ORIGINAL RESEARCH

published: 08 June 2017

doi: 10.3389/fmicb.2017.01036

RNA-Seq Reveals Enhanced Sugar

Metabolism in Streptococcus mutans
Co-cultured with Candida albicans
within Mixed-Species Biofilms
Jinzhi He 1,2† , Dongyeop Kim 2† , Xuedong Zhou 1 , Sang-Joon Ahn 3 , Robert A. Burne 3 ,
Vincent P. Richards 4 and Hyun Koo 2*
1
State Key Laboratory of Oral Diseases, Department of Endodontics, West China Hospital of Stomatology, Sichuan
University, Chengdu, China, 2 Biofilm Research Labs, Levy Center for Oral Health, Department of Orthodontics, School of
Dental Medicine, University of Pennsylvania, Philadelphia, PA, United States, 3 Department of Oral Biology, College of
Dentistry, University of Florida, Gainesville, FL, United States, 4 Department of Biological Sciences, Clemson University,
Clemson, SC, United States

Early childhood caries (ECC), which can lead to rampant tooth-decay that is painful
and costly to treat, is one of the most prevalent infectious diseases affecting children
worldwide. Previous studies support that interactions between Streptococcus mutans
Edited by:
and Candida albicans are associated with the pathogenesis of ECC. The presence of
Ivan Mijakovic, Candida enhances S. mutans growth, fitness and accumulation within biofilms in vitro,
Chalmers University of Technology, although the molecular basis for these behaviors is undefined. Using an established
Sweden
co-cultivation biofilm model and RNA-Seq, we investigated how C. albicans influences
Reviewed by:
Moshe Shemesh, the transcriptome of S. mutans. The presence of C. albicans dramatically altered gene
Agricultural Research Organization, expression in S. mutans in the dual-species biofilm, resulting in 393 genes differentially
Israel
Wiep Klaas Smits,
expressed, compared to mono-species biofilms of S. mutans. By Gene Ontology
Leiden University, Netherlands analysis, the majority of up-regulated genes were related to carbohydrate transport
*Correspondence: and metabolic/catabolic processes. KEGG pathway impact analysis showed elevated
Hyun Koo pyruvate and galactose metabolism, suggesting that co-cultivation with C. albicans
[email protected]
† These
influences carbohydrate utilization by S. mutans. Analysis of metabolites confirmed
authors have contributed
equally as co-first author. the increases in carbohydrate metabolism, with elevated amounts of formate in the
culture medium of co-cultured biofilms. Moreover, co-cultivation with C. albicans altered
Specialty section:
transcription of S. mutans signal transduction (comC and ciaRH) genes associated with
This article was submitted to
Microbial Physiology and Metabolism, fitness and virulence. Interestingly, the expression of genes for mutacins (bacteriocins)
a section of the journal and CRISPR were down-regulated. Collectively, the data provide a comprehensive
Frontiers in Microbiology
insight into S. mutans transcriptomic changes induced by C. albicans, and offer novel
Received: 13 February 2017
Accepted: 23 May 2017
insights into how bacterial–fungal interactions may enhance the severity of dental caries.
Published: 08 June 2017
Keywords: early childhood caries, biofilms, Streptococcus mutans, Candida albicans, transcriptome, RNA-Seq
Citation:
He J, Kim D, Zhou X, Ahn S-J,
Burne RA, Richards VP and Koo H
(2017) RNA-Seq Reveals Enhanced
INTRODUCTION
Sugar Metabolism in Streptococcus
mutans Co-cultured with Candida
Biofilms are associated with many infectious diseases in humans, including those occurring in
albicans within Mixed-Species the mouth (Hall-Stoodley et al., 2004). Early childhood caries (ECC) is a highly prevalent and
Biofilms. Front. Microbiol. 8:1036. difficult to treat biofilm-dependent disease, afflicting mostly underprivileged children worldwide
doi: 10.3389/fmicb.2017.01036 and resulting in estimated annual expenditures of more than $120 billion in the United States

