Journal of Bioscience and Bioengineering VOL. 116 No. 3, 313e318, 2013 www.elsevier.com/locate/jbiosc LuxS affects biofilm maturation and detachment of the periodontopathogenic bacterium Eikenella corrodens Mohammad Minnatul Karim,1, 3 Tatsunori Hisamoto,1 Tetsuro Matsunaga,1, x Yoko Asahi,2 Yuichiro Noiri,2 Shigeyuki Ebisu,2 Akio Kato,1 and Hiroyuki Azakami1, * Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan,1 Department of Restorative Dentistry and Endodontology, Osaka University, 1-8 Yamada-Oka, Suita 560-0871, Japan,2 and Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia 7003, Bangladesh3 Received 8 February 2013; accepted 19 March 2013 Available online 30 April 2013 Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of DluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110e117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Autoinducer; Quorum sensing; Biofilm; Eikenella corrodens; Periodontal disease] Biofilms are sessile, surface-attached communities of microorganisms (1,2). Microorganisms undergo profound morphological changes during the transition from planktonic organisms to cells that constitute complex, surface-attached communities. These changes may occur in response to a variety of environmental signals and are reflected in the new phenotypic characteristics of the biofilm bacteria (3,4). Many bacterial infections are characterized by important factors such as formation of sessile communities and the inherent resistance of these communities to antimicrobial agents (5). Biofilm formation is a complex developmental process requiring a series of discrete and well-organized steps (6). The early stage in biofilm formation involves the attachment of active cells to a solid surface, followed by immobilization on that surface, while the late-stage involves cell adhesion, cell-to-cell interactions, microcolony formation, and formation of a multilayered architecture of the mature biofilm (7,8). * Corresponding author. Tel.: þ81 83 933 5854; fax: þ81 83 933 5820. E-mail address: azakami@yamaguchi-u.ac.jp (H. Azakami). x Present address: Department of Microbiology, Kumamoto University, Kumamoto 860-8556, Japan. Abbreviations: AI, autoinducer; CLSM, confocal laser scanning microscopy; CRM, confocal reflection microscopy; DPD, 4,5-dihydroxy-2,3-pentanedione; HA, hydroxyapatite; MRD, modified Robbins device; QS, quorum sensing; SEM, scanning electron microscopy; TLC, thin layer chromatography; TSB, tryptic soy broth. Human oral bacteria are highly interactive organisms that exist within multi-species dental plaque biofilms (9). Dental plaque is a complex and dynamic microbial community that forms as a biofilm on the surfaces of teeth and oral tissues. It is composed of over 700 species of bacteria (10,11) and is the prime etiological agent of 3 common human oral diseases: dental caries, gingivitis, and periodontal disease (9,12,13). For inter-species communication to occur effectively within dental plaque biofilms, cellecell association and cellecell signaling are required (14,15). Quorum sensing (QS) is a widespread system, used by bacteria for cell-to-cell communication, which regulates expression of multiple genes in a cell density-dependent manner (16,17). QS has been shown to control cell density-dependent behaviors, such as the expression of virulence factors, biofilm formation, and iron acquisition, in a variety of organisms (16,18e20). Thus, QS has been thought to allow bacteria in a biofilm to react concertedly, like a multicellular organism, to changes in the external environment. The unique QS system shared by Gram-positive and Gram-negative bacteria is mediated by autoinducer-2 (AI-2) (21), which is a signaling molecule synthesized by the luxS gene (22). Eikenella corrodens, a facultative anaerobic Gram-negative rod, is predominantly found in sub-gingival plaque samples of patients with advanced periodontitis (23). Previous research has revealed that the monoinfection of germ-free or gnotobiotic rats by E. corrodens can cause severe periodontal disease (24), which 1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.03.013 314 KARIM ET AL. suggests that E. corrodens is a periodontopathogenic bacterium. Additionally, since E. corrodens has been detected in tooth plaque (25), this bacterium may participate in the early stages of biofilm formation in the periodontal pockets. Previously, we reported that E. corrodens includes an