Probing Oral Microbial Functionality – Expression of spxB in Plaque Samples

The Human Oral Microbiome Database (HOMD) provides an extensive collection of genome sequences from oral bacteria. The sequence information is a static snapshot of the microbial potential of the so far sequenced species. A major challenge is to connect the microbial potential encoded in the metagenome to an actual function in the in vivo oral biofilm. In the present study we took a reductionist approach and identified a considerably conserved metabolic gene, spxB to be encoded by a majority of oral streptococci using the HOMD metagenome information. spxB encodes the pyruvate oxidase responsible for the production of growth inhibiting amounts of hydrogen peroxide (H2O2) and has previously been shown as important in the interspecies competition in the oral biofilm. Here we demonstrate a strong correlation of H2O2 production and the presence of the spxB gene in dental plaque. Using Real-Time RT PCR we show that spxB is expressed in freshly isolated human plaque samples from several donors and that the expression is relative constant when followed over time in one individual. This is the first demonstration of an oral community encoded gene expressed in vivo suggesting a functional role of spxB in oral biofilm physiology. This also demonstrates a possible strategy to connect the microbial potential of the metagenome to its functionality in future studies by identifying similar highly conserved genes in the oral microbial community.


Introduction
The Human Oral Microbiome Database (HOMD) contains DNA sequence information from over 1300 genomes [1]. This number is most likely to grow in the future. The wealth of information provided allows the interested researcher to screen the available sequence data for common features and investigate a biological relevant relation to the community structure. For example is a specific gene or a set of genes associated with a healthy community or a predictor of a pathogenic oral biofilm?
One caveat of this approach is that the metagenome only gives a static snapshot of the oral bacterial community potential at the time of sampling [2]. It will not allow determining a dynamic relationship or the functionality of the bacterial community at any given time. While the potential is determined by the metagenome, the functionality of the bacterial community is driven by its metatranscriptome and metaproteome, thus at the RNA and protein level [2,3]. However, investigating the metatranscriptome and the metaproteome comes with certain challenges, including the amount of RNA or protein required to successfully cover the entire metatranscriptome or metaproteome of any given plaque sample. For example detection and quantification of low abundance transcripts by RNA Seq can be challenging [4] since the starting material for RNA isolation, dental plaque, is limited. Furthermore, the amount of species open reading frames would lead to large amounts of diverse sequences reads requiring specific software to adequately manage the data [5][6][7].
Alternatively, the metagenomic information could be used to identify an ecological relevant oral community encoded gene. The expression of this gene in human plaque samples could be determined directly thus probing its functionality in vivo. Ideally several members of the oral microbial biofilm share this gene. We previously identified a high homology of the pyruvate oxidase gene spxB between the oral commensal Streptococcus sanguinis and Streptococcus gordonii [8]. SpxB is an oxido-reductase, catalyzing the conversion of pyruvate to acetyl phosphate, CO 2 and H 2 O 2 under aerobic conditions [9,10]. The gene provides several advantages for the encoding species. Initially, we identified H 2 O 2 as inhibiting substance in the dual species competition with cariogenic Streptococcus mutans [11,12]. Deletion of the spxB gene in both S. sanguinis and S. gordonii rendered them non-competitive against S. mutans demonstrating that SpxB is responsible for competitive H 2 O 2 production [12]. Besides the inhibitory H 2 O 2 action, two additional effects increasing commensals competitiveness are obvious: i) ATP production from acetyl phosphate for energy generation and ii) H 2 O 2 induced release of extracellular DNA (eDNA) [12]. eDNA is a major component of the extracellular polymeric substance (EPS) of biofilms, promoting cell-cell and cell-tooth contact [13,14]. H 2 O 2 induced release of eDNA can also serve in horizontal gene transfer promoting genetic diversity as we demonstrated recently [15]. In addition, the ability to produce inhibitory amounts of H 2 O 2 seems to be limited to oral streptococci [16].
In this pilot study, we demonstrate that spxB is a suitable candidate gene encoded by several important commensal streptococci, identified by homologous sequence search using the HOMD database. Oral streptococcal production of H 2 O 2 as measured with specific indicator plates seems to correlate with the presence of the spxB gene as determined with PCR and spxB specific oligonucleotides. Furthermore, we isolated RNA from freshly isolated human plaque samples and demonstrate that spxB is expressed with Real-Time RT-PCR, suggesting a functional role in the oral biofilm.