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

alone (Kassebaum et al., 2015). Children with ECC are heavily annotation/detection of bacterial transcripts and interpretation
infected with Streptococcus mutans, due in large part to protracted of genomic data (Croucher and Thomson, 2010; Zeng et al.,
feeding of dietary sugars, such as sucrose (Berkowitz et al., 2013), including in mixed-species biofilms (Dutton et al., 2016).
1984; Palmer et al., 2010; Parisotto et al., 2010), which leads Here, we investigate the impact of the presence of C. albicans
to rapid accumulation of virulent biofilms characterized by an on the whole transcriptome of S. mutans using RNA-Seq and
exopolysaccharides (EPS)-rich and highly acidic milieu that cause systems analysis. We first optimized enrichment of S. mutans
rampant destruction of the teeth (Takahashi and Nyvad, 2011; mRNA from bacterial–fungal mixed total RNA, and then
Hajishengallis et al., 2017). used RNA-Seq to transcription profile enriched mRNAs from
Streptococcus mutans has long been regarded one of the key single- and dual-species biofilm. The results show that the
etiologic agents of ECC. S. mutans possesses an exceptional ability presence of C. albicans dramatically altered the transcriptome
to produce EPS using dietary sucrose via secreted exoenzymes of S. mutans. Gene Ontology (GO) and Kyoto Encyclopedia
termed glucosyltransferases (Gtfs), as well as being robustly of Genes and Genomes (KEGG) pathway impact analyses
acidogenic and acid-tolerant (Marsh et al., 2011; Koo et al., 2013). supported that co-culturing of S. mutans with C. albicans
In addition, S. mutans can efficiently cope with environmental enhanced carbohydrate metabolism by S. mutans. Carbohydrate
stresses, which contributes to its ability to establish biofilms, to and metabolites analysis confirmed increased sugar utilization
persist in the host, and to compete with other oral bacteria, and elevated levels of formate in the supernatant fluid of
particularly when conditions are conducive to initiation and dual-species biofilms. Moreover, we also found that C. albicans
progression of dental caries (Lemos and Burne, 2008). However, alters the transcription of two-component signal transduction
S. mutans does not act alone in cariogenic biofilms, as additional systems that are important for fitness and sucrose-dependent
organisms can also contribute to the initiation and/or progression biofilm formation. Conversely, mutacin (bacteriocin) production
of caries (Tanner et al., 2011; Gross et al., 2012). Interestingly, was down-regulated, which could influence the bacterial
results from several clinical studies reveal that Candida albicans composition of the biofilms formed when C. albicans is present.
is often detected in high numbers with S. mutans in biofilms from Collectively, this study provides new insights into the effects of
children with ECC (de Carvalho et al., 2006; Raja et al., 2010; Yang an opportunistic fungus (C. albicans) on the expression of genes
et al., 2012; Klinke et al., 2014; Qiu et al., 2015). that are integral to the persistence and virulence of S. mutans
Candida albicans is a commonly detected opportunistic and how these inter-kingdom interactions may modulate the
fungus in the oral cavity (Ghannoum et al., 2010). This organism pathogenic potential of biofilms in ECC.
interacts actively with commensal (viridans) streptococci and
forms biofilms on acrylic and mucosal surfaces (Jenkinson and
Douglas, 2002; Diaz et al., 2012) to cause oral mucosal infections MATERIALS AND METHODS
(Thein et al., 2009; Xu et al., 2014). In contrast, C. albicans
does not bind well to S. mutans, nor does it colonize teeth Bacterial Strains and Growth Conditions
effectively on its own (Jenkinson et al., 1990; Gregoire et al., Streptococcus mutans strain UA159 serotype c [a cariogenic
2011). However, physical co-adhesion between S. mutans and bacterial pathogen (genome sequence accession number
C. albicans is markedly increased in the presence of sucrose AE014133)] and C. albicans SC5314 (genome sequence accession
(Branting et al., 1989; Gregoire et al., 2011; Metwalli et al., 2013; number CP017630) were used in the present study to generate
Falsetta et al., 2014). S. mutans Gtfs are capable of adhering to single and dual-species biofilm. Both strains were stored at
the surface of C. albicans and producing large amounts of EPS −80◦ C in tryptic soy broth containing 20% glycerol.
in situ using sucrose as substrate (Gregoire et al., 2011; Hwang
et al., 2015). In turn, the EPS on the fungal surface promotes Biofilm Preparation
adhesive interactions and cross-kingdom biofilm development Biofilms were formed using a saliva-coated hydroxyapatite (sHA)
with S. mutans (Falsetta et al., 2014). disk model, as described elsewhere (Koo et al., 2010; Falsetta et al.,
In biofilms formed in vitro, the presence of C. albicans 2014). Briefly, the hydroxyapatite disks (1.25 cm in diameter,
dramatically modifies the physical environment by increasing surface area of 2.7 ± 0.2 cm2 ; Clarkson Chromatography
biomass and EPS production, enhancing biofilm accumulation Products, Inc., South Williamsport, PA, United States) were
and stability (Falsetta et al., 2014). Furthermore, C. albicans coated with filter-sterilized, clarified whole saliva and vertically
appears to activate S. mutans genes associated with biofilm suspended in 24-well plates using a custom-made wire disk
formation and genetic competence (Falsetta et al., 2014; Sztajer holder (Koo et al., 2010). For single-species biofilms, each disk
et al., 2014). Importantly, using a rodent model of the disease was inoculated with approximately 2 × 106 CFU/mL of S. mutans
and a diet rich in sucrose, enhanced levels of S. mutans in ultrafiltered (10-kDa cutoff; Millipore, Billerica, MA, United
in plaque-biofilms were associated with co-infection with States) tryptone-yeast extract broth (UFTYE; 2.5% tryptone and
C. albicans, which lead to onset of rampant caries similar to 1.5% yeast extract, pH 7.0) containing 1% sucrose (37◦ C, 5%
ECC (Falsetta et al., 2014). However, the molecular pathways by CO2 ). For dual-species biofilms, approximately 2 × 104 CFU/mL
which such interactions stimulate S. mutans growth/metabolism, of C. albicans containing predominantly yeast cell forms was also
accumulation and virulence remain unclear. added to the inoculum; the composition of the microorganisms
Recently, RNA sequencing (RNA-Seq) combined with in the inoculum is similar to that found in saliva samples from
integrated gene network-pathway analysis greatly enhanced children with ECC (de Carvalho et al., 2006; Falsetta et al., 2014).

