In this study, we used high-throughput, quantitative proteomics profiling to identify relevant proteins expressed by S. mutans during the development of mixed-species biofilms according to an ecological model. Our results show how S. mutans orchestrates the expression of gene products from distinct pathways (some that overlap) in response to sucrose that facilitates its establishment and optimal survival, in the presence of other bacteria, within a dynamically changing biofilm milieu over time.
S. mutans Survival within Ecological Mixed-species Biofilm Model
Oral biofilms are comprised of mixed microbiota in vivo
. The transition from non-pathogenic to pathogenic biofilm involves environmental changes (i.e. introduction of sucrose) that dramatically influence the microbial and the biochemical composition of biofilm. The mixed-species model used here was designed to mimic the ecological and the biochemical changes associated with cariogenic biofilm assembly 
, which include: 1) microbial population shift between initially low abundance S. mutans
and the early colonizers present in high numbers (e.g. S. oralis
); 2) the introduction of sucrose as environmental challenge, causing biochemical changes (EPS and acid production) and microbial shifts towards dominance of S. mutans
at later stages of biofilm formation; and 3) spatiotemporal changes of 3D architecture and environmental pH (Figure S1
). We selected two specific time points for proteomic analysis that are based on the population shifts, EPS-matrix and microcolony assembly observed in our mixed-species biofilm model. We selected 67 h because at this time point S. mutans
start to shift from initially very low numbers to a major co-habitant while at 115 h S. mutans
become the dominant species (Figure S1B
). Furthermore, we also used single-species S. mutans
biofilms to examine how S. mutans
protein synthesis is affected by the presence of additional organisms.
Overview of S. mutans Proteomic Responses during Biofilm Development
The MudPIT approach detected up to 60% of the proteins encoded by S. mutans
in biofilm samples (; Table S2
), which is significantly higher output than standard proteomic analysis of S. mutans
using 2D gel electrophoresis 
. shows the number of S. mutans
proteins detected per time point and type of biofilm evaluated.
Number of S. mutans UA159 proteins detected in the biofilms.
The distribution of proteins identified in gene ontology (GO) categories of specific biological processes showed that the highest number was detected for proteins involved in S. mutans metabolic process, followed by unannotated (i.e. uncharacterized) proteins (). In addition, the analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways showed that most proteins detected in both mixed- and single-species biofilms belong to (i) starch and sucrose metabolism (carbohydrate metabolism); (ii) purine metabolism (nucleotide metabolism), (iii) pyrimidine metabolism (nucleotide metabolism); and (iv) aminoacyl-tRNA biosynthesis (protein synthesis) metabolic pathways. As an example, we show the proteins differentially expressed in the starch and sucrose metabolism pathway (), which contains some of the critical factors associated with EPS-matrix formation and carbohydrate metabolism.
Distribution of S. mutans UA159 proteins into specific gene ontology (GO) of biological processes.
Sucrose and starch metabolism pathway.
Furthermore, the MudPIT analyses provided quantitative data of the production (i.e. abundance) of each S. mutans
protein detected in the biofilm samples tested. The proteins Tuf (elongation factor Tu), Eno (phosphopyruvate hydratase), and DnaK (molecular chaperone) are among the most abundant proteins in all conditions evaluated (Table S2
). The MudPIT coupled with spectrum counting is advantageous because it is a label-free large-scale and quantitative proteomic approach 
. It is a well-proven method for the identification and quantification of almost all proteins types from complex mixtures 
, such as those found in biofilms despite the inherent limitations of the technique (i.e. spectrum counting is not an absolute quantification and may underestimate proteins that are present in very low amounts 
We also analyzed the temporal effect and the presence of other species on S. mutans
proteome data. In general, a higher number of proteins were detected at 67 h of biofilm formation for both mixed- and single-species communities (). More importantly, the majority of the proteins in most biological processes (such as EPS synthesis and acid tolerance mechanisms) are detected in high abundance at 67 h of mixed-species biofilm development (; Table S2
). The data suggest that S. mutans
cells are highly active with enhanced fitness at 67 h (when S. mutans
is trying to outcompete the other species in our model) than at 115 h (when S. mutans
is already dominant in the biofilm milieu). The proteome profile of S. mutans
grown in a mixed-species community has many differences but also similarities with that from S. mutans
forming biofilms alone over time. For example, S. mutans
proteins involved with EPS metabolism and specific acid tolerance mechanisms are highly abundant in mixed-species biofilms at 67 h, while their profile does not change much over time in single-species biofilm. In contrast, the profile of the proteins associated with sugar uptake and intracellular polysaccharide storage metabolism are similar irrespective of whether S. mutans
is forming biofilms alone or mixed with other bacterial species (; ; 2; S1
Proteins related to EPS synthesis and remodeling.
