The Small Molecule DAM Inhibitor, Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro

Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered and attached to the bottom of the plate when cells were grown in the presence of pyrimidinedione. Scanning electron microscopy analysis demonstrated the absence of an extracellular polysaccharide matrix in pyrimidinedione-grown biofilms compared to control-biofilms. Pyrimidinedione also significantly inhibited MRSA, MSSA, and Staphylococcus epidermidis biofilm growth in vitro. Furthermore, pyrimidinedione does not exhibit eukaryotic cell toxicity. In a microarray analysis, 56 genes were significantly up-regulated and 204 genes were significantly down-regulated. Genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down regulated in pyrimidinedione-grown biofilms. This study demonstrated that the small molecule Dam inhibitor, pyrimidinedione, inhibits pneumococcal biofilm growth in vitro at concentrations that do not inhibit planktonic cell growth and down regulates important metabolic-, virulence-, competence-, and biofilm-related genes. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.


Introduction
Streptococcus pneumoniae (S. pneumoniae) is an important human pathogen. It causes severe and invasive infections, such as pneumonia, septicemia, otitis media, and meningitis, especially in children, the elderly, and immuno-compromised patients [1,2,3]. S. pneumoniae initially colonize the nasopharynx and may persist for months without causing illness, forming specialized structures called biofilms [4,5]. Pneumococci from these biofilms can migrate to other sterile anatomical sites, causing severe biofilm-associated infections such as pneumonia and otitis media [6,7,8]. The planktonic bacteria from these biofilm-associated infections can migrate to other sterile sites, such as the blood stream, causing bacteremia, or to the brain, causing meningitis [9,10,11].
A biofilm is defined as a thin layer of bacteria that adhere to each other and to a living tissue or inert surfaces. These bacteria are surrounded by a self-produced polymeric matrix composed of polysaccharides, proteins, and nucleic acids [12]. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems [13,14]. S. pneumoniae biofilms show increased resistance to common antibiotics, such as penicillin, tetracycline, rifampicin, amoxicillin, erythromycin, clindamycin, levofloxacin, and gentamicin both in vivo and in vitro [15,16,17]. Bacteria within biofilms exhibit altered physiology, metabolism, and gene expression profiles compared to free-floating planktonic cells [18]. Therefore, existing antimicrobial compounds mainly developed to target planktonic bacteria may not be as effective against biofilms. Moreover, the emergence of antibiotic resistant pneumococcal strains necessitates the identification of alternative drug targets and new antimicrobial compounds that could be effective against pneumococcal biofilms. Effective anti-biofilm strategies could inhibit initial bacterial attachment and colonization, interfere with signaling pathways important for biofilm development, or disrupt the biofilm matrix [19,20,21].
Bacterial DNA methyltransferases are generally associated with restriction-modification systems, with the exception of DNA adenine methyltransferase (Dam) and cell cycle-regulated methyltransferase (CcrM) [22]. In bacteria, Dam alters the expression of pathogenic genes involved in several cellular activities, including mismatch repair, initiation of chromosomal replication, DNA segregation, and transposition [23,24]. In bacteria the Dam enzyme catalyzes a methyl group transfer from S-adenosyl-L-methionine (SAM) to the N 6 position of adenine in duplex DNA (Fig 1A). This adenine methylation is unique in bacteria. Therefore, these bacterial enzymes represent excellent antimicrobial target candidates. Moreover, SAM-mediated methylation is an important process in pneumococci, leading to the methylation of DNA and macromolecules, as well as the biosynthesis of quorum sensing (QS) molecules and secondary metabolites, such as polyamine, that play roles in biofilm formation [25,26]. Our previous study showed that 5-azacytidine, a hypo-methylating compound, and sinefungin, a SAM analogue, inhibit S. pneumoniae biofilm growth [27,28]. However, the effect of Dam inhibitor small molecule on pneumococcal biofilm formation has not been studied.
Our results demonstrated that pyrimidinedione inhibited pneumococcal biofilm growth in vitro at concentrations that did not inhibit planktonic cell growth, and it down-regulated the expression of important metabolic-, virulence-, competence-, and biofilm-related genes. Pyrimidinedione is also effective against MSSA, MRSA, and Staphylococcus epidermidis biofilms in vitro, and it is not cytotoxic to eukaryotic cells. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.
Growth curve of S. pneumoniae D39 with pyrimidinedione Cultures of S. pneumoniae D39 were grown in the presence of 1 and 10 μM/ml concentrations of pyrimidinedione in a time course experiment. The cell suspensions were incubated at 37°C in 5% CO 2 , and the optical density at 600nm (OD 600 ) was measured with a spectrophotometer (SpectraMax plus, Molecular Devices, Sunnyvale, CA, USA) at different time points (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10h). The experiments were performed in replicates of five and were repeated three times to obtain statistical significance.
Effect of pyrimidinedione on in vitro biofilm growth S. pneumoniae and Staphylococcus biofilm formation experiments were carried out using a static model in 96-well or 24-well microtiter plates [18,30]. Briefly, bacterial colonies grown overnight on blood agar were scraped and seeded in broth (TSB medium) and further grown until mid-logarithmic phase (1×10 8 cfu/ml). Bacterial cells were diluted 1:1000 in fresh broth, and 200μl or 1 ml cell suspension was used to inoculate a 96-or 24-well microtiter plate. Plates were then incubated at 37°C in 5% CO 2 . Pyrimidinedione was added to each plate, as indicated. Control samples did not contain any supplement, and DMSO-control samples contained 0.01% DMSO (final concentration). After incubation, the medium and planktonic cells were discarded, and the plates were washed three times with sterile PBS. The plates were air dried for 15 min and stained with crystal violet (CV; 0.1%) for 15 min. The CV stain was removed and the plates were washed again. The stained biofilm was dissolved in 95% ethanol, and the OD 570 was measured in an automatic spectrophotometer. All experiments were performed with five replicates, and the average was calculated. To quantify the bacteria within biofilms, plates inoculated as described above were washed, and adherent biofilms were dissolved in sterile water via sonication for 10 s. One hundred micro-liter samples were serially diluted, plated on blood agar plates, and incubated at 37°C in 5% CO 2 for 24 h. After incubation, bacterial colonies were counted and cfu/ml was determined. Planktonic cells (cells in the biofilm supernatant) were counted in a similar manner. measured using the CV-microtiter plate method. Cfu counts of bacteria within biofilms (D39 strain) were conducted as described above. The percent decrease in biofilm biomass was calculated by subtracting the biomass of DMSO-control samples. The half-maximal effective concentration (EC 50 ) of pyrimidinedione was determined as the concentration corresponding to 50% of the maximum biofilm inhibition with respect to DMSO-control biofilms. At each pyrimidinedione concentration, cells suspended within the biofilm supernatant were collected and analyzed. Bacterial growth was detected by measuring optical density at 600nm (OD 600 ), and cfu of planktonic bacteria in the biofilm supernatant of D39 were counted.
To determine whether the biofilm-inhibiting effects of pyrimidinedione extended beyond S. pneumoniae, pyrimidinedione was tested on other biofilm-forming microbial pathogens, such as MSSA, MRSA, and S. epidermidis. Staphylococcus 24 h biofilm assays were carried out in a similar manner as described above, in TSB medium supplied with 1, 5, and 10 μM/ml pyrimidinedione.