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

During the first 18 h, the organisms were grown undisturbed so RNA mix was heated to 70◦ C for 10 min then incubated at 37◦ C
as to allow initial biofilm formation; the culture medium was then for 15 min to hybridize the capture oligos. The RNA/capture
changed twice daily at 8 a.m. and 6 p.m. until the end of the oligo mix was equilibrated with Oligo MagBeads and incubated
experimental period (42 h). at 37◦ C for 15 min. Tubes were placed on a magnet to separate
the supernates containing the enriched bacterial total RNA from
RNA Extraction and Purification the Oligo MagBeads. The enriched bacterial RNA was purified
Biofilms were harvested after 42 h incubation. RNA was and concentrated by ethanol precipitation. The final quality
extracted and purified using protocols optimized for biofilms of enriched bacterial mRNA samples was analyzed using an
formed in vitro (Cury and Koo, 2007). Three separate biological Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA,
replicates for each group (single and mixed-species) were United States). The efficiency of Ribo-ZeroTM rRNA Removal
performed. Briefly, disk sets were incubated in RNALater and MICROBExpressTM Kits were compared and the results are
(Applied Biosystems/Ambion, Austin, TX, United States), then included in the Supplementary Figure S1. Based on experimental
the biomass was removed from the sHA disks. RNAs were data, we selected MICROBEnrich + Ribo-Zero as an optimized
purified and treated with DNase on a column using the Qiagen method to enrich S. mutans mRNA from mixed bacterial–fungal
RNeasy Mini kit (Qiagen, Valencia, CA, United States). The RNA samples.
RNAs were then subjected to a second DNase I treatment cDNA libraries were generated from the enriched mRNA
with Turbo DNase (Applied Biosystems/Ambion) and were samples using NEBNext Ultra directional RNA library prep
purified using the Qiagen RNeasy MinElute cleanup kit kit for Illumina and NEBNext multiplex oligonucleotides
(Qiagen). RNAs were quantified using the NanoDrop ND-1000 for Illumina (New England BioLabs, Ipswich, MA, United
spectrophotometer (Thermo Scientific/NanoDrop, Wilmington, States), following instructions from the supplier. RNA-Seq
DE, United States). RNA quality was evaluated using an Agilent was performed on the NextSeq500 (75-bp single end reads)
2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, by the NextGen DNA Sequencing Core Laboratory of the
United States), and all RNAs used for downstream experiments Interdisciplinary Center for Biotechnology Research at the
were determined to have RNA integrity numbers (RIN) of 9.5 and University of Florida (Gainesville, FL, United States). Read
above. mapping was performed on a Galaxy server hosted by the high-
performance research computing center at the University of
Florida (HiPerGator2.0) using Map with Bowtie for Illumina
Bacterial mRNA Enrichment and (version 1.1.2). Reads were mapped to the S. mutans UA159
RNA-Seq Performance genome. Mapped reads were then counted using the Python
To deplete fungal total RNA, the MICROBEnrichTM Kit (Ambion script htseq-count (Anders et al., 2015).
of Life Technologies, Grand Island, NY, United States) was used
with modifications. Briefly, RNA was combined with binding Statistical Analysis of RNA-Seq Data
buffer and capture oligonucleotide mix. The RNA mix was heated Fold changes and significant differences in gene expression
to 70◦ C for 10 min then incubated at 37◦ C for 1 h to hybridize to between growth conditions were calculated using the
the capture oligos. The RNA/capture oligo mix was equilibrated convergence of three separate approaches: DEseq, edgeR,
with Oligo MagBeads and incubated at 37◦ C for 15 min. and limma (Anders and Huber, 2010; Robinson et al., 2010; Law
Tubes were placed on a magnet to separate the supernatant et al., 2014), as implemented in the R/Bioconductor package
fluids containing the enriched bacterial total RNA from the metaseqR (Moulos and Hatzis, 2015). GO terms were assigned
Oligo MagBeads. The enriched bacterial RNA was purified to genes using Blast2GO v.2.5.0 (Gotz et al., 2008). Relative
and concentrated by ethanol precipitation. Ribo-ZeroTM rRNA enrichment (overrepresentation) of GO terms for up-regulated
Removal Kits for Gram-Positive Bacteria (Epicentre, Madison, genes compared to a background of GO terms for all genes was
WI, United States) and MICROBExpressTM Kit (Ambion) were assessed using Fisher exact tests. The test was performed using
tested for their efficiency of depleting the bacterial ribosomal the Gossip statistical package (Blüthgen et al., 2005) implemented
RNA according to the supplier’s specifications. (1) RNA input within Blast2GO. The false discovery rate (FDR) procedure of
amounts determined the amount of Ribo-Zero rRNA removal Benjamini and Hochberg (1995) was used to correct for multiple
solution to add (10 µL rRNA removal solution for 2.5 to 5 µg, hypothesis testing (FDR = 0.05).
or 8 µL for <2.5 µg total RNA per reaction). Samples in Ribo- To gain further insights into the effects of co-cultivation
Zero rRNA removal solution were incubated at 68◦ C for 10 min of C. albicans on S. mutans, we performed a KEGG pathway
followed by a 15 min incubation at room temperature. To remove impact analysis using the software package Pathway-Express
the hybridized rRNA molecules from the mRNA, the RNA/rRNA as implemented in the R/Bioconductor package ROntoTools
solution reactions were incubated with the prepared microsphere (Calin and Draghici, 2016). A systems biology approach such
beads, mixed well and placed at room temperature for 10 min, as this has the advantage of being able to factor the complex
then at 50◦ C for 10 min. The mRNAs were separated from the interactions among genes. It combines evidence from traditional
microspheres bound with rRNAs by a filter column provided in expression level data with information regarding the dynamics
the kit. The final purification of eluted mRNA was performed by of gene–gene interaction and the relative position of the
ethanol precipitation. (2) For MICROBExpressTM Kit, RNA was gene within a pathway. Positional information is important
mixed with binding buffer and capture oligonucleotide mix. The as the action of genes up-stream in a pathway can propagate

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

further down-stream and amplify effects of changes in gene gene expression when co-cultured with C. albicans in the
expression. By combining all evidence, pathway impact can presence of sucrose (Falsetta et al., 2014; Sztajer et al., 2014).
be calculated. FDR procedure of Benjamini and Hochberg However, whole bacterial transcriptome characterization of
(1995) was again used to correct for multiple hypothesis testing effects of the presence of C. albicans in the dual-species
(FDR = 0.05). biofilm milieu in combination with gene network pathway
analysis is needed to gain a comprehensive picture of the
Data Validation underlying molecular mechanisms. To achieve this, we used our
To validate the RNA-Seq data, quantitative real-time PCR extensively optimized RNA extraction and mRNA enrichment
(qRT-PCR) was employed to measure changes in the amount protocol. Total RNA was isolated either from S. mutans
of mRNA of selected genes. The cDNA was synthesized from single-species or S. mutans–C. albicans co-cultivation biofilms,
1 µg of purified RNA with Bio-Rad iScript cDNA synthesis and prokaryotic mRNA was enriched as described above and in
kit (Bio-Rad Laboratories, Inc., Hercules, CA, United States), the Supplementary Figure S1. A total of 587909293 reads were
and quantitative amplification condition using Bio-Rad iTaq produced from the six samples. The sequence reads of all samples
Universal SYBR Green Supermix and Bio-Rad CFX96 system were deposited in the NCBI sequence read archive (SRA) as a
(Bio-Rad Laboratories, Inc.). Standard curves for each primer study under the accession number of (SRR5116699-5116704).
were used to determine the relative number of cDNA molecules, We first applied Multi-Dimensional Scaling (MDS) to provide a
and relative expression was calculated by normalizing to the visual representation of the transcriptomic similarities between
gyrA gene transcripts, which is a validated reference gene for dual- and single-species biofilms. Samples marked with distinct
normalization of qRT-PCR (Rocha et al., 2015; Zeng and Burne, colors were projected to a two-dimensional space and clustered
2016). The minimum information for publication of qRT-PCR separately (Figure 1A), indicating high levels of correlation
experiments (MIQE) guidelines were followed for quality control and reproducibility among samples, as well as distinctive
of the data generated and for data analysis (Bustin et al., transcriptome profiles from S. mutans in the presence or absence
2009). of C. albicans. Three statistical methods, including DEseq, edgeR,
and limma, were used to pinpoint differentially expressed genes
(DEGs) between groups.
Carbohydrates and Metabolites Analyses
Overall, 393 genes showed significant differences in expression
The biofilm and the respective surrounding culture medium were
between single and dual-species biofilm for all three statistical
collected at 42 h, homogenized via sonication and centrifuged
methods with log2 (fold change) > 0.6 or < −0.7 (Supplementary
at 5,500 × g for 10 min at 4◦ C. The supernatant was filtered
Table S1), accounting for ∼20% (393/2042) of the total genes
through 0.2 µm-pore-size membrane filter (ultra-low protein
annotated in S. mutans UA159. Among the DEGs, 134 were
binding, surfactant-free cellulose acetate, Nalgene, Rochester, NY,
up-regulated, 259 were down-regulated, and about 40% (158/393
United States). The amount of sucrose, glucose, and fructose
genes) were of unknown function or hypothetical. The genes
in the supernatant were quantified using high-performance
encoding the four-enzyme pyruvate dehydrogenase complex
anion-exchange chromatography (HPAEC; Dionex, Sunnyvale,
[pdhD (SMU_1424)-pdhA (SMU_1423)-pdhB (SMU_1422)-
CA, United States) and the biofilm-derived metabolites were
pdhC (SMU_1421)] and adhE (SMU_148) showed the highest
identified and quantified through 1 H nuclear magnetic resonance
up-regulation in mixed-species biofilms (fold change > 4.5,
(1 H-NMR; Bruker Avance III HD NMR spectrometer, Bruker
Figure 1B). All of these five genes are part of the pyruvate
Biospin, Billerica, MA, United States) as described previously
metabolism pathway, converting pyruvate to acetyl-CoA in
(Kim et al., 2017). The significance was determined by direct
cells growing in aerobic conditions. The gene (SMU_2133c)
comparison with concentration in the blank (original UFTYE
marked with a question mark in Figure 1B is not part of
medium) and substrates and metabolites are characterized by the
the pyruvate pathway and has an ambiguous annotation:
calculation of fold changes (log2 ) relative to the blank (UFTYE
hypothetical protein, transmembrane protein, or phage infection
medium). The concentrations of glucose, fructose, formate, and
protein. Based on the RNA-seq data, we selected 10 DEGs
lactate in the supernatant of single- and dual-species biofilms
(three down-regulated and seven up-regulated) showing a broad
were subtracted with the values of blank. A pairwise comparison
range of differential expression for validation by using qRT-PCR
(non-parametric Mann–Whitney U test or parametric t-test)
analysis. Consistent with the RNA-Seq data, the qPCR data
was performed using SPSS 18.0 software (IBM Co., Armonk,
showed significant differential expression of all genes tested
NY, United States). Differences are considered significant with
∗ P < 0.05 or ∗∗∗ P < 0.001. (Table 1) and a linear-correlation with RNA-seq data (r2 = 0.98).
We noted that the level of differential expression of some genes
(e.g., gtfB, gtfC) was not entirely similar to that reported in our
previous work (Falsetta et al., 2014). Differences between the
RESULTS AND DISCUSSION two studies may be due to several factors, including different
RNA sources/preparation (total RNA vs. rRNA depleted/mRNA
Transcriptomic Changes of S. mutans enriched) and algorithm/data analysis to calculate fold changes.
within Mixed-Species Biofilms Despite differences in the level of gene expression, both studies
Results from previous studies have shown enhanced S. mutans confirmed up-regulation of gtfBC in dual-species biofilm
growth and biofilm formation as well as alterations in (vs. single-species S. mutans biofilm).