It is critical to keep in mind that sucrose has dual function in assembling virulent biofilms: energy generation resulting in acid production and EPS matrix formation. The acid produced by bacterial cells entrapped within the EPS-enmeshed microcolonies cannot be readily neutralized, creating various low pH microenvironments within the complex biofilm 3D architecture (Figure S1
). Thus, the acidic niches create favorable conditions for acid-tolerant bacteria to prosper, ensuring continued localized acid production. To thrive in this highly organized, acidic environment and changing ecosystem, S. mutans
has sophisticated mechanisms to cope with fluctuation of dietary nutrients and environmental stresses, such as pH 
. Therefore, we focused on essential metabolic pathways that may be important for S. mutans
fitness in a cariogenic model, including proteins related to: (i) EPS synthesis and remodeling (); (ii) intracellular polysaccharide storage (IPS) and lipoteichoic acid metabolism (); and (iii) acid tolerance response mechanisms ( and ).
Proteins related to IPS and lipoteichoic acid (LTA) metabolism.
Proteins related to acid and stress tolerance response: F1F0-ATPase and fatty acid metabolism (linked to membrane composition).
Proteins related to acid and stress tolerance response: Proteins responsible for regulation of intracellular pH - branched chain amino acids (BCAA), malolatic fermentation (MLF), and agmatine diamenase system (AgDS).
Proteins Related to EPS Synthesis and Remodeling
The profile of abundance of S. mutans
proteins implicated in EPS matrix synthesis (GtfB, GtfC, GtfD, Ftf), remodelling (DexA, FruA), adhesion (GbpA, GbpB, GbpC and GbpD), and regulation (VicRS, GcrG, LuxS, RpoA, CcpA and ManL) are shown in . Among them, GtfB, GtfC, and GbpB are detected in elevated amounts particularly at 67 h. GtfB synthesizes mostly insoluble glucans (rich in α1,3-linked glucose) while GtfC forms a mixture of insoluble and soluble glucans (mostly α1,6-linked glucose) 
. Secreted GtfC is primarily incorporated into the tooth-pellicle whereas GtfB preferably attaches to the bacterial surfaces 
. The insoluble and rigid α1,3-linked glucans produced by these surface-adsorbed Gtfs are essential for the assembly and structural integrity of the matrix and microcolonies, as well as for the maintenance of acidic pH microenvironments within biofilms 
. GtfB and GtfC are also recognized virulence factors associated with pathogenesis of dental caries in vivo
We also observed that specific regulators of expression of these exoenzymes were detected in S. mutans
at 67 h of biofilm development. The two component system VicRK and the LuxS-based signaling system were identified, which have been shown to directly regulate glucan synthesis by GtfB and GtfC 
. Furthermore, CcpA and ManL were detected in high levels at early stage of biofilm development; they are also implicated with up-regulation of EPS synthesis 
). Altogether, these observations are congruent with the pattern of Gtfs detected in our biofilm system.
