, Tris-HCl, pH 8.0 (Cisar et al., 1979). Equal amounts of each P.
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aurantiaca strain were incubated together with F. nucleatum, either in the presence of 0, 30,
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90, or 120 µg mL-1 estradiol, or, 10 nM, 100 nM, 1 µM, and 10 µM of DPD or its three
190
analogs. Physical coaggregation was recorded at time 0 and after 30 minutes using a
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spectrophotometer (SHIMADZU UV-visible, BioSpec-mini, Kyoto, Japan) (Fteita et al.,
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2014). During the incubation period, the test cuvettes were kept covered and stored at 37°C.
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2.7.
Measurement of fluorometric DPPIV enzyme activity/inhibition
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The OD adjusted WCE from each P. aurantiaca strain was loaded in a 96-well plate
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containing Tris-HCl buffer (pH 8) with 0.05% Triton X-100. The buffer was added to
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enhance the enzyme solubility of the WCE. For detecting enzyme activities in the DPPIV
198
fluorometric assay, 1 mM of the fluorogenic substrate H-Ala-Pro-7-amido-48
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buffer (pH 8), followed by an immediate enzyme activity measurement of all wells (Fteita et
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al., 2015). The experiment was performed in pentaplicate. To ensure the inhibition capacity of
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the serine protease inhibitor, di-isopropylfluorophosphate (DFP), against DPPIV, the above-
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mentioned protocol was applied using WCE to DPPIV inhibitor as 4:1 (Koreeda et al., 2001;
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Shibata et al., 2003). The DPPIV activity/inhibition measurements were recorded between 0-
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45 minutes and, to confirm the steadiness of the results, the final reading was done after 1
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hour after starting the reaction. All measurement steps of the fluorescence release by the
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substrate in both activity and inhibition conditions were performed kinetically (Fteita et al.,
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2015).
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2.8.
Analysis of DPPIV enzyme activity/inhibition by zymography
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Bacterial crude samples for zymography were prepared with fresh WCEs. The DPPIV
212
fluorogenic activity and inhibition were examined with a DPPIV-specific zymography using
213
8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels including a
214
fluorogenic substrate, H-Ala-Pro-7-amido-4-trifluromethylcoumarin (Bachem, Bubendorf,
215
Switzerland). A pre-stained low molecular weight SDS-PAGE standard with a range of 17-
216
102 kDa (Bio-Rad, USA) was used as a reference. The gel was run at 110V for 1-1.5 hours.
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After electrophoresis, the gels were washed twice followed by an overnight incubation for
218
enzyme activation. The enzyme activity was seen as dark bands in the UV-fluorescein device
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setting and as light bands in the stain-free gel setting of the imaging system used (Bio-Rad
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ChemiDoc™ MP Imaging System).
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2.9.
Determination of the role of DPPIV in biofilm formation
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the Bradford protein assay of biological samples (Hammond and Kruger, 1988) and
225
incorporating the assay enhancement method using microwave (Akins and Tuan, 1995; Fteita
226
et al., 2014). A saliva-coated 96-well plate was prepared as previously described (Fteita et al.
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2014), and each P. aurantiaca strain on the plate was incubated anaerobically with either
228
estradiol (0, 30, 90, and 120 nmol L-1) or with estradiol and 1 mM of DPF (DPPIV inhibitor)
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in pentaplicates for 48 hours (Koreeda et al., 2001; Shibata et al., 2003). The wells were
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rinsed twice with PBS to eliminate unbounded cells and 0.2N of NaOH was loaded to each
231
well. Sonication and microwave heating were performed, followed by the Bradford protein
232
assay (Bio-Rad, USA) with the necessary incubation and shaking procedures (Fteita et al.,
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2015). A colorimetric detection of the amount of proteins in biofilms was performed at an
234
absorbance of 595 nm using a micro-plate reader. To obtain actual protein levels, datasets
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were standardized using bovine serum albumin (Sigma-Aldrich, USA) concentrations (Fteita
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et al., 2014).
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2.10. TEM imaging
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After the incubation of the OD-adjusted P. aurantiaca strains with DPD and its analogs for 24
240
hours, each suspension was centrifuged at 10 000 g for 5 minutes. During centrifugation, the
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centrifuge internal temperature was adjusted to 37°C simulating the anaerobic atmosphere.
242
Once centrifugation was done, the supernatants were discarded and the bacterial pellets were
243
immediately collected and fixed with 5% glutaraldehyde in 0.16 mol 1-1 s-collidine–HCl
244
buffer, pH 7.4 and sent to TEM facility for imaging, as described previously (Musrati et al.,
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2014).