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

FIGURE 1 | Overall transcriptomic changes of S. mutans within S. mutans–C. albicans dual biofilm. (A) Multi-Dimensional Scaling (MDS) plot based on Euclidean
distances derived from a sample variance parameter, showing the level of correlation and reproducibility among samples. Red circles (D) show dual biofilm, whereas
blue triangles (S) show single. Samples from single and dual biofilm group clustered together, respectively, indicating the different transcriptome pattern of S. mutans
with and without C. albicans. (B) Plot showing fold change and levels of significance for differential expression for all genes.

Gene Ontology terms were assigned to all genes in the biofilm (vs. single-species S. mutans biofilm) as determined by
S. mutans genome. We compared terms for the up- and chromatographic analyses (Figure 2A). Since Candida is rather
down-regulated genes to a background of all terms to obtain an inefficient in metabolizing sucrose (Williamson et al., 1993),
overall insight into the impact of C. albicans on S. mutans when S. mutans can cross-feed sucrose break-down products (glucose
growing together in dual-species biofilms. Forty-two GO terms and fructose) to C. albicans (Sztajer et al., 2014; Kim et al., 2017).
were overrepresented (enriched) (Supplementary Table S2): We observed that the concentrations of glucose and fructose in
among them, 22 were involved with biological processes, of the supernatant of dual-species biofilm are significantly lower
which 10 with up-regulated genes and 12 with down-regulated than those in single-species S. mutans biofilm (Figure 2A-a2;
genes (Table 2). Notably, all the GO terms for up-regulated P < 0.05). The data indicate that S. mutans co-cultured with
genes belonged to the biological process domain and were C. albicans utilized most of the fermentable sugars, while also
involved in carbohydrate transport and metabolic/catabolic increasing the levels of formate (Figure 2B). Hence, once they
process. These findings are interesting since sugar catabolism are together within biofilm, these organisms may cooperate with
is a key risk factor for dental caries (Selwitz et al., 2007), and each other for provision of sugar substrates and metabolites.
are consistent with enhanced sugar utilization in dual-species Conversely, enhanced sugar utilization can also cause localized
carbohydrate limitation in the presence of Candida that could

TABLE 1 | Validation of RNA-Seq data by qPCR.

TABLE 2 | GO terms for biological processes with up and down-regulated genes
Gene Fold change (dual/single) for S. mutans grown with C. albicans.

RNA-seq qPCRa (normalized by gyrA) Up regulated biological process Down regulated biological process

comC 0.46 0.30 ± 0.03 Disaccharide metabolic process Translation

luxS 0.60 0.60 ± 0.04 Oligosaccharide metabolic process Multi-organism process
atpB 0.62 0.64 ± 0.03 Cellular carbohydrate catabolic process Cellular protein metabolic process
hrcA 1.57 1.57 ± 0.09 Oligosaccharide catabolic process Response to external biotic stimulus
SMU.104 1.80 1.77 ± 0.14 Disaccharide catabolic process Response to other organism
ciaR 2.04 2.06 ± 0.21 Phosphoenolpyruvate-dependent sugar Response to biotic stimulus
lacC 2.25 1.83 ± 0.09 phosphotransferase system Defense response to other organism
gbpC 2.26 2.36 ± 0.16 Lactose metabolic process Response to external stimulus
adhE 4.49 3.90 ± 0.14 Carbohydrate metabolic process Defense response
pdhA 11.39 8.12 ± 1.60 Carbohydrate transport Protein metabolic process
a Changesin transcript levels were determined using gyrA as an internal control and Defense response to bacterium
qRT-PCR results are presented as averages ± standard deviations. Response to bacterium