ortholog of the luxS gene on its genome, and secretes AI-2 in its culture supernatant. Moreover, the mutation DluxS was found to enhance biofilm formation in E. corrodens (26), which suggests that AI-2 signal plays a role in biofilm formation by E. corrodens. In fact, a number of studies have described the role of AI-2 in biofilm formation. For example, synthesized AI-2 directly stimulates Escherichia coli biofilm formation and controls biofilm architecture by stimulating bacterial motility (27). Additionally, several studies have indicated that AI-2 controls biofilm formation (28,29). Conversely, other studies have shown that the addition of AI-2 failed to restore the biofilm phenotype of the parental strain (30e36), due to either the central metabolic effect of LuxS [S-ribosylhomocysteine lyase (EC 4.4.1.21)] or the difficulty in complementation of AI-2 (37). AI-2 is produced by the LuxS enzyme, which converts S-ribosyl homocysteine into 4, 5-dihydroxy-2,3-pentanedione (DPD), and DPD is subsequently converted into AI-2. This LuxS-catalyzed reaction is also an integral part of the activated methyl cycle, which is an important metabolic pathway that serves to recycle homoserine from S-adenosyl methionine to maintain methionine biosynthesis. Therefore, whether enhanced biofilm formation by the DluxS mutation in E. corrodens is responsible for either effect is not clear. In this study, we investigated the direct effect of AI-2 on biofilm formation. Moreover, we compared the structure and mechanism of biofilm formation between the luxS mutant and wild type. The results of this study suggest that LuxS may facilitate the biofilm maturation and detachment in E. corrodens. MATERIALS AND METHODS Bacterial strains and growth conditions E. corrodens 1073 was provided by S. S. Socransky (Forsyth Dental Center, Boston, MA, USA). E. corrodens cells were statically cultured at 37 C in tryptic soy broth (TSB) containing 2 mg/mL of KNO3 and 5 mg/mL of hemin. The luxS mutant cells (26) of E. corrodens were cultured in a medium supplemented with 50 mg/mL kanamycin. An E. corrodens strain harboring a plasmid pLESluxS, which contains the luxS gene, was cultured in a medium supplemented with 30 mg/mL carbenicillin. Adherence assay for the quantitation of biofilm production by E. corrodens strains (static assay) E. corrodens strains formed a macroscopically visible biofilm that was firmly attached to the wells of tissue culture plates (96-well cell culture plate, nontreated polystyrene, flat-bottom with lid; BD Bioscience, San Jose, CA, USA), and the biofilm production was determined by a previously described assay (38). This assay is used to measure the degree of primary attachment and accumulation of multilayered cell clusters and the subsequent biofilm production on a polystyrene surface. Briefly, following growth of the biofilm in the TSB medium for 36 h at 37 C, the plates were gently washed 4 times with distilled water and the adherent bacterial cells were stained with crystal violet. The stain was then dissolved by ethanol and optical density was measured at 595 nm (OD595) using a spectrophotometer (Tecan GENios Microplate Reader, Männedorf, Switzerland). AI-2 purification from culture supernatant Purification of AI-2 from culture supernatant of E. corrodens was performed as described previously (39). For AI2 extraction, the log-phase culture of E. corrodens 1073 was inoculated in fresh TSB medium and then statically cultured for 15 h at 37 C. After that, the bacterial cells were removed by centrifugation (12,000 g, 5 min, 4 C), and the supernatant was filtered by performing decompression filtration using filter papers with 0.22 mm pore size (Membrane Filters, ADVANTEC, Japan). AI-2 was extracted 3 times by mixing the filtrated supernatant with ethyl acetate at a ratio of 4:3 (supernatant:ethyl acetate). The obtained ethyl acetate phase was dried in a rotary evaporator at 40 C. The resulting sample was dissolved with a small amount of ethyl acetate and then stored at 30 C. A solution of AI-2 in ethyl acetate was applied to a silica gel thin layer chromatography (TLC) plate (TLC Silica gel 60 RP-18F, Merk Ltd. Germany) and developed with 60% methanol. Individual bands that appeared were observed under UV at 254 nm, and the section corresponding with each band was cut out. The compounds obtained from each silica gel section for a specific band were eluted using ethyl acetate, filtered, and