Abstract: The present study investigated the influence of synthetic N-acyl homoserine lactones (N-acyl HSLs), signaling compounds for quorum sensing, on the growth and production of proteins in periodontopathic bacteria Porphyromonas gingivalis. N-butyryl HSL, N-hexanoyl HSL, N-heptanoyl HSL, N-octanoyl HSL and N-tetradecanoyl (myristoyl) HSL were used as synthetic N-acyl HSLs (Sigma-Aldrich). N-tetradecanoyl HSL inhibited the growth of all P. gingivalis strains used in this study in a dose-dependent manner. When we compared the SDS-PAGE profiles of sonicated samples of P. gingivalis bacterial cells cultured with/without N-tetradecanoyl HSL, we found that protein production was changing. The growth of P. gingivalis was inhibited by myristoyl coenzyme A (CoA) but did not by lauroyl CoA. These results indicated that P. gingivalis responded to auto-produced N-tetradecanoyl HSL-like molecule and slowed their growth.
Introduction
Recently, dental plaque has characterized as biofilm and the formation is associated with intercellular communication; quorum sensing (QS) (McNab et al., 2003). QS is widespread among gram negative and gram positive bacteria (Fuqua et al., 1994; 1996; Miller and Bassler, 2001; Whitehead et al., 2001). QS bacteria synthesize and secrete extracellular signaling molecules. When a critical threshold concentration of autoinducer is attained, a signal transduction cascade is triggered, resulting in the density dependent regulation of gene expression and a change in behavior of the organism for facilitating environmental adaptation (Engebrecht et al., 1983; Fuqua and Greenberg, 1998). The predominant signaling molecules of gram-positive bacteria are peptides, while in gram-negative bacteria, different system of QS, which use different type of signaling molecules, has so far been described (Bassler, 1999). Vibrio harveyi is free-living gram-negative marine bacterium that possesses some different systems (Bassler et al., 1994). The autoinducer of gram-negative bacteria has been identified as an N-acyl homoserine lactone (N-acyl HSL) (Cao and Meighen, 1989; Bassler et al., 1993; Shaw et al., 1997), whereas the structure of the autoinducer for one of other QS systems has been called AI-2. In the case of V. harveyi the hydroxybutyryl homoserine lactone is the autoinducer. Frias et al. (2001) suggested that the gram negative periodontal organisms do not possess N-acyl HSL-dependent signaling systems. However, several of these organisms such as Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans secrete signals functionally related to AI-2 (Fong et al., 1993, 2001; Chung et al., 2001; Burgess et al., 2002).
P. gingivalis has frequently been isolated from periodontal lesions and shown to be related to the onset and progression of periodontal disease (Slots et al., 1986; Slots and Listgarten, 1988; Mayrand and Holt, 1988; Van Winklehoff et al., 1988). Although previous researchers were unable to obtain any evidence to suggest that the gram negative anaerobe P. gingivalis employs N-acyl HSL-mediated QS, they have demonstrated that this periodontopathic bacteria produces a diffusible signal molecule capable of activating the V. harveyi AI-2 biosensor (Chung et al., 2001; Frias et al., 2001; Burgess et al., 2002). The objective of the present study was to investigate whether P. gingivalis responds to N-acyl HSLs. Therefore we investigated the influence of synthetic N-acyl HSLs on the growth and production of proteins in this organism.
Materials and Methods
Bacterial Strains and Culture Conditions
P. gingivalis ATCC 33277, ATCC 53977, Su63 and W50, Prevotella
intermedia ATCC 25611, Prevotella loescheii ATCC 15930, Fusobacterium
nucleatum ATCC 25586 and A. actinomicetemcomitans Y4 were used in
this study. These strains were maintained anaerobically on blood agar plates
containing trypticase soy agar (Becton Dickinson Microbiology System, Cockeysville,
MD, USA) supplemented with 10% defibrinated horse blood, hemin (5 μg mL-1;
Sigma Chemical Co., St. Louis, MO, USA) and menadione (0.5 μg mL-1;
Wako Pure Chemical Industries, Osaka, Japan). The bacterial strains were cultured
anaerobically in trypticase soy broth (Becton Dickinson Microbiology System)
supplemented with hemin and menadione at 37.
Effect of Synthetic N-acyl HSLs or Acyl Coenzyme A on the Growth
N-butyryl-DL-homoserine lactone (HSL), N-hexanoyl HSL, N-heptanoyl
HSL, N-octanoyl HSL and N-tetradecanoyl (myristoyl) HSL were used
as synthetic N-acyl HSLs (Sigma-Aldrich Co., Steinheim, Germany). Each
N-acyl HSL was added to the culture broth at various concentrations
(100 , 10 and 1 μM) and its influence on the growth of tested bacteria
evaluated. Growth was monitored by optical density (OD) at 660 nm. In order
to confirm survival for 48 h, 100 μL aliquots were taken from cultures
with N-acyl HSL showing no bacterial growth, inoculated onto blood agar
plates and cultured for one week under anaerobic conditions.