Ethics Statement
The Institutional Review Board of University of Oklahoma HSC approved the study protocol for human subjects. IRB protocol # 1934. Participants signed a written consent form following an approved procedure by the IRB.

Bacterial Strains and Growth Conditions
Bacterial strains are listed in Tab. 1. Bacteria were routinely grown aerobically in 5% CO 2 at 37uC overnight in BHI medium (Brain Heart Infusion; Difco, Sparks, MD) or on BHI plates, or as otherwise indicated.

Subjects and Plaque Sampling
The present study sole intention was to determine the feasibility of measuring spxB gene expression in dental plaque samples. Therefore, no subject related data were collected from the 8 volunteers asked to donate plaque samples. From each individual, supragingival dental plaque samples were recovered between 16 and 18 hours after tooth brushing, from interproximal, vestibular and lingual surfaces of all teeth using a dental probe. The collected plaque samples were immediately removed from the dental probe after a visible amount has built up using a sterile tip and resuspended in 1 ml TRIzol (lifetechnologies). The sampling procedure lasted not longer than five minutes. Resuspended plaque samples in TRIzol were immediately frozen at 280uC until further processing the next day. Plaque samples for plating were removed from the teeth using a cotton swap and directly plated onto the H 2 O 2 indicator plates.

RNA Isolation and cDNA Synthesis
To isolate RNA, cells were disrupted three times for 30 seconds each using 0.1 mm zirconia/silica beads (BioSpec, Inc) in a FastPrep FP210 Homogenizer (Thermo Scientific) using the highest speed setting. Total RNA isolation from TRIzol was carried out according to the manufacturer's instructions (Isolation of total RNA using TRIzol, lifetechnologies). RNA samples were treated with Turbo DNase to remove traces of chromosomal DNA after manufacturers recommendations (Ambion). RNeasy Mini-Elute cleanup kit (Qiagen) was used to purify RNA samples after DNase treatment. 2 ml RNA was immediately run on a 1% agarose gel to check for integrity. cDNA was synthesized using qScript TM cDNA synthesis kit (Quanta Biosciences) according to the manufacturer's protocol.

RNA Integrity
RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). The yield of the extracted RNA and intactness of rRNA was assayed from 1 ml RNA after DNase treatment and RNeasy MiniElute cleanup in a 2100 BioAnalyzer using the protocol of the RNA 6000 Nano Lab Chip Kit (Agilent Technologies, Inc.). The chip allows for analyzing 12 samples simultaneously. RNA was therefore collected first and stored at 280uC until all 8 samples were available for the RNA 6000 Nano Lab Chip run.

RT-PCR and Real-Time PCR
RT-PCR was performed as described earlier [17]. Real-time RT PCR was performed to determine specific cDNA copies with the comparative threshold cycle (CT) method using a MyiQ single-color real-time PCR detection system (Bio-Rad) and PerfeCtaTM SYBR H Green SuperMix for iQ TM (Quanta Biosciences). Relative changes in cDNA copies representing differential gene expression were calculated using the DC T method described previously [18]. The 16S rRNA gene was used as the housekeeping reference gene. Oligonucleotides were synthesized by IDTDNA. Oligonucleotide sequences are: 16S rRNA F -59-AAGCAACGCGAAGAACCTTA-39; 16S rRNA R -59-GTCTCGCTAGAGTGCCCAAC-39; universal spxB F -59-CATCATGGGTGACGGTGCAT-39; universal spxB R -59-GCGTTAGGGAAGTCACAACC-39.