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The TEM grid sections were visualized by a JEOL JEM-1400 Plus transmission electron
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microscope (JEOL, Tokyo, Japan) with an operation power of 80 kV acceleration voltage.
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section; the first magnification (x2,500) was to evaluate the section validity to be included in
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the study (bacterial contamination or artifacts), the second magnification (x12,000) was to
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visualize the fimbria formation changes among different bacterial cells, while the third
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magnification (x25,000) was mainly to run fimbria thickness calculations for obtaining
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quantitative data of the three P. aurantiaca strains under the effect of graded concentrations of
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DPD and its analogs. Bacterial fimbriae thickness was measured from five different sites of
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each bacterial cell using the software ImageJ 1.48v (Wayne Rasband National Institute of
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Health, USA) (Musrati et al., 2014).
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2.11. Statistical analysis
The data distribution from each experiment was checked for normality using the
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Kolmogorov-Smirnov and Shapiro-Wilk standard normality tests. Since all data were
261
normally distributed, multiple comparisons between the estradiol concentration groups were
262
performed by using the single factor “one-way ANOVA” with a two-tailed probability level
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of statistical significance. When applicable, each experiment was performed at least twice
264
independently in five replicates. P-value below 0.05 was accepted as statistically significant.
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3. RESULTS 3.1.
Planktonic growth and coaggregation
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Elevated estradiol concentrations enhanced significantly not only the planktonic growth of the
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clinical P. aurantiaca strains (AHN 37505 and AHN 37552) (Fig. 1a) but also the
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coaggregation capability of AHN 37505 strain with F. nucleatum (Fig. 1b).
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3.2.
DPPIV activity of WCEs
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DPPIV activities of the three strains of P. aurantiaca showed a transitional increase over
281
time. The strain AHN 37505 had the highest level of enzyme activity followed by the type
282
strain CCUG 57723 and the other clinical strain (AHN 37552). A significant inhibition was
283
observed in all three strains when 1 mM of DFP was added in each bacterial WCE (Fig. 2a).
284
Based on CFU values, the 15-minute exposure of P. aurantiaca cells to DFP had no
285
significant effect on their viability except for one strain, AHN 37505, where a significant
286
decrease in the number of colonies was observed (Fig. 2b). In the zymogram, clear bands of
287
DPPIV activity corresponded to the crude WCE of the three P. aurantiaca strains between the
288
molecular weight markers of 102-79 kDa. In the wells containing the same WCE
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preincubated with 1 mM of DFP, no visible bands of DPPIV activity were observed in any of
290
the tested strains (Fig. 2c).
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3.3.
Inhibition of DPPIV activity by DFP inhibited the biofilm formation
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Biofilm mass of all P. aurantiaca strains significantly increased with elevated estradiol
294
concentrations of different peaks of protein production. This estradiol-regulated enhancement
295
was totally depleted when the strains were incubated with 1 mM DFP; no significant change
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was seen in biofilm formation between different estradiol concentrations, except for P.
297
aurantiaca AHN 37505, which exhibited a slight increase in its biofilm mass (Fig. 3).
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3.4.
Regulation of DPPIV enzyme activity by DPD and its analogs
300
Fluorometric enzyme activity measurements showed significant differences in the DPPIV
301
activity among the three P. aurantiaca strains incubated with graded concentrations of DPD
302
and analogs in a strain- and dose-dependent response (Fig. 4). For all strains, the peak of
303
inhibition in the enzyme activity was reached among DPD and its analogs at the
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concentrations of 100 nM and 1 µM.
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3.5.
307
analogs
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Biofilm formation (Fig. 5) and coaggregation capabilities (Fig. 6) of the three strains of P.
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aurantiaca were slightly but significantly decreased when exposed to DPD, butyl DPD, and
310
isobutyl DPD but not to ethyl DPD (P<0.05). Fimbriae thickness showed a transient and
311
significant increase with increasing the DPD and its analogs concentrations followed by a
312
significant decrease in the two highest concentrations (1 µM and 10 µM) in all strains (Fig. 7
313
and Fig. 8). Although not considered quantitatively, TEM micrographs revealed clear
314
changes in the number and color intensity of intracellular inclusions and granules of all
315
strains with a clear visibility in the type strain P. aurantiaca CCUG 57723 (Fig. 8a, b, and c).
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Regulation of biofilm mass, coaggregation, and fimbriae thickness by DPD and its
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4. DISCUSSION
317
In these in vitro experiments, biofilm formation of P. aurantiaca (which was treated with
318
estradiol) was found to be dependent on DPPIV activity. To our knowledge, the present study
319
is the first to demonstrate such a modulatory effect on this novel species within the P.