In order to study the effect of acyl coenzyme A (acyl CoA) on P. gingivalis autoinducer synthesis, P. gingivalis ATCC 33277 cells were cultured in the broth containing myristoyl CoA or lauroyl CoA (100 and 10 μM; Doosan Serdary Research Laboratories, Toronto, Canada). Growth was monitored by OD at 660 nm.
SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis
P. gingivalis ATCC 33277 and ATCC 53977 were cultured with/without
N-tetradecanoyl HSL for 24 h. Cells were harvested by centrifugation.
Harvested cells were suspended in distilled water and disrupted by sonication.
The protein content of sonicated sample was adjusted to 1 mg protein per ml.
The adjusted samples were suspended in SDS-PAGE loading buffer (Laemmli, 1970)
and boiled for 10 min. The samples were loaded on the gel. SDS-PAGE was done
in 10 to 20% gradient micro slab gels (Daiichi Pure Chemical Co., Tokyo, Japan)
and stained with Coomassie brilliant blue (CBB; Amersham Biosciences AB, Uppsala,
Sweden).
Results
Effects of Synthetic N-acyl HSLs on the Growth
N-tetradecanoyl HSL completely inhibited the growth of all P.
gingivalis strains tested in this study at 100 μM (Table
1). Growth inhibition of this N-acyl HSL was dose-dependent (Fig.
1). P. gingivalis cells from cultures showing no growth formed many
colonies (data not shown), indicating that this microorganism survived at least
48 h in the broth containing N-tetradecanoyl HSL. Also N-tetradecanoyl
HSL slightly inhibited the growth of P. intermedia and F. nucleatum
at 100 μM (Table 3). In this study, no synthetic N-acyl
HSL affected the growth of any other oral bacteria tested.
Effects of Acyl CoA on the P. Gingivalis Growth
Myristoyl CoA inhibited the growth of P. gingivalis in a dose- dependent
manner (Table 2). However lauroyl CoA showed no inhibitory
effect on the growth of P. gingivalis.
Effect of N-tetradecanoyl HSL on the Protein Expression
When we compared the SDS-PAGE profiles of sonicated samples of P. gingivalis
bacterial cells cultured with or without N-tetradecanoyl HSL. The effects
of N-tetradecanoyl HSL on the protein expression of P. gingivalis
ATCC 33277 and ATCC 53977 are shown in Fig. 2. We found
that SDS-PAGE profile of P. gingivalis ATCC 33277 obviously changed by
addition of N-tetradecanoyl HSL. In ATCC 53977, some protein bands disappeared
by addition of this synthetic homoserine lactone. We confirmed that the alterations
of the protein profile were not due to contamination by gram staining and culture
as black-pigmented gram-negative coccobacillus.
| Table 1: | Effects of N-acyl HSLs on the growth of P. gingivalis strains |
| Growth was monitored by Optical Density (OD) at 660 nm, ++: 0.3≤OD660, +: 0.15≤OD660 <0.3, ±: 0.08≤OD660<0.15, -: OD660<0.08 | |
| Table 2: | Effects of acyl CoA on the growth of P. gingivalis ATCC 33277 |
| Growth was monitored by Optical Density (OD) at 660 nm, ++: 0.3≤OD660, +: 0.15≤OD660<0.3, ±: 0.08≤OD660<0.15, -: OD660<0.08 | |
| Table 3: | Effects of N-acyl HSL on the growth of oral bacteria |
| Growth was monitored by Optical Density (OD) at 660 nm, ++: 0.3≤OD660, +: 0.15≤OD660<0.3, ±: 0.08≤OD660<0.15, -: OD660<0.08 | |
| Fig. 1: | Inhibitory effect of N-tetradecanoly HSL on the growth of P. gingivalis ATCC 33277 |
| Fig. 2: | SDS-PAGE profile of P. gingivalis sonicated samples (CBB staining), a) ATCC 33277, b) ATCC 53977. Lanes, 1 Sonicated sample of P. gingivalis intact cells, 2 Sonicated sample of P. gingivalis cells cultured with 10 μM N-tetradecanoly HSL, 3 Sonicated sample of P. gingivalis cells cultured with 1 μM N-tetradecanoly HSL |
Discussion
Since bacteria within the biofilms reach a high density, it has been suggested that QS might play a key role in bacterium-bacterium communication and, therefore, in the formation of biofilm (Schripsema et al., 1996; Fong et al., 2001). QS mechanisms control the production of virulence factors in some species of bacteria (Passador et al., 1993; De Kievit and Iglewski, 2000; Winzer and Williams, 2001). P. gingivalis has been long considered one of the main periodontopathic bacteria, playing an important role in bone and tissue destruction (Holt et al., 1999). It is absent in health and during disease reaches an important portion of the total population and has the capability of producing a large number of virulence factors. In the periodontal pocket, P. gingivalis is found predominantly as a component of complex biofilm containing multiple bacterial species. To facilitate adaptation to life within the oral cavity, P. gingivalis must be capable of sensing and responding to prevailing environmental conditions, including variations in temperature, oxygen tension, pH, nutrient availability and the presence of other cells (Xie et al., 1997; Lamont and Jenkinson, 1998).