dried under a continuous flow of N2 gas. The resulting samples J. BIOSCI. BIOENG., were dissolved in dimethyl sulfoxide (DMSO), and AI-2 activity was confirmed by performing the AI-2 assay using Vibrio harveyi BB170 as a sensor strain (40). Biofilm formation of the E. corrodens in flow cell biofilm model using modified Robbins device (MRD) The E. corrodens biofilm formation in flow cell model using MRD and hydroxyapatite (HA) disks was used as described previously (41) with some modifications. Three separate MRDs and HA disks (10 disks/ MRD), processed with saliva for 8 h, were prepared for each strain. E. corrodens biofilms were formed by anaerobic perfusion of culture medium containing bacterial cells for 7 days. Culture medium was perfused through 3 separate MRDs for a 7-day period. HA disks were removed aseptically from each MRD. HA disks were observed by scanning electron microscopy (SEM) and the number of viable bacterial cells was counted. HA disks were washed and ultrasonicated in 1.0 mL distilled water at 4 C for 30 min to remove the E. corrodens biofilms. The collected bacteria were diluted and spread over sheep-blood agar plates. After culturing at 37 C for 2 days, CFUs were counted. For SEM examination, HA disks were first washed 3 times with 0.1 M cacodylate buffer and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 30 min at room temperature, and then subsequently washed 3 times in 0.1 M cacodylate buffer. After fixation, all samples were dehydrated in a graded series of aqueous ethanol, dried, ion coated with platinum, and examined by SEM (JSM-6390LV; JEOL, Japan) as previously described (42). Three-dimensional observations of E. corrodens flow cell biofilms To observe the E. corrodens biofilms three-dimensionally, flow cell biofilm samples on celluloid disks (Celltight; Sumitomo Bakelite Co., Tokyo, Japan) were prepared using an MRD as described above (43). Biofilm samples were stained with the Live/Dead BacLight bacterial viability kit L7007 (Invitrogen, Carlsbad, CA, USA) for 15 min at room temperature. The samples were observed by confocal laser scanning microscopy (CLSM; LSM 510; Carl Zeiss, Oberkochen, Germany). Images were processed by image analysis software (Imaris; Bitplane AG, Zurich, Switzerland). Observation of biofilm by confocal reflection microscopy (CRM) For biofilm flow cultures, biofilms were maintained using the flow-reactor method, as previously described (44). Briefly, stationary-phase cultures were diluted to an OD660 of 0.1 with TSB media, and 300 mL of the cell suspension was injected into a flow cell (Stovall Life Science, Greensboro, NC, USA) with channel dimensions of 1  4  40 mm (H  W  L). After static incubation for 1 h, a flow of 1/10 TSB medium was initiated at a rate of 0.2 mL/min. Biofilms were grown at 37 C for 48 h. A Carl Zeiss LSM 510 META (Carl Zeiss) was used to acquire confocal micrographs. For the CRM observation, cells were illuminated with a 514-nm argon laser, and the reflected light was collected through a 505e530 nm band-pass filter to avoid the influence of autofluorescence. An NT 80/20 half mirror (Carl Zeiss) was used as a beam splitter. Confocal images were analyzed using the Carl Zeiss LSM 5 PASCAL Software (Version 3.5, Carl Zeiss). The thickness of each image stack was 0.8 mm. For bio-volume quantification, confocal images were analyzed using the COMSTAT computer program, which functions as a script in MATLAB software (Mathworks, Natick, MA, USA). Statistical analysis The results of each series of experiments are shown as mean (standard deviation). The significance of intergroup differences was analyzed using Student’s t-test (unpaired t test). RESULTS AND DISCUSSION AI-2 enhances biofilm formation directly in E. corrodens Previously, we reported that the biofilm-forming efficiency of the DluxS mutant was approximately 1.3-fold greater than that of wild type and suggested that AI-2 signal plays a role in the biofilm formation by E. corrodens (26). Although the LuxS enzyme catalyzes the reaction that leads to the synthesis of the AI-2 precursor, DPD, this reaction is also a part of the activated methyl cycle. Therefore, whether the enhancement of biofilm formation observed in the DluxS mutant was due to the direct effect of AI-2 deletion or a defect of the C1 metabolism remains unclear. To evaluate this, it was necessary to add purified AI-2 into the culture of E. corrodens, and