PCR
Colony PCR was performed by scraping a small amount of cells from the respective agar plate using a sterile pipet-tip and resuspending the cells in a pre-aliquoted PCR reaction mix. Alternatively, cells were inoculated in 2 ml BHI overnight; 1 ml was transferred into tubes containing lysing matrix B (MP Biomedicals, Solon, OH) and cells were homogenized in a FastPrep FP210 homogenizer (Thermo Scientific) (speed setting of 6.5). After centrifugation for 10 min at 13.200 rpm in a tabletop centrifuge, 2 ml supernatant containing chromosomal DNA was used as PCR template. PCR was performed with a G-Storm GS1 thermocycler (Gene Technologies) according to the manufacturer's protocol. GoTaq-DNA polymerase was obtained from Promega, and oligonucleotides specific for spxB and 16S rRNA were the same as listed above. S. mutans UA159 [32] S. mitis 12261 [33] S. gordonii DL1 [34] S. gordonii V288 [35] S. oralis MC3-1 [36] S. oralis J22 [36] S. sanguinis SK36 [37] S. sanguinis 133-79 [38] S.

Detection of H 2 O 2 Production
Indicator plates for H 2 O 2 production were prepared and used as described [19]. This indicator plates allow for the detection of bacterial H 2 O 2 production resulting in a blue pigment (Prussian blue, ferric ferrocyanide) that forms in the presence of H 2 O 2 . Plates were aerobically cultured in 5% CO 2 at 37uC overnight.

Results
Prevalence of the spxB Gene in the Oral Biofilm Community A BLAST search was performed using the S. gordonii CH1 nucleotide sequence as query against all available oral microbial genomes on the HOMD server. In addition to S. gordonii, the following oral Streptococci encode spxB homologs: Streptococcus sanguinis, Streptococcus mitis, Streptococcus infantis, Streptococcus oralis, Streptococcus oligofermentans and Streptococcus cristatus (Tab. 2). Furthermore, Streptococcus pneumoniae encodes also spxB as reported in the literature [20] and found during the BLAST search, but was omitted since it is usually not found in the dental associated oral biofilm. The homology of the spxB genes encoded by the oral streptococcal community is high ranging from 93% to 97% on the nucleotide level (against S. gordonii CH1) with a core sequence of about 1700 bp when compared to the 2308 bp of S. gordonii CH1. This suggests that the spxB gene is highly conserved among oral Streptococci. Interestingly, no homolog was found in cariogenic Streptococcus mutans confirming our previous observation that S. mutans does not produce competitive amounts of H 2 O 2 [11].

Detection of spxB with spxB Universal Oligonucleotides
The available sequence data for spxB from oral Streptococci was used to design an spxB specific set of oligonucleotides. Initially, the oligonucleotides were tested for their ability to amplify spxB from several Streptococci present in our laboratory culture collection. The H 2 O 2 production potential of the here used streptococci was assessed first by inoculating 10 ml of an overnight culture on specific H 2 O 2 indicator plates. The plates were incubated overnight to allow for growth and H 2 O 2 production. As shown in Fig. 1 A, with the exception of S. mutans and S. salivarius, all other Streptococci produced H 2 O 2 evident from the formation of a blue pigment. Subsequently, cells were removed and chromosomal DNA isolated to perform PCR with 16S rRNA and spxB specific oligonucleotides. The 16S rRNA control showed amplification for all strains tested. A clear correlation was evident between positive spxB amplification in S. mitis, S. gordonii, S. oralis, S. sanguinis, S. infantis and S. parasanguinis and the formation of the blue pigment (Fig. 1B). No significant amplification was observed for all S. mutans strains and S. salivarius, but we did recognize weak bands after longer integration during picture documentation (Fig. 1B).
To determine if the observed faint bands would interfere with our intention to measure spxB expression by giving false positive amplification products, RNA was isolated from S. mutans, S. gordonii and S. salivarius and cDNA synthesized. RT-PCR with spxB oligonucleotides showed only amplification for S. gordonii in the RT-PCR reaction, but no amplification for the no RT control (Fig. 2). 16S rRNA amplification was positive in all three RT-PCR reactions (data not presented). This suggests that the newly designed oligonucleotides are able to amplify spxB from H 2 O 2 positive oral Streptococci and are suitable to be used in gene expression analysis.