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intermedia group. Moreover, we present evidence that a disruption in QS signaling with C1-
321
alkyl analogs of DPD may modify the biofilm-related virulence factors of P. aurantiaca.
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DPPIV activity of the P. aurantiaca strains was confirmed by zymography where clear bands of activities corresponded to a known molecular weight on the standard ladder.
324
The observed enzyme activity bands were considered to be solely obtained from DPPIV with
325
a high specificity based on previous reports where DFP totally inhibited the DPPIV activity as
326
seen in the disappearance of the corresponding bands (Koreeda et al., 2001; Shibata et al.,
327
2003). The 100% inhibition rate of DPPIV with no cytotoxic effect on the P. aurantiaca cells
328
was performed using the gold standard method, i.e., counting the bacterial growth as CFUs.
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Only one clinical strain of P. aurantiaca (AHN 37505) showed a significant decrease in its
330
proliferation when incubated with DFP.
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Several studies have addressed the impact of eukaryotic DPPIV in various biological,
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physiological, and pathological interactions, including the activation of immune cells, cancer
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pathogenesis, and metabolic disorders (Augustyns et al., 1999; Beckenkamp et al., 2016;
334
Bellé et al., 2011). However, limited knowledge exists about the involvement of bacterial
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DPPIV in such systemic interactions in general, and in the oral cavity in particular. Among
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oral Gram-negative anaerobes, P. gingivalis and P. intermedia are known as significant
337
producers of DPPIV (Gazi et al., 1997; Banbula et al., 2000; Clais et al., 2014; Fteita et al.,
338
2015). Biofilm formation of P. gingivalis, a major pathogen in periodontal disease
339
pathogenesis, was recently found to increase the bacterial DPPIV activity (Clais et al., 2014).
340
Being a novel species isolated from the oral cavity and with scarce data available in the
341
literature, P. aurantiaca gained our interest to investigate its potential pathogenicity under the
342
effect of estradiol. In our previous studies dealing with members of the P. intermedia group, it
343
was demonstrated that estradiol regulates various virulence characteristics potentially being
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involved in pregnancy-associated gingivitis (Fteita et al., 2014; 2015). In our previous study,
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346
of the other P. intermedia group organisms) in the presence and absence of estradiol (Fteita et
347
al, 2015). The present results confirm our previous finding in which P. aurantiaca exhibited a
348
significant DPPIV enzyme activity, which seems to play a key role in its estradiol-regulated
349
capability to form biofilms. The latter observation was confirmed when the DPPIV inhibition
350
by DFP (DPPIV inhibitor) abolished the significant differences between biofilm mass of P.
351
aurantiaca grown in different estradiol concentrations.
In addition to host-derived molecules, such as estradiol, bacteria are exposed to
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bacterially-derived QS molecules. QS is a crucial process where bacteria can communicate
354
with each other and respond to autoinducers in order to modify their gene expression and
355
virulence properties (Fuqua et al., 1994). P. intermedia is one of the periodontal pathogens
356
tested for their ability to produce extracellular autoinducer-like activities (Frias et al., 2001).
357
Although complementation experiments were not achievable due to limited gene sequencing
358
data in the database, Frias et al. (2001) was able to demonstrate the production of AI-2 by
359
several P. intermedia strains in a glucose- and growth phase-independent manners. Disruption
360
of QS plays a key role in modifying bacterial motility, adhesion, and biofilm-related
361
virulence. Development of anti-QS agents through a pro-drug approach, targeting chemical
362
structure alterations of analogs and bacterial membrane permeability, resulted in the
363
production of next-generation non-bactericidal antimicrobial agents, the ester derivatives of
364
DPD analogs (Kamaraju et al., 2011; Guo et al., 2012). Up to date, data on the modulatory
365
effect of DPD and its analogs on bacterial cells are scarce; nevertheless, Guo et al. (2012)
366
found that DPD and isobutyl DPD inhibit QS in Escherichia coli. In the present study, it is
367
shown for the first time that DPD and its analogs have a dose-dependent inhibitory effect on
368
fimbria thickness and a slight but significant inhibition of DPPIV enzyme activity,
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coaggregation, and biofilm formation.
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ACCEPTED MANUSCRIPT In conclusion, the results of our in vitro experiments indicate that estradiol regulates
370
the proliferation and biofilm formation of P. aurantiaca and these events significantly depend
372
on DPPIV activity. Biofilm and DPPIV enzyme activity-related virulence of P. aurantiaca
373
may be inhibited, at least partly, by the disruption of QS signaling with C1-alkyl analogs of
374
DPD.