compare the biofilm-forming efficiency with and without AI-2. Partially purified AI-2 was collected from the culture supernatant of E. corrodens, grown until mid-exponential phase, when the amount of secreted AI-2 reached a maximum level (39). The high-purity AI-2 was obtained by extraction from the culture supernatant with ethyl acetate and separation by TLC (39). The band exhibiting AI-2 activity on the TLC plates was extracted (39) and used as partially purified AI-2 in the subsequent experiment. As shown in Fig. 1, the addition of 0.1 mg/mL of the partially purified AI-2 resulted in enhanced biofilm formation in the DluxS VOL. 116, 2013 LuxS AFFECTS BIOFILM MATURATION IN E. CORRODENS * * 315 * 5.6 5.4 2.5 (Log of CFU) Biofilm formation (OD at 595 nm) 5.2 2 1.5 5 4.8 4.6 4.4 4.2 1 E. corrodens E. corrodens luxS E. corrodens luxS (luxS) FIG. 2. Enumeration of viable bacteria in biofilm. Counting was performed in at least 9 wells; log of mean values and standard deviations have been indicated. Asterisk indicates significant difference, P < 0.05. DluxS, DluxS mutant strain of E. corrodens 1073; DluxS (luxS), DluxS mutant strain complemented by the luxS gene. 0.5 0 0 µg 0.1 µg/mL E. corrodens 1073 E. corrodens 1073 luxS FIG. 1. Effect of purified AI-2 on biofilm formation by E. corrodens. Purified AI-2 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 0.1 mg/mL. DMSO was used as a negative control. Biofilm formation with or without the purified AI-2 was assayed using the static method. Assays were performed in at least eight wells; mean values and standard deviations have been indicated. Asterisk indicates significant difference, P < 0.05. mutant strain (w1.3-fold), while the addition of AI-2 did not affect biofilm formation in the wild-type strain. Previously, we have reported that the DluxS mutant strain can produce no AI-2 (26) and that the purified AI-2 has AI-2 activity using V. harveyi BB170 (39). Thus, these results suggest that the addition of AI-2 enhanced biofilm formation directly in E. corrodens. However, this result contradicts our previous result that the biofilm-forming efficiency of the DluxS mutant was greater than that of the wild type. From our previous results, we assumed that AI-2 reduces biofilm formation in E. corrodens. In the current study, the biofilm of wild type appeared to be detached from the bottoms of the microtiter plate wells, whereas the biofilm of the luxS mutant remained firmly attached. This observed detachment may reflect a problem with the static biofilm assay, which requires multiple cycles of solution exchange and washing. Therefore, results of the static assay may not reflect actual biofilm-forming efficiency. DluxS mutation decreases bacterial viability in E. corrodens biofilm The static assay using microtiter plate is widely used biofilm assay. However, it is difficult to analyze the biofilm formation over time owing to batch cultivation. To investigate the actual biofilm, we observed continuous biofilm formation in a flow cell system. The biofilms were formed on HA disks fixed in MRD using flow cell system. Following biofilm formation, the bacterial cells were removed by sonication. The number of viable bacteria in biofilm was determined by calculating CFU. As shown in Fig. 2, total bacterial CFU in the DluxS mutant biofilm was lower than that from wild type, but this difference was restored by the complementation of the luxS gene. These results indicate a reduction in the number of viable bacteria in the DluxS mutant biofilm. As described above, the reaction by LuxS enzyme is also a part of the activated methyl cycle, which plays role to maintain methionine biosynthesis by recycling homoserine from S-adenosyl methionine. It has been reported that luxS mutation affects cellular levels of fermentation products, fatty acids, nucleic acids, and amino acids (45). Prior research has suggested a relationship between luxS mutation and bacterial viability in biofilm. For example, Maria et al. reported that low levels of luxS expression seem to provide an advantage for bacterial survival during infections by the group A streptococci (46). Moreover, Ahmed et al. reported that the level of viability in the luxS mutant biofilms is lower than that in the wild-type biofilm of Streptococcus intermedius (47). Therefore, we hypothesized that decreasing metabolic levels for C1 compounds in the DluxS mutant might lead to a reduction in