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

FIGURE 2 | The composition of extracellular carbohydrates and metabolites. (A) HPAEC chromatograms of selected carbohydrates profile (a1) and the
concentrations of glucose and fructose as the main carbohydrates (sucrose is not detected in both supernatants) in biofilm-culture supernatants (a2). In the box
whisker plots, whiskers represent minimum and maximum, and the box represents the 25th and 75th percentiles (n = 3). ∗ P < 0.05. (B) Profiles of extracellular
metabolites (e.g., organic acids, alcohols, sugar alcohols, amino acids) (b1) and the concentrations of formate and lactate (red arrowheads) as the main organic
acids, which is associated with cariogenic properties of S. mutans, in biofilm-culture supernatants (b2). Substrates and metabolites are characterized by the
calculation of fold changes (log2 ) relative to the blank (fresh UFTYE medium). Data represent mean ± standard deviations (n = 3). ∗∗∗ P < 0.001.

influence the gene expression profile and the bacterial metabolic and the transcriptome pattern could be compartmentalized
pathways. within the biofilm. It is apparent that when S. mutans and
We also performed a KEGG pathway impact analysis based C. albicans are together within biofilms when conditions
on the sequencing data. The analysis detected eight KEGG are conducive for ECC, the presence of Candida modulates
pathways that were significantly impacted: pyruvate metabolism, carbohydrate utilization while also creating carbohydrate-
galactose metabolism, butanoate metabolism, glycine, serine limiting conditions, both of which can activate the PDH
and threonine metabolism, propanoate metabolism, glyoxylate pathway.
and dicarboxylate metabolism, oxidative phosphorylation, and
amino sugar and nucleotide sugar metabolism (Figure 3 and
Supplementary Table S3). Highly consistent with GO analysis,
Co-culturing with C. albicans Modulates
most of the impacted pathways were involved in carbohydrates Carbohydrate Utilization by S. mutans
metabolism. The end products of sugar fermentation are The increased sugar metabolism can explain in part the
energy generation and predominantly organic acids, which increased carriage of S. mutans and C. albicans and enhanced
can provide advantages for S. mutans survival and growth, virulence of plaque-biofilms in vivo (Falsetta et al., 2014).
while acidification of the environment helps both S. mutans Sucrose, in particular, has long been considered the most
and C. albicans (highly acid-tolerant organisms) to outcompete cariogenic of all carbohydrates. This disaccharide serves as
beneficial commensal bacteria (Burne and Marquis, 2001; both a readily metabolizable carbon and energy source and
Klinke et al., 2009). This metabolic cooperation provides as an essential substrate for the synthesis of the adhesive
an effective mechanism that promotes co-existence while extracellular glucan matrix by S. mutans, which strengthens
enhancing S. mutans accumulation (Falsetta et al., 2014; Kim the interactions between S. mutans and C. albicans and
et al., 2017). At the same time, the bacterial population is augments the stability of biofilms containing these organisms
probably heterogeneous with respect to carbohydrate utilization, (Gregoire et al., 2011; Falsetta et al., 2014). In addition to

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

FIGURE 3 | Plot showing result of a pathway impact analysis as implemented in Pathway-Express. The y-axis shows evidence for over-representation of differentially
expressed genes in a pathway and the x-axis shows perturbation evidence (measured expression changes propagating across the pathway topology).
A combination of the factors on each axis equates to the level of impact and the diagonal line represents a significance threshold (α = 0.05). After FDR correction,
eight KEGG pathways (red dots) remained significant. Two pathways (pyruvate and galactose metabolism) at the extremes of the axes showed the most impact.

sucrose utilization extracellularly (Bowen and Koo, 2011), single species S. mutans; Supplementary Table S1). The scrA
S. mutans rapidly transports sucrose into the cell by the gene encodes a high-affinity sucrose PTS permease, EIIScr , that
phosphoenolpyruvate:sugar phosphotransferase system (PTS) internalizes sucrose as sucrose-6-phosphate (Sato et al., 1989).
(Ajdić and Pham, 2007; Moye et al., 2014). Here, we observed The ScrB enzyme is a sucrose-6-PO4 hydrolase that produces
that scrA (SMU_1841), scrB (SMU_1843), and scrK (SMU_1840) glucose-6-PO4 and fructose (Figure 4). After phosphorylation
were significantly up-regulated in dual-species biofilms (vs. of fructose to fructose-6-phosphate by a fructokinase (scrK),

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

FIGURE 4 | KEGG galactose metabolism pathway map (smu:00052) for S. mutans UA159. S. mutans genes involved in the pathway are shown in green. Of these,
eleven showed differential expression. The nine bordered in red were up-regulated and the one bordered in blue was down-regulated. Six of the seven genes
comprising the galactose pathway for lactose catabolism (lacE, lacF, lacG, lacA, lacB, lacC, and lacD) were up-regulated, and two genes of Leloir pathway (galK and
galT) were up-regulated. Numbers inside boxes are enzyme commission numbers.

the phosphorylated products are channeled into the glycolytic (GlcNAc) (Kamthan et al., 2013), which is a ubiquitous dietary
pathway (Chassy and Victoria Porter, 1979). Furthermore, pttB sugar and also produced through bacterial biosynthesis (Moye
(SMU_2038), encoding a trehalose PTS permease, was also et al., 2014). We found detectable amounts of GlcNAc in
up-regulated in dual-species biofilms (Supplementary Table S1). the supernatant of biofilm cultures of S. mutans–C. albicans
Notably, the trehalose-PTS (i.e., EIITre ), the primary transporter [∼20 µM; compared to S. mutans alone (∼10 µM) (data
for trehalose (Poy and Jacobson, 1990), is also able to transport not shown)], which may help to explain, at least in part
sucrose, as mutants derived from S. mutans UA159 that this observation. S. mutans can metabolize galactose by two
lacked ScrA could still internalize sucrose via the PTS if an distinct pathways: the tagatose 6-phosphate pathway (de Vos and
intact EIITre was present (Zeng and Burne, 2013). Besides the Vaughan, 1994) and Leloir pathway (Ajdić et al., 1996). Previous
PTS, the multiple-sugar metabolism system (Msm) (Tao et al., studies have shown that S. mutans can efficiently metabolize when
1993) and the maltose/maltodextrin ABC transporter (Kilic both the tagatose 6-phosphate pathway and Leloir pathways
et al., 2007) have been also implicated in sucrose uptake by are functional, while the tagatose pathway is responsible
S. mutans, albeit not nearly as effectively as the sucrose PTS. predominantly for the utilization of the phosphorylated galactose
Still, both malG (SMU_1570) and malF (SMU_1569) encoding moiety that comes from the breakdown of lactose 6-phosphate
maltose/maltodextrin ABC transport permease, as well as malX (Abranches et al., 2004; Zeng et al., 2010).
(SMU_1568) encoding maltose/maltodextrin-binding protein In S. mutans, the genes encoding the tagatose 6-phosphate
were up-regulated in the presence of C. albicans (Supplementary pathway are arranged as part of the lac operon. When
Table S1). co-culturing with C. albicans, six of the seven genes comprising
Genes for galactose metabolism by S. mutans were also tagatose 6-phosphate pathway [lacE (SMU_1491), lacF
up-regulated in the presence of C. albicans, which could (SMU_1492), lacA (SMU_1496), lacB (SMU_1495), lacC
simply reflect relief of catabolite repression. Another possible (SMU_1494), and lacD (SMU_1493)] were up-regulated
explanation is that C. albicans can produce galactose via (Figure 4 and Supplementary Table S4), which are consistent
up-regulation of its metabolic pathway by N-acetylglucosamine with their sequential role in the galactose metabolism. The