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Funding
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This work was supported by the Finnish Doctoral Program in Oral Sciences (FINDOS) (D.F.),
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Turku University Foundation (D.F. and U.K.G.), and Finnish Dental Society Apollonia
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(U.K.G.). XCM thanks China Scholarship Council (No.201406140119) for financial support.
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Conflict of interests
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The authors declare no conflict of interest.
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Sintim HO, Smith JA, Wang J, Nakayama S, Yan L (2010) Paradigm shift in discovering next-generation anti-infective agents: targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules. Future Med Chem 2:10051035.
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ACCEPTED MANUSCRIPT Sintim HO, Gürsoy UK (2016) Biofilms as "Connectors" for oral and systems medicine: A new opportunity for biomarkers, molecular targets, and bacterial eradication. OMICS 20:3-11. Tateda K, Ishii Y, Horikawa M, Matsumoto T, Miyairi S, Pechere JC, et al. (2003) The
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Pseudomonas aeruginosa autoinducer N-3-oxododecanoyl homoserine lactone
accelerates apoptosis in macrophages and neutrophils. Infect Immun 71: 5785-5793. Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR (2005) Making ‘sense’ of
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metabolism: autoinducer-2, LuxS and pathogenic bacteria. Nat Rev Microbiol 3: 383-
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ACCEPTED MANUSCRIPT Figure legends Fig. 1a) Planktonic growth properties of three Prevotella aurantiaca strains within gradually increased estradiol concentrations (0, 30, 90, and 120 nmol L-1). Data represent the colony forming unit (CFU) mL-1. 1b) Spectrophotometrically evaluated coaggregation between
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Fusobacterium nucleatum ATCC 25586 and the P. aurantiaca strains in different estradiol concentrations (0, 30, 90, and 120 nmol L-1). Data represent the optical density (OD) readings of 0.7 at 660 nm. Asterisks indicate significant differences with the control (*P < 0.05,
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Fig. 2) DPPIV activity, zymography, and proliferation rate of three P. aurantiaca strains
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without and with DPPIV inhibition by di-isopropylfluorophosphate (DFP-/+). a) For DPPIV activity, data are presented as fluorescence excitation and emission intensity values, and b) for proliferation measurements, data are represented as colony forming unit (CFU) mL-1. c) For zymography, the enzyme assay was performed twice, only one representative gel is
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presented. Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01). Fig. 3) Protein levels representing the biofilm mass of the P. aurantiaca strains in different estradiol concentrations (0, 30, 90, and 120 nmol L-1) without and with dipeptidyl peptidase
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IV (DPPIV) inhibition by di-isopropylfluorophosphate (DFP-/+). Data are presented as mg mL-1. Solid bars represent the groups with no DFP, while the dashed bars represent the groups
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with DFP. Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01). Fig. 4) DPPIV enzyme activity of the P. aurantiaca strains in concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM). Data are presented as excitation and emition OD values. Asterisks indicate significant differences with the control (*P<0.05, **P<0.01, and ***P<0.001).
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ACCEPTED MANUSCRIPT Fig. 5) Protein levels representing the biofilm mass of the P. aurantiaca strains in different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM). Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01). Fig. 6) Coaggregation between Fusobacterium nucleatum ATCC 25586 and the P. aurantiaca
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strains in different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM). Data represent the optical density (OD) readings of 0.7 at 660 nm. Asterisks indicate significant differences with the control (*P < 0.05, **P<0.01).
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Fig. 7) Fimbriae thickness measurements of the three P. aurantiaca strains incubated in
different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100
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nM, 1 µM, and 10 µM). Data are presented in nanometers scale (nm). Measurements were taken from images of 25,000x magnification. Asterisks indicate significant differences with the control (*P<0.05, **P<0.01, and ***P<0.001).
Fig. 8a, b, and c) TEM micrographs reveal the difference in Fimbriae thickness and
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intracellular changes in bacterial cell morphology between the control and different concentrations of DPD, ethyl DPD, butyl DPD, and isobutyl DPD (0, 10 nM, 100 nM, 1 µM, and 10 µM) of the three study strains: (a) P. aurantiaca AHN 37505, (b) P. aurantiaca AHN
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37552, and (c) P. aurantiaca CCUG 57723. Scale bars indicate 200 nm.
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ACCEPTED MANUSCRIPT Highlights
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Quorum sensing (QS) and estradiol effect on P. aurantiaca virulence were evaluated. Biofilm mass and DPPIV enzyme activity were used for the evaluation. Estradiol enhanced the growth, coaggregation, and biofilm mass of P. aurantiaca. QS Signaling disruption may, partly, inhibit virulence of P. aurantiaca.
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1. 2. 3. 4.