It is also noteworthy that the presence of dextranase (DexA) and fructosyltransferase (Ftf) (albeit less abundant than GtfB and GtfC) may have direct implications on the initial assembly of an insoluble EPS-matrix and with the acidification of the biofilm microenvironment. DexA digest soluble α1,6-linked glucans which provides (i) small dextrans which serve as acceptors for synthesis of insoluble glucans by GtfB, and (ii) provide additional substrates for acid production 
. Fructans formed by Ftf provide storage of extracellular nutrients, and have high water regain value that help to keep biofilm hydrated 
Conversely, synthesis of glucan binding proteins (Gbps) may enhance the ability of S. mutans
to interact with the EPS-rich matrix 
. The adhesion between the bacterial cells and the EPS-matrix may be mediated in part through cell-surface GbpC, and possibly GbpB whereas secreted GbpA and GbpD may be cross-linked with the matrix contributing to the maintenance of the biofilm architecture 
. The elevated amounts of GbpB observed here may have direct implications for the biofilm morphogenesis and structural integrity, because a conditional mutant for this protein has impaired biofilm accumulation 
The presence of proteins associated with EPS synthesis in one hand with others involved with glucan binding processes illustrates how S. mutans builds up the biofilms after the introduction of sucrose, particularly at 67 h (a critical time point where the microbial population shifts occurs towards S. mutans dominance in our biofilm model). We conducted RT-qPCR analysis of selected genes at 67 and 115 h as well as the preceding time points of 43 and 91 h to examine the dynamics of gene expression associated with the proteins of interest. Clearly, the expression of gtfB, gtfC and gbpB were highly induced as the biofilm transits from 43 to 67 h (P<0.05) while their expression declines as S. mutans become the dominant species in the mature 115 h-biofilm (P<0.05), which agrees well with the quantitative proteome data. Although the fold of change in protein synthesis and gene expression does not present the same magnitude in all cases, the trend is conserved (either induction or repression over time).
Furthermore, genes related to EPS synthesis, remodeling and regulation are more highly expressed by S. mutans
when grown in a mixed-species community that mimics the ecological plaque model than alone (P
<0.05; Figure S2
), confirming the proteomic profile between these two biofilm systems. Such differences could explain the structural disparity in the EPS-matrix and the size of microcolonies between mixed- and single-species biofilms (Figure S1
Thus, the interplay of the gene products involved with glucan synthesis, degradation and binding provide an opportunity for S. mutans, which are initially in low number, to thrive in a mixed-species community by: 1) assembling EPS matrices on which the organism binds avidly through several Gbps, 2) providing additional carbohydrate sources for acid production, and 3) constructing highly cohesive bacterial islets (microcolonies) enmeshed in EPS, which facilitates the creation of acidic niches throughout the biofilm 3D architecture.
Proteins Related to Intracellular Polysaccharide Storage (IPS) and Lipoteichoic Acid (LTA) Metabolism
IPS are glycogen-like storage polymers important for S. mutans
virulence and are associated with the pathogenesis of dental caries 
. IPS provide S. mutans
with endogenous source of carbohydrates that can be metabolized when exogenous fermentable substrates have been depleted in the oral cavity. Proteins related to IPS metabolism are abundant at 67 h in mixed-species biofilms (). In particular, glycogen phosphorylase, a key enzyme in IPS metabolism/synthesis (see ), is detected in high levels at the earlier time point. The expression of gene glgP
is significantly higher at 67 h than at the later time points (P
<0.05, ), which agrees with the temporal trend seen in the proteome data for mixed-species biofilms. These findings indicate increased storage of IPS by S. mutans
after introduction of sucrose as the biofilm start to accumulate. A similar profile of protein abundance and gene expression changes between 67 and 115 h was observed with single-species biofilms (, Figure S2
), although some differences in the type of proteins were detected (e.g. high levels of glycogen synthase in single-species vs. high levels of glycogen phosphorylase in mixed-species).
Dynamics of S. mutans gene expression during mixed-species biofilm development.
We also observed another factor that may contribute with IPS accumulation by S. mutans
. Proteins DltA, DltC, and DltD, involved with metabolism of LTA, were more abundant at 67 h, and were particularly elevated in mixed-species biofilms (). This observation is relevant because disruption of expression of dltABCD
induced the synthesis of IPS 
. Whether these proteomic changes can actually increase the amounts of stored IPS in S. mutans
within biofilms and how they are triggered by the presence of other organisms awaits further investigation. Furthermore, D-alanyl-LTA is involved with bacterial adhesion to hydroxyapatite and artificial surfaces, and initial biofilm formation process, possibly by incorporating LTA into the extracellular matrix 
. Interestingly, the defective expression of dltABCD
reduced acid tolerance of S. mutans
grown in planktonic cultures 
Overall, the detection of proteins associated with IPS and LTA metabolism, which are particularly elevated at 67 h in mixed-species system provide additional insights on how S. mutans could establish themselves, survive and respond to an increasingly acidic and EPS-rich microenvironment following the introduction of sucrose.