PCR Amplification of spxB from H 2 O 2 Positive Plaque Colonies
To further evaluate the spxB amplification potential of the newly designed spxB specific oligonucleotides, dental plaque was collected from five subjects. The plaque samples were inoculated on H 2 O 2 indicator plates to separate single colonies. Fig. 3A represents plaque samples from two subjects and illustrates that colonies with and without blue pigment can be distinguished. Ten blue colonies from each subject were picked and used for colony PCR with the spxB specific oligonucleotides (Fig. 3B). PCR amplification was successful for all colonies in the five subjects. Obvious, however, was the difference in PCR efficiency. While subject 1 had strong bands for all ten colonies, subject 4 showed some heterogeneity in the band intensity (Fig. 3B). In addition, 45 white colonies were subject to PCR amplification with the spxB oligonucleotides. No amplification was observed (Fig. 3C). In summary, the spxB specific oligonucleotides are able to amplify spxB from freshly isolated H 2 O 2 positive plaque bacteria suggesting that the oligonucleotides could be used in general to evaluate the presence of this gene in human plaque samples.

RNA Isolation from Plaque Samples
The goal of this study is to detect the expression of spxB from freshly isolated dental plaque samples to evaluate the in vivo expression of this gene. The major challenge in the detection of gene expression from host-derived biofilms is the isolation of sufficient amounts of high quality RNA [21]. To test feasibility of RNA isolation, plaque was sampled from 11 subjects asked to refrain from tooth brushing in the morning to collect a sufficient amount of plaque in the afternoon. The plaque samples were immediately processed for RNA isolation. Isolated RNA was analyzed and RIN (RNA Integrity Number) determined. Fig. 4A and 4B showing agarose gel-electrophoresis and Bioanalyzer gel visualization. The respective RIN numbers and RNA concentrations are presented in Fig. 4C. Sample 1 and 2 showed very low RIN and were not further used. The remaining samples ranged from RIN 4.8 to RIN 7.8. The RNA concentration range was from 50 ng/ml to 418 ng/ml.

Detection of spxB Expression from Plaque Samples
Initially the expression of spxB in 9 individual plaque samples was compared using 16S rRNA as housekeeping reference. The expression was normalized to the subject with the highest spxB expression relative to the others. The result presented in Fig. 5 demonstrates that there is a high degree of interpersonal variation in spxB expression. The observed fold difference reached about 88 fold when the highest and the lowest expression were compared (subject 8 vs. subject 3). Contamination by chromosomal DNA was excluded by running a no RT control along with a RT-PCR (data not shown).
In addition, the expression of spxB from a single subject on five different time-points was determined to learn how stable the expression is on a day-to-day basis. As shown in Fig. 6, time point 1 and 2 as well as 3 and 4 were taken on two subsequent days, one hour apart while time point 5 was taken several days later. Although slight variations are visible, the expression of spxB in one subject seems to be constant over time. Furthermore spxB expression can be detected repeatable from RNA isolated from human plaque samples.