Effect of pyrimidinedione on biofilms grown at different time points
In vitro S. pneumoniae biofilm growth varies at different time points. Therefore, we analyzed the effects of pyrimidinedione on S. pneumoniae D39 biofilms at different time points in vitro. S. pneumoniae biofilm was grown at different concentrations of pyrimidinedione [0 (DMSO control), 0.5, 1, 5, and 10 μM/ml] for 5, 10, 15, and 20 h.

Effect of pyrimidinedione on established biofilms
To analyze the inhibitory effects of pyrimidinedione on established biofilms, S. pneumoniae D39 biofilms were grown for 15 h. These established biofilms were then treated with different concentrations (1−400μM/ml) of pyrimidinedione and further incubated at 37°C in 5% CO 2 for 6 h. The biofilms were washed, and the biofilm biomass was detected by CV-microtiter plate assay.

Visualization of pneumococcal (D39 strain) biofilm growth by confocal microscopy
In vitro biofilm growth, with and without pyrimidinedione, was analyzed by confocal microscopy. Biofilms were grown on microdiscs for 15 h with 7μM/ml pyrimidinedione and stained using LIVE/DEAD Biofilm Viability staining kit (Invitrogen). A control sample was grown with 0.01% DMSO (final concentration). Biofilms were examined with a Nikon A1 confocal microscope (Nikon Instruments Inc., NY, USA) using fluoresce in (green) and Texas red (red) band pass filter sets. The live bacteria with intact cell membranes appear green, and those with damaged membranes appear red.

Visualization of biofilm morphology by scanning electron microscopy (SEM)
Pneumococcal (D39 strain) biofilms grown with 7μM/ml pyrimidinedione in 24-well tissue culture plates for 15 h were analyzed by SEM. The control sample was grown in 0.01% DMSO. Planktonic cells were removed, and the plates were gently washed twice with sterile PBS (pH 7.4). The samples were pre-fixed for 2 h in a 2% glutaraldehyde and paraformaldehyde solution, followed by 2 h post-fixation in 1% osmic acid. The samples were then treated with a graded series of ethanol (from 60% to 100%), washed three times with t-butyl alcohol, and were then immersed in t-butyl solution at -20°C. The samples were dried in a freeze dryer (ES-2030, Hitachi, Tokyo, Japan) and platinum coated using an IB-5 ion coater (Eiko, Kanagawa, Japan). The samples were visualized using a S-4700 field emission scanning electron microscope (Hitachi).