Proteins Related to Acid Stress Tolerance Response Mechanisms
The assembly of an insoluble EPS matrix and its spatial arrangement with bacterial cells creates acidic and protective microenvironments inside the microcolonies 
. S. mutans
have several mechanisms to cope with stressors such as low external pH and acidification of cytoplasm 
. Our data showed that S. mutans
mounts an intricate yet interconnected response to adapt and to survive acidic stress, which are influenced by the presence of other organisms and biofilm age.
All proteins that encode the F1F0-ATPase system 
for proton extrusion and ATP generation were detected in mixed-species biofilms (). Among them, AtpD was the most abundant protein, which has a critical function in the assembly of ATPase complex and is highly induced at low pH 
. This complex helps to maintain the ΔpH across the bacterial membrane by pumping protons out of the cell. The temporal expression of gene atpD
showed that from 43 to 67 h the expression is similar, and then significantly declines at 115 h (P
<0.05), confirming the quantitative proteome data ().
Low pH triggers changes in the membrane fatty acid composition and also affects the permeability of the membrane to protons 
. All proteins encoded by the fatty acid biosynthetic gene cluster were also detected (). This cluster may be connected with ATPase system because the fatty acid composition is important for the optimal function of ATPpase, which is anchored to the membrane. In particular, FabM was detected in high levels at 67 h of mixed-species biofilm development (). FabM is responsible for the synthesis of monounsaturated fatty acids and is critical for S. mutans
survival at low pH 
. The expression profile of gene fabM
confirmed the trend of the protein detection between 67 and 115 h (). Thus, the data suggest that S. mutans
modulates specific changes in fatty acid profile in the membrane and the assembly of F1F0-ATPase system to ensure an optimal condition to control the protons level in the cytoplasm (and as a result the intracellular pH).
In addition, the proteins directly responsible for cytoplasm alkalinization are also detected in elevated amounts at 67 h of biofilm development (), which include: 1) the metabolism of branched chain amino acids (BCAA) 
, 2) the malolatic fermentation (MLF) system 
, and 3) agmatine diamenase system (AgDS) (which also produces ATP that can be used for growth or to extrude protons via F1F0-ATPase system 
). Among them, metabolism of BCAA may have a significant role as its components are abundant (particularly IlvC), and they may have a synergistic role with F1F0-ATPase system and fatty acid composition in the membrane to enhance S. mutans
survival in a low pH environment within biofilms. This may occur because S. mutans
senses the low pH and modulate the carbon flux from acid production to BCAA biosynthesis 
. The abundance of MLF related proteins is rather low, and only one protein from the AgDS was detected in our analyses (), which is not surprising because S. mutans
express AgDS at relatively low levels 
. Thus, the MLF and AgDS systems may have comparatively minor roles in S. mutans
tolerance to acidic environment in the biofilms tested.
In summary, the F1F0-ATPase system, the membrane fatty acid biosynthesis and the BCAA metabolism appear to play major roles on acid tolerance, particularly when S. mutans
is shifting from a minor to become a major resident within an increasingly acidic milieu found in the interior of microcolonies of mixed-species biofilm. However, the other mechanisms for acid stress adaptation (i.e. MLF and AgDS), even if having a minor role (based on protein abundance), may be also important for overall S. mutans
fitness by helping to increase the cytoplasmic pH and generate ATP. The ATP generated can be used by F1F0-ATPase system to extrude protons from the cytoplasm. The expression of such interconnected mechanism is particularly important because the loss of one or more of the stress adaptive mechanisms can lead to a substantial reduction in S. mutans
Other Proteins Related to Responses to Acid Stress
The exposure to acidic environment and other insults found in the biofilms may lead to accumulation of abnormal proteins. S. mutans
(and other organisms) uses molecular chaperones and proteases to modulate the stability of proteins and prevent the accumulation of abnormal proteins by overseeing the correct folding 
. The synthesis of chaperones GroEL, GrpE, DnaJ, DnaK and HtpX was elevated at 67 h of mixed-species biofilm development (Table S3
). This finding makes sense because in our analyses the majority of the proteins are highly abundant at this time point, when S. mutans
start to become a major co-habitant in the mixed-species biofilm. Therefore, the augmented production of chaperones ensures the quality of proteins being expresses, enabling S. mutans
to thrive in this biofilm.