bacterial viability in the biofilm. AI-2 facilitates biofilm maturation in E. corrodens To compare wild-type and DluxS mutant biofilms in more detail, we observed biofilm formations on HA disks by using MRD by SEM. Results of this analysis revealed that wild-type E. corrodens formed dense biofilms, whereas the DluxS mutant formed sparse biofilms (Fig. 3). Studies of several bacterial species indicate that both the initial adherence stage and the later stages, including biofilm maturation, are affected, when quorum-sensing pathways are inhibited (7,8). Thus, biofilm maturation in the DluxS mutant might be inhibited by the lack of AI-2, and conversely, AI-2 might enhance the maturation of E. corrodens biofilm. Biofilms of both strains formed on celluloid disks by CLSM (Fig. 4) were also observed. Similar to what was observed during the SEM analysis, the wild-type strain was again observed to form a dense biofilm, whereas the luxS mutant formed a sparse biofilm, although the thicknesses of both biofilms were nearly equivalent (30 mm). These results support our hypothesis that AI-2 can enhance the maturation of E. corrodens biofilm. Results of live/dead staining showed that live (green) bacteria dominated the wild-type biofilm, whereas dead (red) bacteria were the dominant component in the luxS mutant biofilm (Fig. 4), which supports our suggestion that viable bacteria decreased in luxS mutant biofilm. AI-2 facilitates biofilm maturation and detachment in E. corrodens The SEM technique requires dehydration of the sample prior to analysis, which leads to major distortions and artifacts in the actual images in the case of wet biological samples. In CLSM observation, fluorescence staining is required after biofilm formation. Therefore, CRM was used to analyze the difference of intact biofilm structures between the 2 strains (Fig. 5). We observed over time that wild-type biofilm masses were increasingly projected outward into the surrounding medium, where they gradually became thinner. This growth projection was not observed in the DluxS mutant biofilm, and the biofilm continued to mature without detachment at 48 h. Thus, the results suggest that in wild-type strain, AI-2 facilitated both the maturation and the detachment of biofilm. 316 KARIM ET AL. J. BIOSCI. BIOENG., FIG. 3. Scanning electron microscopy (SEM) images of biofilm formation formed by E. corrodens 1073 (A) and E. corrodens 1073 DluxS (B). Scale bars are 5 mm (upper) and 20 mm (lower), respectively. A single example from the 5 images is presented. Biofilm development can be divided into 3 distinct stages: attachment of cells to a surface, growth of the cells into a sessile biofilm colony, and detachment of cells from the colony into the surrounding environment (48). The final stage of biofilm development, which is the detachment of cells from the biofilm colony and B A 1 their dispersal into the environment, is an essential stage of the biofilm life cycle that contributes to biological dispersal, bacterial survival, and disease transmission. Like other stages of biofilm development, dispersal can be a complex process that involves numerous environmental signals, signal-transduction pathways, 20 µm 3 2 4 20 µm 1 3 2 4 Thickness : 30 µm 1 2 Live bacteria: Green 3 4 Dead bacteria: Red Confocal position for biofilm FIG. 4. Confocal laser scanning microscopy (CLSM) of biofilm formation on celluloid disks by E. corrodens 1073 (A) and E. corrodens 1073 DluxS (B). Live cells and dead cells are stained green and red, respectively. Panels 1e4 correspond to confocal positions in the biofilm (30, 20, 10, and 0 mm from the surface of disks, respectively). A single example from the 5 images is presented. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) VOL. 116, 2013 LuxS AFFECTS BIOFILM MATURATION IN E. CORRODENS 317 FIG. 5. Confocal reflection microscopy (CRM) of biofilm formation by E. corrodens 1073 (A) and E. corrodens 1073 DluxS (B) at 24 h and 48 h. A field of 140  140 mm (x, y) is shown, as indicated in the panels. A single example from the 5 images is presented. and effectors (49). Relative to this, it has been reported that AI-2 regulates biofilm maturation and dispersal in some bacteria. For example, in Vibrio cholerae, AI-2 mutants form thicker biofilms than wild-type strains on glass coverslips (50), and are also deficient in biofilm detachment (51). 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