Effect of pyrimidinedione on biofilm global gene expression
In situ synthesis of oligonucleotide microarrays was used to compare global gene expressions between the S. pneumoniae D39 strain within biofilms grown in the presence and absence of pyrimidinedione. Biofilms were grown with 7 μM/ml pyrimidinedione, as described above, in 24-well plates and washed with sterile water. The adherent biofilm cells at the bottom and side of plate were scraped, pelleted, and subjected to lysozyme 100 μl (3 mg/ml) treatment for 4 min. Total RNA was isolated using the RNeasy Total RNA Isolation System Kit (Qiagen, Valencia, CA, USA) following the manufacture's protocol. Contaminated genomic DNA was removed on a column by RNAase-free DNAse (Qiagen) treatment at 20-25°C for 10 min. RNA was quantified by Nano-Drop, and its integrity was checked by capillary electrophoresis using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA).
cRNA probes synthesis and hybridization were performed using a Low Input Quick Amp WT Labeling Kit (Agilent Technologies USA) as per manufacture's protocol. Briefly, 100ng total RNA was mixed with WT primer mix and incubated for 10 min at 65°C. A cDNA master mix (0.1M DTT, 5X first strand buffer, RNase-Out, 10mM dNTP mix, and MMLV-RT) was prepared separately, and mixed with the RNA with WT primers mix and incubated at 40°C for 2 h and thereafter at 70°C for 10min. Transcription of dsDNA was performed by adding the transcription master mix (NTP mix, 4X transcription buffer, 50% PEG, 0.1 M DTT, RNase-Out, T7-RNA polymerase, inorganic pyrophosphatase, and cyanine 5-CTP) to the dsDNA reaction samples and incubating the samples at 40°C for 2 h. Amplified and labeled cRNA was purified on RNase mini columns (Qiagen) and quantified using a spectrophotometer. The cyanine 5-labeled cRNA mix was fragmented by incubating at 60°C for 30 min with 10X blocking agent and 25X fragmentation buffer. The fragmented cRNA were dissolved in 2X hybridization buffer and pipetted on the assembled Streptococcus pneumoniae D39 (MYcroarray.com) 6× 7k Microarray. The hybridization reaction was performed in hybridization oven at 57°C for 17 h and the arrays were washed as per manufacturer's instructions.
Hybridization images were analyzed with DNA Microarray Scanner (Agilent Technologies) and the data quantification was performed using Agilent Feature Extraction Software version 10.7 (Agilent Technologies). The average fluorescence intensity for each spot was calculated, and the local background was subtracted. All data normalization and the selection of differentially expressed genes were performed using GenoWiz 4.0 (Ocimum Biosolutions, India). Genes were filtered by removing flag-out genes from each experiment. Global normalization was performed. The average normalized signal channel intensities were divided by the average normalized control channel intensities to calculate the average normalization ratio.
Microarray experiments were performed in three biological replicates. Statistical significance was determined by Student's t-test. A p value less than 0.05 was considered significant. A 1.4-fold change in each gene, and in each microarray experiment, was considered significant and was included in the final results. The functional annotation (molecular and biological function) and gene ontology of the two sets of differentially expressed genes were determined using the UniPortKB database (http://www.uniport.org/uniport/P0A4M0) and STRING version 9.1. Microarray data were deposited in NCBI's Gene Expression Omnibus (GEO) database (http:// www.ncbi.nlm.nih.gov/geo/info/linking.html) and are accessible through GEO Series accession number GSE65339.