Although we recognize the importance of oxidative and osmotic stresses in the S. mutans
physiology in biofilms, we did not analyze the data in greater detail to keep the focus on the acid stress processes. Nevertheless, we did detect high levels of NADH oxidase Nox (a major contributor to oxidative stress response 
) and transcriptional repressor Rex (linked to coping with oxidative stress 
), which may indicate that the access of oxygen to the S. mutans
cells within the biofilms may be limited likely due to increase of thickness of the biofilms following introduction of sucrose.
In general, the profile of proteins and expression of genes associated with acid tolerance responses (and to other stresses) were different between mixed- and single-species biofilms ( and ; Figure S2
). Most of the proteins are detected in elevated levels in mixed-species biofilms. The expression of selected S. mutans
genes (atpD, fabM, groES, nox
) was significantly higher in mixed-species biofilms (vs. single-species) at all time points (P
<0.05; Figure S2
). These differences are congruent with the overall observations between these two biofilm systems. Clearly, S. mutans
growing in mixed-species biofilms has a distinctive fitness, allowing the bacterium to out-compete other co-habitants and to optimally survive the acidic milieu.
In addition, several uncharacterized proteins detected in this study could have an important role in S. mutans
fitness and tolerance to environmental stresses within cariogenic biofilms (Table S2
). Our data provide opportunities to investigate the function of these proteins in the expression of virulence by this pathogen, especially in the context of ecological biofilm concept. For example proteins encoded by genes SMU.1760 to SMU.1763, SMU.1337, SMU.210 are promising candidates for future studies 
. The genes SMU.1760 to SMU.1763 are organized in o operon-like gene cluster, and their encoded proteins may be involved in stress response. SMU.1760, SMU.1761, SMU.1762 and SMU.1763 genes are all up-regulated in S. mutans
lacking functional SpxA and SpxB 
, which have a global regulatory role in S. mutans
stress response to acidic and oxidative environment. The proteins encoded by SMU.1337 (alpha/beta superfamily hydrolases with unassigned function) and SMU.210 (hypothetical protein with unknown function) are particularly abundant at 67 h (during microbial shifts favoring S. mutans
) and 115 h (when S. mutans
is the dominant species) of biofilm development, respectively. Each may have a distinctive role; SMU.1337 may be an important hydrolytic enzyme for S. mutans
fitness and survival, while SMU.210 could be involved with persistence and stress adaptation. These proteins present homology to conserved hypothetical proteins from other bacteria (e.g. Streptococcus pyogenes
, Streptococcus gallolyticus
; Streptococcus anginosus
; Streptococcus downei
), and could have biological relevance to some pathogenic Streptococcus
strains, deserving future investigation with defective mutant strains to pinpoint their exact role 
The proteome analysis using multidimensional protein identification technology (MudPIT) revealed how S. mutans optimizes its metabolism and adapts, while enhancing its virulence and competitiveness, in response to a dynamically changing environment induced by sucrose within mixed-species biofilms. Moreover, the proteome data matched very well with the results from gene expression analyses using RT-qPCR, demonstrating the usefulness of this label-free quantitative proteomics approach to study the pathophysiological stage of microorganisms within complex biofilms over time.
Our study showed a complex interplay between gene products involved with EPS matrix assembly, remodeling and binding in one hand with specific processes associated with acid stress tolerance mechanisms, which are particularly induced when S. mutans is trying to outcompete other organisms (e.g. S. oralis) present in the biofilm system. In a simplified manner, the augmented production of EPS synthesis/remodeling and glucan-binding proteins helps to assemble a highly insoluble matrix that are uniquely arranged with bacterial cells forming microcolony complexes. At the same time, up-regulation of F1F0-ATPase system (e.g. AtpD), membrane fatty acids byosynthesis (e.g. FabM), and BCAA (e.g. IlvC) overlapping with molecular chaperones appears to be major responses by S. mutans (based on protein abundance and gene expression) to survive and adapt inside the microcolonies, which are highly acidic at 67 h of biofilm development in our system. These biological processes may be the major driving forces behind S. mutans successful establishment in mixed-species biofilms. Thus, novel therapies to control biofilm virulence expression should target them as a whole rather than a single pathway.
Clearly, the spatiotemporal regulation of this intricate yet interconnected network of pathways is highly complex and dynamic, and deserves further investigation both in vitro and in vivo.