N-acyl HSL dependent QS systems exist in many gram negative bacteria (Fuqua et al., 1994; 1996; Miller and Bassler, 2001; Whitehead et al., 2001), however several investigators (Chung et al., 2001; Frias et al., 2001; Burgess et al., 2002) suggested that periodontal bacteria including P. gingivalis do not possess N-acyl HSL dependent signaling circuits. In their reports, those bacteria were tested for production of extracellular autoinducer-like activities that stimulates the expression the genes in biosensors of V. harveyi or Chromobacterium violaceum. It suggests that P. gingivalis does not employ N-acyl HSLs which are able to stimulates the biosensors. The objective of the present study was to investigate whether P. gingivalis possesses N-acyl HSL dependent QS system by the assay using synthetic N-acyl HSLs. N-tetradecanoyl HSL inhibited the growth and affected on protein production in P. gingivalis. The growth of P. gingivalis was inhibited by myristoyl CoA but did not by lauroyl CoA, suggesting that P. gingivalis responds to auto-produced N-tetradecanoyl (myristoyl) HSL-like molecule(s) and slows their growth.
The growth of some strains of Rhizobium leguminosarum bv. viciae is inhibited by N-(3-hydroxy-7-cis tetradecanoyl) HSL, which was previously known as the small bacteriocin (Van Brussel et al., 1985; Schripsema et al., 1996; Thorne and Williams, 1999; Wilkinson et al., 2002). The cinRI locus is at the top of a regulatory network QS loci in R. leguminosarum bv. viciae (Lithgow et al., 2000). CinI, LuxI homologue, produces the N-(3-hydroxy-7-cis tetradecanoyl) HSL and CinR is a LuxR-type regulator. CerI, which is also LuxI homologue, in Rhodobacter sphaeroides synthesizes N-(7-cis-tetradecanoyl) HSL (Puskus et al., 1997). Laue et al. (2000) demonstrated that Pseudomonas fluorescens F113 makes at least three different N-acyl HSLs including N-(3-hydroxy-7-cis tetradecanoyl) HSL and identified a gene hdtS which does not belong the LuxI or LuxM family of N-acyl HSL synthases. Interrogation of the P. gingivalis W83 genome sequence database (www.tigr.org) for homologues of N-acyl HSL synthases belonging to either the LuxI or LuxM family failed to identify any candidates. However, one open reading frame (ORF) with significant homology with HdtS amino acid sequence was identified (TIGR locus: PG1249, 1-acyl-sn-glycerol-3-phosphate acetyltransferase, putative). The ORF showed 25% identity and 48% amino acid similarity with the HdtS over 201 amino acids. The ORF possesses two motifs which are conserved in the lysophosphatidic acid acyltransferases (West et al., 1993; Shih et al., 1999). It is possible that the ORF is an acyltransferase which transfers acyl-chains onto a substrate to generate some N-acyl HSLs. Further study is required to define its function for N-acyl HSL formation. N-octanoyl HSL showed weak inhibitory effect on growth of P. gingivalis (Table 1). Wilkinson et al. (2002) suggested that the growth inhibition of N-(3-hydroxy-7-cis tetradecanoyl) HSL in R. leguminosarum required the presence of other N-acyl HSLs such as N-octanoyl HSL. In the case of P. gingivalis, N-octanoyl HSL may be associated with the growth inhibition of N-acyl HSLs.
The present study suggests that P. gingivalis produces N-tetradecanoyl HSL-like signaling molecule and responds to this N-acyl HSL as if there had been an increase in cell density and changed protein production to become more fit for habitation in the worse environment.
Acknowledgements
This study was supported by High-Tech Research Center Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2001-2005, by Oral Health Science Center Grant 5A04 from Tokyo Dental College and by Grant-in -Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (17592165).