Quantification of gene expression by real-time RT-PCR
Microarray gene expression results were confirmed by real-time RT-PCR. Thirteen differentially expressed genes, along with the control gyrB gene, were analyzed by real-time RT-PCR. The primers used are shown in Table 1. cDNA amplification was carried out with real-time PCR in a reaction mixture of 20 μl (total volume). The reaction mixture consisted of 10 μl 2× SYBR Green PCR Master Mix (Roche Applied Science, Indianapolis, IN, USA), 3 pmol of each forward and reverse primers, and 4 μl cDNA. The PCR reaction conditions were as follows: initial denaturation at 95°C for 10 min, followed by 45 cycles of DNA denaturation for 15 s at 95°C, primer annealing for 10 s at 56°C, and extension for 15 s at 72°C; and a final extension step at 72°C for 5 min. To verify that cDNA samples were not contaminated with genomic DNA, a control reaction where no reverse transcriptase was added was included in each RT-PCR experiment. The relative quantification of gene expression was performed using the 2 -ΔΔCT method as described elsewhere [31]. Gene expression normalization was performed using a housekeeping gene, gyrB, and the standard condition was a biofilm grown without pyrimidinedione.
Evaluation of pyrimidinedione-mediated eukaryotic cellular cytotoxicity by Cell-Counting Kit (CCK-8) The cytotoxicity of pyrimidinedione was tested on the human middle ear epithelium cell (HMEEC) line using a CCK-8 kit (Dojindo, MD, USA). HMEECs were kindly provided by Dr.  David J. Lim (House Ear Institute, LA, USA) and were maintained in DMEM and BEBM medium (1:1) with required supplements [32,33]. HMEECs (1×10 4 /well) were seeded in 96-well plates and incubated at 37°C overnight in presence of 5% CO 2 . After overnight culture, HMEECs were treated with pyrimidinedione (1, 5, or 10 μM/ml) for 24 h in triplicate. Positive and negative control wells were supplemented with 0.01% DMSO (final concentration) or 2% Triton X-100, respectively. After incubation, 10 μl CCK8 solution was added to each well, and the cells were incubated for a further 2-3 h. Mitochondrial dehydrogenase within the cells reduced CCK8 solution to a yellow product called formazan. The amount of formazan produced in the reaction sample is positively correlated with cell viability. Absorbance at 450nm was measured using a microplate reader.

Statistical analysis
Individual experiments were carried out in triplicates or five-replicates, and mean values were calculated. The mean value differences were assessed by Student's t-test, and the statistical significance was set at a p-value of less than 0.05.

Results
Growth curve of S. pneumoniae supplemented with pyrimidinedione The planktonic cell growth of S. pneumoniae D39 in the presence of pyrimidinedione (1μM/ml or 10 μM/ml) was not significantly inhibited during the time course experiment. At 6 h incubation, 12% of the cells were inhibited, however, at stationary phase (10 h post-inoculation), planktonic cell growth was not inhibited (Fig 2). This result indicated that pyrimidinedione is partially bacteriostatic, and it has no effect on final bacteria planktonic growth.
Pyrimidinedione inhibits S. pneumoniae (serotype 2, 3, 19 and 11) biofilm growth in vitro Addition of pyrimidinedione inhibited pneumococcal biofilm growth in vitro. A significant decrease (p<0.05) in biofilm biomass of S. pneumoniae serotypes 2, 3, 19, and 11 were detected in biofilms grown in the presence of pyrimidinedione ( Fig 3A). The inhibitory effects of pyrimidinedione was concentration dependent, and the calculated EC 50 (S. pneumoniae D39) was 1 μM. Addition of 1 μM/ml pyrimidinedione significantly decreased S. pneumoniae D39 biofilm biomass by 54% (p< 0.05, Fig 3A) and cfu counts by 83% (p< 0.05, Fig 3B) in comparison to DMSO-control biofilms. However, the planktonic cell growth of S. pneumoniae serotypes 2, 3, 19, and 11 in the presence of pyrimidinedione was not significantly decreased ( Fig 3C). Similarly, no significant decrease in planktonic bacterial cfu counts was detected in the D39 strain ( Fig 3D). This result indicated that pyrimidinedione significantly inhibits biofilm growth but has no effect on planktonic growth. The inhibitory effect of pyrimidinedione was significant in all S. pneumoniae serotypes tested, indicating that the inhibitory effect of pyrimidinedione was independent of serotypes, and the application of this molecule could be extent to other serotypes biofilms as well. Pyrimidinedione significantly inhibited in vitro MSSA, MRSA, and S. epidermidis biofilm growth in a dose-dependent manner, similar to that observed in pneumococci (Fig 4). S. epidermidis, MSSA 29213 and MRSA CCARM 3903 are strong biofilm producing strains. 1 μM/ml concentration of pyrimidinedione significantly (p< 0.05) decreased biofilm biomass of S. epidermidis, MSSA 29213 and MRSA 3903 by 50, 55 and 53% respectively. This indicates that pyrimidinedione is equally effective against both antibiotic resistance and sensitive strong biofilm producing staphylococcus.