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

galactose moiety of lactose, and possibly galactose alone, changes that enhance carbohydrate utilization (vs. single-species
can be transported and phosphorylated by a lactose-specific S. mutans biofilm). Such a scenario would also be consistent with
(LacEF) PTS. The resultant galactose 6-phosphate generated activation of the PDH pathway, since S. mutans growing with
by an intracellular 6-phospho-β-galactosidase is converted into excess carbohydrate predominantly shunt carbon through LDH.
tagatose 6-phosphate, then to tagatose-1,6-bisphosphate, and Notably, the inactivation of pdh impairs the survival of S. mutans
then to glyceraldehyde 3-phosphate and dihydroxyacetone by in limiting sugar conditions in stationary phase (Busuioc et al.,
the enzymes galactose-6-phosphate isomerase (lacAB), tagatose 2010), such that activation of pdh could be important for survival
6-phosphate kinase (lacC), and tagatose-1,6-bisphosphate or persistence of S. mutans or a sub-population of S. mutans in
aldolase (lacD), respectively (Abranches et al., 2004). the mixed-culture system employed here.
Furthermore, two genes of Leloir pathway [galK (SMU_886) and In addition to producing energy for growth and anabolic
galT (SMU_887)] were also up-regulated (Figure 4), although processes, the pdh operon has also been shown to be vital
the GalK pathway is minor and not as efficient as the tagatose for survival of sugar-starved S. mutans (Busuioc et al.,
pathway in S. mutans (Abranches et al., 2004). In the Leloir 2010) and its acid tolerance (Korithoski et al., 2008), which
pathway, galactose enters the cell via an unidentified permease, are critical virulence properties within cariogenic biofilms.
where it is phosphorylated by galactokinase (galK) to yield When carbohydrates are in excess, the LDH enzyme is
galactose 1-phosphate, which is then converted into glucose allosterically activated by fructose-1,6-biphosphate, a glycolytic
1-phosphate by hexose1-phosphate uridyltransferase (galT) intermediate, to catalyze the conversion of pyruvate to lactate
and UDP-glucose epimerase [galE (SMU_888)]. The resulting via generating NAD+ from NADH (homolactic fermentation).
glucose 1-phosphate can enter the glycolytic pathway. Notably, LDH would be less active in carbohydrate-limiting
conditions and utilization of galactose via the tagatose pathway
(which is activated in dual-species) bypasses the production
Pyruvate Metabolism of S. mutans in of the intermediates that regulate carbohydrate regulation
Dual-Species Biofilms (F-1,6-bP, G-6-P) (Zeng et al., 2010). However, in carbohydrate-
The transcriptomic data suggest that C. albicans promotes liming conditions, the metabolic shift between pyruvate and
S. mutans sugar utilization, leading to induction and/or formate is controlled by PFL, which can convert pyruvate to
derepression of genes for multiple catabolic pathways via inputs formate and acetyl-CoA via up-regulated pyruvate metabolism
from global and specific regulatory systems. Among them, (heterolactic fermentation). Acetyl-CoA can be further converted
pyruvate metabolism is an important mechanism for S. mutans to acetyl-phosphate (acetyl-P), which can be used for the
survival and expression of virulence within cariogenic biofilms production of ATP via acetate kinase. Importantly, PFL appears
that balances the need for ATP with maintenance of NAD/NADH to have a key role in pyruvate metabolism of S. mutans residing
ratios and for carbon for amino acid biosynthesis (Kim et al., within natural dental biofilm (Abbe et al., 1982).
2015). Our pathway impact analysis shows that the pyruvate We detected higher concentrations of extracellular formate
metabolism is substantially altered when S. mutans is growing in in dual-species biofilms (∼five-fold increase vs. single-species)
the presence of C. albicans, compared to single-species biofilms as determined via 1 H-NMR (P < 0.001, Figure 2B-b2), which
of S. mutans (Figures 1B, 5 and Supplementary Table S4). All was consistent with the increased expression of the genes for
the genes in the pdh operon (pdhD–pdhA–pdhB–pdhC), as well as PFL. In contrast, ldh expression between dual-species and single-
pfl2 (SMU_493) encoding PFL (Figure 5) and pflA (SMU_1692) species S. mutans biofilms was unaffected and similar amounts
encoding PFL activating enzyme (Supplementary Table S4) of lactate were found in the culture medium (Figure 2B-b2).
were up-regulated in dual-species biofilm (vs. single-species Formate, a stronger acid (pKa = 3.75 vs. pKa of lactate = 3.86),
S. mutans). It is conceivable that pyruvate metabolism was has been detected in significant amounts in resting cariogenic
different between single- and dual-biofilms since both the glucose plaque in humans (Distler and Kröncke, 1986). Thus, induction
and fructose from sucrose and galactose catabolism lead to of S. mutans PDH and PFL by the fungal presence within
pyruvate production. biofilms may contribute to the enhanced cariogenicity observed
Pyruvate sits at an intersection of key pathways of sugar in vivo (Falsetta et al., 2014). Enhanced sugar metabolism induced
metabolism, and is converted by (1) pyruvate dehydrogenase by co-culturing of C. albicans combined with transcriptomic
complex (PDHc) into acetyl-CoA and CO2 , (2) pyruvate changes in PDH and PFL may offer at least one explanation
formate lyase (PFL) yielding acetyl-CoA and formate, and (3) for the carbohydrate limitation (Figure 2) and the observed
lactate dehydrogenase (LDH) into lactate that can be further gene expression patterns in dual-species biofilms. Moreover,
metabolized. PDH and PFL pathways are activated when PFL is inactivated by oxygen, whereas PDH production is
carbohydrates are not present in excess, suggesting that at least enhanced by growth in air. The up-regulation of PDH and
some cells in the dual-species biofilm might experience limitation PFL pathways also suggest the potential for complex and
for carbohydrate (Figure 2). Both GO and KEGG results indicate spatially heterogeneous gene expression patterns, whereby cells
S. mutans and C. albicans may be competing for the fermentable are carbohydrate-limited, but differences in exposure of cells
sugar available in the culture medium when grown together within certain regions of the biofilm may influence whether the
in biofilms, which could trigger derepression or activation of PFL pathway (anaerobic) is active or cells predominantly use
alternative transport and catabolic pathways of S. mutans, as PDH (aerobic) to favor acetate production and generation of
well as other adaptive mechanisms in response to environmental additional ATP.