Discussion
The HOMD contains DNA sequence information from over 1300 genomes (www.homd.org). An eminent question is what kind of information can be extracted from the deposited sequence data which goes beyond the determination of what is present in the oral cavity or what kind of potential metabolic pathways are encoded [3,22]. The next logical step is to move away from the static information of sequence data to determine if the microbial potential of the chromosomal sequences is converted to a  functional response in vivo. The here presented study uses the sequence data provided by HOMD to identify an oral microbial community shared gene, spxB and demonstrates its functional expression in freshly isolated human plaque samples.
Several oral commensal Streptococci encode the spxB gene as we identified in this study. Up to 80% of the detected initial colonizers belong to the genus Streptococcus. Some species are even discussed as constant members constituting a core group for initial biofilm formation [23,24]. The spxB gene is therefore highly abundant during initial biofilm formation and might be considered as core gene involved in oral biofilm formation. The production of an antimicrobial substance like H 2 O 2 could therefore be regarded as an important protection mechanism of the initial colonizers of the resident biofilm community against invading and competing species like the extremely H 2 O 2 sensitive S. mutans [11]. More importantly, it might also be a mechanism to shape the colonization process towards a specific species composition. Only species co-evolved with oral streptococci and therefore adapted to withstand H 2 O 2 can integrate or colonize in close proximity to the initial colonizers and extend the developing biofilm community [8]. The gene is expressed in the different subjects suggesting a function in the biofilm mode of growth of oral streptococci. The next step is to design a large-scale study to relate the expression of spxB with an assessment of the oral health status. A positive association of spxB expression with oral health would allow for a better caries risk assessment in the future. Expression studies on infection relevant genes in vivo in humans and animal models have been done before as summarized in [25]. Three in vivo studies are relevant in the context of our study: i) An investigation of in vivo gene expression in the human host was carried out with Streptococcus pyogenes (Group A Streptococcus, GAS) [26]. The author's demonstrated GAS host-pathogen interactions by analyzing the expression of 17 GAS genes in throat swab specimens sampled from 18 pediatric patients with pharyngitis. Several known and putative virulence genes and regulatory genes were highly expressed during infection [26]; and ii) In a study with the periodontal pathogen Porphyromonas gingivalis, Shelbourne et al. demonstrated a clear correlation between periodontal disease status and elevated expression of dnaK and htpG encoding general stress response proteins [27]; iii) while another study established the expression of an uncharacterized gene in P. gingivalis (G1334) as more frequent in diseased sites compared to healthy sites [28]. The importance of the G1334 gene in virulence was confirmed in a mouse abscess model of infection [28]. The here presented expression of a community-encoded core gene of oral biofilm formation and the single-species focus by other groups detecting virulence and stress related gene expression in vivo demonstrate the feasibility to specifically determine the expression of genes of interest in their ecological context.
Limitations of the study became apparent when the RIN where determined. Although all samples where processed after the same protocol, RNA degradation was a problem for some samples. Two RNA samples were severely degraded and not further considered, while others showed varying degrees of degradation. In general, an RIN of 10 would indicate no degradation. RIN greater than five indicates good total RNA quality for reverse transcription [29]. Some of the samples were close to 5 and therefore would be considered not ideal for spxB expression quantification. Nonetheless, we used the total RNA of subject 3 and 4 for cDNA synthesis to determine spxB expression. The respective relative expression of spxB from subject 3 and 4 were among the lower spectrum, however, subject 5 and 10 showed a similar relative expression level albeit higher RIN. Another caveat of this study lies in the fact that the oral microbial diversity among subjects varies [30] and the spxB sequences will have sequence dissimilarities. This will result in nucleotide mis-pairing of the spxB oligonucleotides used for spxB expression. As a consequence, non-ideal PCR amplification conditions will occur due to different primer efficiencies when plaque samples with undefined species composition are used. This might be prevented in the future if more spxB sequences become available to optimize oligonucleotides for amplification. In addition, the main advantage we take in our approach is the wide distribution of a highly conserved gene among the most prominent genus in the oral biofilm. If other similar important genes can be identified needs to be determined.
In summary, we showed in vivo expression of an oral biofilm community encoded gene in its ecological context, thus suggesting an active role of spxB in oral biofilm physiology. This study demonstrates how the HOMD database can be used to determine a dynamic relationship or the functionality of the potential encoded in the metagenome of the oral biofilm.