Pyrimidinedione inhibits S. pneumoniae D39 biofilm growth at both early and late stages
The growth of biofilms under different pyrimidinedione conditions was inhibited at each time point analyzed. A dose-dependent decrease in biofilm biomass was detected in biofilms grown with pyrimidinedione compared to DMSO-control samples ( Fig 5). In the DMSO control, there was an increase in biofilm growth at 5 h of incubation, maximal growth occurred at 10 h of incubation, and then biofilm growth declined. A 70% decrease in biofilm growth was observed with the addition of 5 μM pyrimidinedione at 10 h of incubation. These results indicated that pyrimidinedione is an effective inhibitor of S. pneumoniae biofilm growth in vitro at both early (5 h) and late (20 h) stages. Pyrimidinedione has no effect on established biofilm biomass Established S. pneumoniae biofilms were treated with different concentrations of pyrimidinedione. Biofilm quantification by CV-microtiter plate assay demonstrated no significant decrease in biofilm biomass following a 6-h treatment with pyrimidinedione ( Fig 6A). There was also no difference observed in cfu counts between control and pyrimidinedione-treated biofilms ( Fig 6B). This indicates that pyrimidinedione is unable to dismantle biofilms or kill bacteria within biofilms. Hence, this small molecule cannot eradicate an established biofilm.

Visualization of biofilm growth by confocal microscopy
Confocal microscopy analysis revealed a significant difference in the morphology of biofilms grown in the presence of pyrimidinedione compared to control biofilms (DMSO alone). Control biofilms were compact, thick, and had a well-organized three-dimensional structure ( Fig  7A & 7B). In contrast, biofilms grown with pyrimidinedione were thin, the cells were scattered and attached to the bottom of disc, and their three-dimensional structure was not well Visualization of biofilm morphology by scanning electron microscopy SEM analysis revealed that control biofilms (DMSO-control) were thick, organized, and heterogeneous with micro-colonies. The cells were surrounded by extracellular matrix and were connected to the bottom of the plate and to each other, forming a three-dimensional organized biofilm structure of significant depth ( Fig 8A, 8B, 8C & 8D). The extracellular polysaccharide matrix (EPS) was clearly visible on the cell surface ( Fig 8D, arrow). In contrast, pyrimidinedione-exposed biofilms were thin, disorganized, and devoid of micro-colonies. Cells were attached only to the base of the plate, while cell-cell adherence was absent (Fig 8E, 8F, 8G & 8H). The cell surfaces were smooth and devoid of matrix and EPS. These results indicated that in presence of pyrimidinedione, pneumococci were unable to form an organized biofilm.

Effect of pyrimidinedione on biofilm global gene expression
Upon microarray analysis, it was determined that the expression of 56 S. pneumoniae genes was significantly (p< 0.05) up-regulated, while expression of 204 genes was significantly (p< 0.05) down-regulated in pyrimidinedione-grown biofilms, as compared to controls. Among the 56 up-regulated genes, 22 encoded uncharacterized and hypothetical proteins, and 34 encoded functional proteins (Table 2). Of the 204 down-regulated genes, 45 encoded hypothetical proteins and 159 encoded functional proteins (Table 3). Genes involved in some functional protein categories were exclusively down-regulated or up-regulated in pyrimidinedione-grown biofilms (Fig 9). The functional annotation of the differentially regulated genes revealed that genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down-regulated. The fold change values of relative gene expression and predicted protein functions are detailed in Tables 2 and 3.
The microarray results detected that expression of 11 genes belonging to the fatty acid synthesis (FAS) locus was significantly down-regulated (more than 1.4-fold) in pneumococcal biofilms grown in the presence of pyrimidinedione. No FAS pathway genes were up-regulated. The FAS pathway is made up of 13 genes arranged in a single locus that are involved in FAS initiation and product elongation (Fig 11).
Five genes involved in signal transduction were also down-regulated in pyrimidinedionegrown biofilms. Expression of the ciaR and ciaH gene, encoding a DNA-binding response regulator protein and a sensor histidine kinase respectively were down-regulated. These two genes encode the two-component regulatory system CiaH/CiaR, which is involved in the early steps of competence regulation. Expression of the comC gene, encoding the competence-stimulating peptide type 1, essential for pneumococcal competence and biofilm formation, was down-regulated 1.5-fold. Similarly, expressions of the ptsH gene, encoding the phosphocarrier protein HPr, and the SPD_0082 gene, encoding a sensor histidine kinase, were down-regulated 1.6and 1.4-fold, respectively.
A large number of genes involved in transcription and DNA binding were down-regulated in pyrimidinedione-grown biofilms. Expression of 23 transcription and DNA binding genes was down-regulated, while five genes were up-regulated. Important down-regulated genes include, ccpA, rpoC, comX2, bplS, spx, and cps2A. The ccpA gene encodes catabolite control protein A, which is a negative repressor protein with a regulatory role in carbohydrate metabolism. The comX2gene encodes a sigma factor that functions as a competence-specific global transcription modulator involved in bacterial competence.
Other down-regulated genes encoded cell membrane proteins, as well as proteins involved in amino acid synthesis, catalytic activity, cell wall organization and biogenesis, homeostasis, response to stress, and thiamine and riboflavin biosynthesis.