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

FIGURE 5 | KEGG pyruvate metabolism pathway map (smu:00620) for S. mutans UA159. S. mutans genes involved in the pathway are shown in green. Of these,
eight showed differential expression. The six bordered in red were up-regulated and the one bordered in blue was down-regulated. At the center of the pathway,
showing strong up-regulation is the four-enzyme pyruvate dehydrogenase complex (pdhD–pdhA–pdhB–pdhC) (PDH).

C. albicans Can also Promote S. mutans competence stimulating peptide (CSP), as well as several
Fitness and Virulence through Signal late competence genes, including comYB (SMU_1985), comYD
(SMU_1983), comEA (SMU_625), and comEC (SMU_626) were
Transduction System down-regulated [log2 (fold change) < −0.7]. Factors regulating
Two-component signal transduction systems (TCSTS) involve the development of genetic competence have been shown to
phosphotransfer events between transmembrane sensor kinases influence acid tolerance, biofilm formation, eDNA release and
and cytoplasmic response regulators, which are transcription stress tolerance in general. In parallel, a recent microfluidic
factors that bind DNA to repress and/or activate gene expression study revealed that CSP signaling to induce competence is
(Stock et al., 2000). Currently, 14 TCSTS have been identified that
highly sensitive to pH and can be turned-off even in mildly
are able to enhance the ecological fitness and cariogenic potential
acidic conditions (<pH 6.0, Son et al., 2015). Thus, biofilm
of S. mutans (Smith and Spatafora, 2012). Genes encoding CiaRH
microenvironmental changes may down-regulate genes involved
[i.e., ciaR (SMU_1129) and ciaH (SMU_1128)] were up-regulated
in the presence of C. albicans (two-fold), and this system has with the development of genetic competence.
been implicated in acid tolerance, sucrose-dependent adherence
and biofilm formation by S. mutans (Qi et al., 2004; Ahn et al., Other Potential Interactions between
2006; Biswas et al., 2008). Although in a different bacterial-fungal S. mutans and C. albicans
biofilm system, Dutton et al. (2016) also observed that ciaR Another notable difference between mono- and dual-species
gene in S. gordonii was up-regulated in early-stage of interaction biofilms was the marked down-regulation of genes associated
with C. albicans. The gene comC (SMU_1915) encoding with the production of a suite of small antimicrobial peptides

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

FIGURE 6 | A snapshot of some of the transcriptome changes in S. mutans when co-cultured with C. albicans. Overall, the data reveal that the presence of
C. albicans enhances sugar metabolism and metabolic fitness, and alters competence gene expression in S. mutans. (A) Up-regulation of the scrA gene encoding a
high-affinity sucrose PTS permease (EIIScr ) and possibly in concert with the product of the pttB gene encoding the trehalose-PTS (EIITre ) facilitates internalization of
sucrose as sucrose-6-phosphate, with the glucose moiety carrying the phosphate group. Glucose-6-phosphate and fructose are produced via the action of ScrB,
and ScrK used ATP to convert the fructose to fructose-6-phosphate. Both of these phosphohexoses can enter the glycolytic pathway. When S. mutans is growing in
the presence of C. albicans, pyruvate metabolism can be affected by the availability of glycolytic intermediates. (B) The presence of C. albicans also appears to
up-regulate the production of the CiaRH two component signal transduction system (TCSTS), which modulates acid tolerance, sucrose-dependent adherence and
biofilm formation in S. mutans. (C) Another signaling system in S. mutans, the CSP-ComDE pathway, is affected by C. albicans. ComC (N) is secreted and
processed by ComAB to produce CSP (see text for more detail) and this peptide is then detected by the ComDE TCSTS. ComE directly activates bacteriocin
production and CSP indirectly stimulates the development of genetic competence, as well as influencing cell. Indeed, changes of CSP-comDE pathway via
down-regulation of comC can lead to down-regulation of downstream bacteriocin genes (e.g., nlmAB) and decreases in expression of late competence genes
(comYABCD). Red letters indicate up-regulated genes, blue letters show down-regulated genes.

(bacteriocins) termed mutacins by S. mutans when co-cultured has indeed been correlated with lower mutacin biological
with C. albicans (Table 2 and Supplementary Table S1). activity.
Since the expression of mutacin genes, which is regulated We observed significant down-regulation of comC, the
mainly by the CSP-ComDE system, is considered to be precursor for competence-stimulating peptide (CSP) that is a
important in the competition with early colonizers, including direct activator of mutacins via ComDE (van der Ploeg, 2005). It
S. sanguinis and S. gordonii (Merritt and Qi, 2012), a less was previous shown that C. albicans activated S. mutans comS and
robust activation of mutacin gene expression in S. mutans sigX (Sztajer et al., 2014), so the down-regulation of comC might
could alter the microbial composition of oral biofilms in a be perceived as inconsistent with this previous study. However,
way that promotes a symbiotic relationship between S. mutans ComCDE do not directly activate comS or sigX (comX). Indeed,
and C. albicans. Furthermore, previous studies reveal that the observed decrease of comC (the CSP precursor structure
deletion of genes [nlmA (SMU_150) and nlmB (SMU_151)] gene) expression is completely consistent with the fact that
encoding mutacins IV and V in S. mutans results in lower SMU_1914c, SMU_299c, SMU_1889c, SMU_423, SMU_150, and
antimicrobial activity against S. pyogenes (Hale et al., 2005; SMU_151, which encode bacteriocins or products required for
Hossain and Biswas, 2011), so decreased mutacin gene expression bacteriocin production, were also down-regulated. Bacteriocin