Quantification of gene expression by real-time RT-PCR
Thirteen differentially expressed genes from our microarray analysis were confirmed by realtime RT-PCR, and their differential expression levels were in agreement with the microarray data (Table 4).

Pyrimidinedione does not exhibit eukaryotic cell toxicity
The CCK8 cell viability experiment revealed no significant difference in mean absorbance of HMEECs treated with 1, 5, or 10 μM pyrimidinedione with respect to untreated or DMSOcontrols (p>0.45). The absorbance of 2% triton X-100 treated HMEECs was significantly (p<0.002) lower than control-or pyrimidinedione-treated cells, indicating no acute cellular cytotoxicity at the tested concentrations (Fig 12). Thus, pyrimidinedione showed no evidence of acute toxicity to human HMEECs at a concentration of 10 μM, which was 10-fold greater than the established EC 50 .

Discussion
S. pneumoniae are known to cause various biofilm-related infections in human. The physiology, metabolism, and gene expression profile of biofilm bacteria are different than planktonic bacteria (18). In S. pneumoniae biofilms, the quorum sensing (QS) signal generated by competence stimulating peptide (CSP) plays an important role in coordinating the spatial distribution of cells and the aggregation of exopolysaccharides [34,35]. Autoinducer-2 (AI-2) is the only QS molecule in pneumococci synthesized through activated methyl cycle (AMC), where Dam enzyme catalyze the transfer of a methyl group from SAM to macromolecules and adenine within DNA duplex [36,37,38]. Therefore, we hypothesized that interfering in Dam activity could have adverse effect on S. pneumoniae biofilms growth. Several studies reported that DNA adenine methylation regulates the expression of various virulence-related genes in        numerous pathogens [39,40,41]. Our previous study demonstrated that interfering with methylation activity, either using the hypo-methylating agent (5-azacytidine) or a SAM analogue (sinefungin), inhibited pneumococcal biofilm growth. In this study, we examined the effect of the small molecule Dam inhibitor, pyrimidinedione, on S. pneumoniae biofilm growth. We then evaluated global gene expression changes within biofilms grown in the presence of pyrimidinedione. Planktonic cell growth of S. pneumoniae D39 was not inhibited in the presence of different pyrimidinedione concentrations. The CV-microtiter plate assay and cfu counts detected a significant decrease in biofilm formation in samples treated with pyrimidinedione, and this inhibitory effect was concentration-dependent in all serotypes tested. The normal growth of planktonic cells and decreased biofilm formation in presence of pyrimidinedione indicated that pyrimidinedione selectively inhibits pneumococcal biofilms. Similarly, the Dam mutant strains of Yersinia enterocolitica and Haemophilius influenza showed reduced adhesion and host cell invasion capacity [42,43]. Pyrimidinedione was effective in inhibiting pneumococcal biofilm growth at both early and late stages [44]. Similar inhibitory effects of 5-aza-cytidine and sinefungin on S. pneumoniae biofilms as well as a small molecule adenosine mimetic on Salmonella enteric biofilms were previously reported [27,28,45]. However, pyrimidinedione was not effective in dismantling biofilms nor was it cytotoxic to bacteria within biofilms. S. aureus and S. epidermidis are important pathogens implicated in a wide variety of biofilmrelated infections, including infections present within medical devices. The inhibitory effects of pyrimidinedione on S. aureus (MSSA and MRSA) and S. epidermidis biofilms indicated broadspectrum anti-biofilm activity against antibiotic-resistant bacteria.
To understand biofilm changes at the microscopic level, we examined biofilms grown with and without pyrimidinedione by confocal microscopy and SEM. In microscopic analysis, the control and pyrimidinedione-biofilms demonstrated a significantly different morphology. The control biofilms were well organized and compact with significant thickness, and the cells were interconnected with each other and to the base of the plate [46]. A remarkable feature of these control biofilms was the presence of EPS [47]. EPS was completely absent in biofilms grown with pyrimidinedione. Due to lack of EPS, the cells were scattered, attached only to the bottom of the plate, and were unable to form an organized biofilm structure [48]. EPS is important for biofilm development; the absence of this structure indicated that bacteria were attached to the bottom of the plate. Therefore, it is possible that they could be easily washed away, decreasing biofilm biomass calculations and cfu counts [49].
In-order to examine the changes in gene expression of pyrimidinedione-grown biofilms, we evaluated global gene expression by microarray analysis. The overall gene expression pattern demonstrated that more genes were down-regulated in pyrimidinedione-grown biofilms compared to control biofilms. The gene expression analysis of 13 differentially expressed genes identified by microarray was confirmed using real-time RT-PCR. A functional annotation In pneumococci, lactose and galactose are metabolized by the tagatose-6-phosphate pathway (light orange box) and the Leloir pathway (left; light green box). Tagatose-6-phosphate pathway genes include lacA, lacB, lacC, and lacD, and Leloir pathway genes include galM, galK, galT-1, and galE-2. Along with these the lacF2 gene (encodes the PTS system, lactose-specific IIA component) and lacE2 gene (encodes the PTS system, lactose-specific IIBC component) are required for galactose transport. The gap gene (encoding glyceraldehyde-3-phosphate dehydrogenase), eno gene (encoding phosphopyruvate hydratase), and pyk gene (encoding pyruvate kinase) are involved in glycolysis (central blue box). The lacA, lacB, lacC, lacD, galT-1, galE-2, lacE-2, gap, eno and pyk genes were down-regulated by more than 1.4 folds in this study. The relative fold-changes in gene expression are highlighted. demonstrated that 17 functional gene groups were exclusively down-regulated, and four clusters were exclusively up-regulated in pyrimidinedione-grown biofilms.
The down-regulation of genes involved in DNA replication, cell division, cell organization and biogenesis, response to stress, homeostasis, and protein folding indicated that cell division may be perturbed and that cells were stressed in the presence of pyrimidinedione. In support of this hypothesis, 13 genes encoding ribosomal proteins showed reduced transcription in pyrimidinedione-grown biofilms, indicating that the pneumococci had reduced its translational capacity [50]. Similarly, bacterial transcriptional, signaling, and transport capacity may also be obstructed by down-regulation of transcription, DNA binding, transport, and signaling protein-encoding genes in pyrimidinedione-grown biofilms.
Fatty acid biosynthesis is essential for bacterial membrane integrity and cellular physiology, and the fatty acid biosynthesis gene mutant strains were unable to survive [51]. Pneumococcal fatty acid biosynthesis genes are collectively known as type II fatty acid synthase, which are clustered at a single location. The down-regulation of fatty acid biosynthesis genes in pyrimidinedione-grown biofilms indicated that disruption of fatty acid biosynthesis and product elongation may be affected.
A striking observation in our microarray and real-time RT-PCR gene expression results was the up-regulation of galactose metabolic pathway genes and down-regulation of glycolysis pathway genes. In pneumococci, lactose and galactose are metabolized by the tagatose-6-phosphate and Leloir pathways, respectively [52,53,54]. Our results demonstrated up-regulation of tagatose pathway genes, Leloir pathway genes, lactose-specific IIA & IIBC component encoding genes (lacF2and lacE2), and the lacT gene, encoding a transcriptional antiterminator. Conversely, we found down-regulation of glycolysis pathway genes in pyrimidinedione-grown biofilms. The up-regulation of galactose metabolism genes indicated that cellular carbohydrate metabolism was changed, and cells adapted to an alternative pathway. In pneumococci, the galactose and lactose metabolism pathway genes are up-regulated in the presence of sugars other than glucose [55]. The precise reason for the up-regulation of the galactose metabolic pathway genes in the presence of pyrimidinedione remains to be elucidated. However, it was reported that the transcription factor CcpA (carbon catabolite protein A), ptsH, encoding the phosphocarrier protein HPr, and the SPD_0082 gene, encoding a sensor histidine kinase, facilitates pneumococci utilization of diverse carbohydrate sources during colonization, multiplication, and biofilm formation [56,57]. Moreover, the regulation of the central carbohydrate metabolic pathway genes is under the control of carbon catabolite repression (CCR), which is mediated by the transcription factor CcpA and the histidine phosphoprotein HPr [58,59,60].
The down-regulation of five phosphate-specific transport system genes indicates that the transportation of molecules from the periplasm to the cytoplasm may be hindered by pyrimidinedione. Mutagenesis of the pst ABC genes in pneumococci resulted in decreased rates of phosphate uptake, decreased growth rates, decreased transformation, and reduced pathogenicity [61,62].
Here we detected the down regulation of virulence-related genes (ply, srtA, ptrA, lytB, nrc, and SPD_1295) in pyrimidinedione-grown biofilms. The ply gene is a virulence gene encoding the toxin pneumolysin, which causes eukaryotic cell lysis and plays a major role in pneumococcal invasion [63,64]. In S. pneumonia, the srtA gene is another virulence gene, and the srtA gene mutant strain showed low virulence and low adherence towards human pharyngeal cells [65]. The lytB gene encodes a choline-binding protein, and nrc and SPD_1295 encode Table 4. Gene expression analysis by real-time RT-PCR. Fold changes in gene expression of biofilms grown with pyrimidinedione with respect to control.