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

production is dominantly regulated by CSP, which acts directly benefits to C. albicans, such as enhanced colonization on the
through the ComDE TCS to activate bacteriocin gene expression tooth surface and cross-feeding sucrose break-down products
(van der Ploeg, 2005). Previous studies showed that CSP can (e.g., glucose) for fungal utilization. Conversely, S. mutans
regulate C. albicans growth and morphogenesis (Jarosz et al., could also impact C. albicans transcriptome based on recent
2009). It is possible that Candida, in addition to promoting observations that S. gordonii activate fungal genes associated
an acidic microenvironment, can produce several proteases that with filamentation and proteases (Dutton et al., 2016). We are
may inhibit CSP signaling system, similar to the inhibition of optimizing a protocol for both bacterial and fungal mRNA
S. mutans bacteriocin production by S. gordonii challisin (Wang enrichment from mixed-species biofilms for dual RNA-Seq
and Kuramitsu, 2005). Hence, the presence of C. albicans and its studies, which combined with metaproteomics, may provide
effects on the biofilm milieu could subvert mutacin production additional mechanistic explanations. Clearly, this bacterium–
to enhance its own persistence. Of note, the low pH created by fungus interaction is complex and multifaceted, and could induce
the combination of C. albicans and S. mutans may diminish the additional cross-kingdom responses and alter the surrounding
need for S. mutans to produce bacteriocins that mainly target biofilm microenvironment to modulate the cariogenic potential
comparatively acid-sensitive commensal streptococci. of biofilms. We are currently exploring how the fungal infection is
We also detected genes associated with clusters of acquired and how Candida responds to the presence of S. mutans
regularly interspaced short palindromic repeats (CRISPRs, i.e., in cariogenic biofilms. Enhanced colonization and increased
SMU_1760c, SMU_1761c, and SMU_1762c) of S. mutans that carriage of C. albicans in plaque biofilms may also provide a
were down-regulated in dual-species biofilms. Lack of CRISPR fungal reservoir that could promote Candida infections of oral
activity could be a direct reflection of decreased competence mucosal surfaces. Thus, inclusion of antifungals may be an
observed in mixed-species biofilms, which would decrease the important factor for devising more effective therapies to control
amount of DNA being internalized by S. mutans. CRISPR are ECC and its consequences.
involved primarily in antiviral defenses in prokaryotes (Brouns
et al., 2008). However, the role of S. mutans CRISPR in caries
development or in C. albicans–S. mutans interactions is presently AUTHOR CONTRIBUTIONS
unknown, but may warrant further investigation if the change in
CRISPR expression does reflect some response to the presence of JH, DK, S-JA, RB, VR, and HK conceived the experiments; JH,
fungi. DK, S-JA, and VR performed the experiments; JH, DK, XZ, S-JA,
In summary, the present study provides a comprehensive RB, VR, and HK analyzed the results and data interpretation; JH,
insight into S. mutans transcriptomic changes associated with DK, XZ, S-JA, RB, VR, and HK drafted and co-wrote the paper;
the presence of C. albicans within mixed-species biofilm. GO JH, DK, XZ, S-JA, RB, VR, and HK final approval of the version
term and KEGG pathway impact analysis support an active to be published; JH, DK, XZ, S-JA, RB, VR, and HK agreed to be
influence on S. mutans at the transcriptional level. The fungal accountable for all aspects of the work in ensuring that questions
presence modulates the expression of genes involved in S. mutans related to the accuracy or integrity of any part of the work are
sugar metabolism, fitness and survival within biofilms, offering appropriately investigated and resolved.
plausible explanations for the enhanced bacterial accumulation
and virulence of the bacterial-fungal biofilms in the context
of ECC (Figure 6). RNA-Seq provides a detailed ‘snapshot’ of FUNDING
the overall transcriptome changes, but there are limitations.
For example, RNA-Seq provide an average gene expression This work was supported in part by the National Institute for
profile at a given time-point without taking into consideration Dental and Craniofacial Research (NIDCR) grants DE025220 and
the substantial spatio-temporal heterogeneity that exists within DE018023 (HK).
complex biofilms. Nevertheless, future studies using specific
mutants of S. mutans can now be designed based on the
RNA-Seq data with the goal of comparing the behaviors of the ACKNOWLEDGMENTS
strains to interact and persist in co-culture system. This will
include testing different mutant strains with altered abilities We thank Dr. Arjun Sengupta and Dr. Byung-Hoo Lee for
in carbohydrate metabolism and/or respond to changes in metabolomics analyses. The authors are also grateful to Dr.
the environment caused by C. albicans presence, including Geelsu Hwang, Dr. Yuan Liu, and Dr. Yong Li for helpful
CcpA as well as mutacin production (nlmAB) and cell–cell discussions during the manuscript preparation.
communication (comDE). Furthermore, future studies using
recently developed single-cell in situ RNA-Seq (Lovatt et al., 2014)
and a bacterial–fungal nanoculture system (Kim et al., 2017) SUPPLEMENTARY MATERIAL
for localized gene expression may facilitate description of the
spatio-temporal transcriptome patterns within biofilms. The Supplementary Material for this article can be found
Although we focus on the influence of the presence of online at: http://journal.frontiersin.org/article/10.3389/fmicb.
C. albicans on S. mutans transcriptome, S. mutans also provides 2017.01036/full#supplementary-material

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

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He et al. Transcriptomic Changes of Streptococcus mutans Co-culturing with Candida albicans

Zeng, L., and Burne, R. A. (2016). Sucrose- and fructose-specific effects Conflict of Interest Statement: The authors declare that the research was
on the transcriptome of Streptococcus mutans probed by RNA conducted in the absence of any commercial or financial relationships that could
sequencing. Appl. Environ. Microbiol. 82, 146–156. doi: 10.1128/AEM. be construed as a potential conflict of interest.
02681-15
Zeng, L., Choi, S. C., Danko, C. G., Siepel, A., Stanhope, M. J., and Burne, R. A. Copyright © 2017 He, Kim, Zhou, Ahn, Burne, Richards and Koo. This is an open-
(2013). Gene regulation by CcpA and catabolite repression explored by RNA- access article distributed under the terms of the Creative Commons Attribution
Seq in Streptococcus mutans. PLoS ONE 8:e60465. doi: 10.1371/journal.pone. License (CC BY). The use, distribution or reproduction in other forums is permitted,
0060465 provided the original author(s) or licensor are credited and that the original
Zeng, L., Das, S., and Burne, R. A. (2010). Utilization of lactose and galactose publication in this journal is cited, in accordance with accepted academic practice.
by Streptococcus mutans: transport, toxicity, and carbon catabolite repression. No use, distribution or reproduction is permitted which does not comply with these
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