Gene
Mean fold change p-value hemolytic proteins. The prtA gene encodes the pneumococcal cell wall-associated serine protease A, which is important in virulence in intraperitoneal infections [66]. The significantly decreased biofilm growth observed in the presence of pyrimidinedione may be due to the decreased expression of competence and biofilm-related genes (Fig 13). Here, the competence stimulating peptide-1 (CSP-1) precursor encoding gene comC, the twocomponent regulator encoding genes ciaH and ciaR, and the alternative sigma factor encoding gene comX were down-regulated in response to pyrimidinedione treatment. In S. pneumoniae, the CSP-mediated QS system initiates the regulation of genetic competence, which involves the expression of early gene products encoded by comAB and comCD genes, and the two-component regulatory system CiaH-CiaR [67,68]. The pneumococcal comC gene encodes the CSP precursor and the comDE genes encode the CSP receptor and response regulator protein. The CiaH-CiaR negatively regulates comCDE expression and thus affects the development of competence. The response regulator comE binds to the early gene promoter and initiates transcription, as a results accumulation of CSP, ComD, phosphorylated ComE and ComX (a global transcription modulator) increases. The ComX alternative sigma factor initiates the transcription of late competence-specific operon, which facilities in DNA uptake and recombination of DNA [68,69]. Previous studies have reported that QS plays an important role in biofilm formation and a ciaR/H gene mutant strain was unable to form biofilms [49]. Oggioni et al. (2006)  The cytotoxicity of pyrimidinedione was tested on the HMEEC line using a CCK-8 kit. The absorbance of the reaction was measured at 450 nm, and was compared among HMEECs exposed to pyrimidinedione (1 μM, or 5 μM, or 10 μM), 2% Triton X-100 (complete lysis), control (medium alone), and DMSO-control. No significant difference in mean absorbance was detected for HMEECs treated with various concentrations of pyrimidinedione versus untreated or DMSO controls (p> 0.43). However, all were significantly different from triton X-100-treated cells (p< 0.002), indicating no acute cellular cytotoxicity at the tested concentrations. The results were compared by Student's t-test (*** corresponds to p< 0.002, # corresponds to p>0.43). The error bars represent the SD. detected up-regulation of the comC gene in biofilms and demonstrated that S. pneumoniae comC mutants were less virulent and unable to form biofilms in vitro. They further reported that, when supplemented with external CSP-1, wild type levels of biofilm formation were restored in the mutant strain [18]. Similarly, in S. mutans, a comC mutant strain was unable to produce the signal peptide and biofilm formation was disrupted.
Our results indicate that the Dam inhibitor, pyrimidinedione, down-regulates the expression of various pathway genes including those involved in cellular metabolism, translation, transcription, cell division, amino-acid synthesis, virulence, and DNA replication. Previous pneumoniae. Induction of genetic competence in pneumococci is regulated by a CSP-mediated quorum-sensing system. The precursor of CSP is encoded by the comC gene, and the ComAB (secretary and transporter) protein, facilities extracellular accumulation of mature CSP. Mature CSP then binds to ComD receptor, resulting in ComD auto-phosphorylation and phosphoryl group transfer to the response regulator, ComE. Phosphorylated ComE binds to the early gene promoter and activates the transcription of early genes. As a result of ComE binding, the transcription of the comCDE operon, and the production of CSP, ComD and phosphorylated ComE levels increase. ComE binding also initiates the accumulation of ComX (alternative sigma factor). ComX binds to the late gene promoter and stimulates the expression of late protein-encoding genes which facilitates recombination and DNA uptake. CiaH-CiaR is the second two-component regulatory system affecting the development of competence via regulation of comCDE expression. The fold-changes of gene expression are highlighted in red. studies report that disruption of Dam or Dam activity affects bacterial fitness and alters gene expression [39,40,41,70]. These perturbations were postulated to be indirect secondary effects of basic cellular fitness. As a result, pneumococcal planktonic cell growth was reduced but not completely inhibited. It is likely that the down-regulation of competence and biofilm-related genes resulted in lower levels of biofilm growth. These bacteria were unable to build organized biofilms or aggregate biofilm matrix [49].

Conclusion
This study demonstrated that a small molecule Dam inhibitor, pyrimidinedione, perturbed pneumococcal biofilm growth in vitro at concentrations that did not inhibit planktonic cell growth and down-regulated the expression of important metabolic-, virulence-, competence-, and biofilm-related genes. Pyrimidinedione is also effective against MSSA, MRSA, and Staphylococcus epidermidis biofilm growth in vitro, and it is not cytotoxic to mammalian cells. Pyrimidinedione has potential for the development of new anti-biofilm compounds, and a ideal candidate molecule which require further study for in vivo biofilm prevention.