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. 2017 Jun 12;96(10):1129–1135. doi: 10.1177/0022034517714414

Bacterial GtfB Augments Candida albicans Accumulation in Cross-Kingdom Biofilms

K Ellepola 1, Y Liu 2, T Cao 1, H Koo 2,✉, CJ Seneviratne 1,✉
PMCID: PMC5582686  PMID: 28605597

Abstract

Streptococcus mutans is a biofilm-forming oral pathogen commonly associated with dental caries. Clinical studies have shown that S. mutans is often detected with Candida albicans in early childhood caries. Although the C. albicans presence has been shown to enhance bacterial accumulation in biofilms, the influence of S. mutans on fungal biology in this mixed-species relationship remains largely uncharacterized. Therefore, we aimed to investigate how the presence of S. mutans influences C. albicans biofilm development and coexistence. Using a newly established haploid biofilm model of C. albicans, we found that S. mutans augmented haploid C. albicans accumulation in mixed-species biofilms. Similarly, diploid C. albicans also showed enhanced biofilm formation in the presence of S. mutans. Surprisingly, the presence of S. mutans restored the biofilm-forming ability of C. albicans bcr1Δ mutant and bcr1Δ/Δ mutant, which is known to be severely defective in biofilm formation when grown as single species. Moreover, C. albicans hyphal growth factor HWP1 as well as ALS1 and ALS3, which are also involved in fungal biofilm formation, were upregulated in the presence of S. mutans. Subsequently, we found that S. mutans–derived glucosyltransferase B (GtfB) itself can promote C. albicans biofilm development. Interestingly, GtfB was able to increase the expression of HWP1, ALS1, and ALS3 genes in the C. albicans diploid wild-type SC5314 and bcr1Δ/Δ, leading to enhanced fungal biofilms. Hence, the present study demonstrates that a bacterial exoenzyme (GtfB) augments the C. albicans counterpart in mixed-species biofilms through a BCR1-independent mechanism. This novel finding may explain the mutualistic role of S. mutans and C. albicans in cariogenic biofilms.

Keywords: Streptococcus mutans, glucosyltransferase, candidiasis, glucans, dental caries, dental plaque

Introduction

Streptococcus mutans is an important cariogenic bacteria in the oral cavity (Ajdić et al. 2002; Kutsch and Young 2011). S. mutans colonize the tooth enamel and form pathogenic biofilms associated with dental caries, particularly in early childhood caries (ECC). Frequent consumption of dietary sugars such as sucrose is a major factor associated with ECC etiology (Hajishengallis et al. 2017). In this context, S. mutans becomes an important biofilm modulator capable of effectively converting dietary sucrose into acids and producing extracellular glucans using exoenzymes termed glucosyltransferases (Gtfs) (Bowen and Koo 2011). Specifically, insoluble glucans produced by glucosyltransferase B (GtfB) provide bacterial binding sites and form the core of the extracellular matrix of cariogenic dental plaque biofilm in vivo (Mattos-Graner et al. 2000; Bowen and Koo 2011).

However, S. mutans is not the sole agent associated with ECC as other organisms can contribute to its pathogenesis (Hajishengallis et al. 2017). Interestingly, Candida albicans is the most frequently isolated fungal organism in cariogenic plaque-biofilm associated with ECC (de Carvalho et al. 2006; Raja et al. 2010; Yang et al. 2012; Qiu et al. 2015). It has been demonstrated that C. albicans presence can enhance S. mutans growth, accumulation, and biofilm formation both in vitro and in vivo (de Carvalho et al. 2006; Pereira-Cenci et al. 2008; Raja et al. 2010; Klinke et al. 2011; Metwalli et al. 2013; Falsetta et al. 2014). Yet, the role of S. mutans on C. albicans accumulation in this interkingdom interaction remains unclear. Biofilm formation is a major virulence attribute of C. albicans (Naglik et al. 2008; Dongari-Bagtzoglou et al. 2009). Expression of agglutinin-like sequences (ALS) and hyphal-wall protein (HWP) genes is important for biofilm formation (Nailis et al. 2006; Zhu and Filler 2010; Alves et al. 2014) and under the regulation of biofilm and cell-wall regulator (bcr1), a critical zinc-finger transcription factor that is essential for C. albicans biofilm development (Nobile et al. 2006).

Previously, we demonstrated that S. mutans–derived GtfB has high affinity and binding strength to the C. albicans cell surface (Gregoire et al. 2011; Hwang et al. 2015). The GtfB bound on the C. albicans surface produces glucans, which enhance the ability to harbor more viable S. mutans cells than single-species biofilms and accrue more biomass during mixed-species biofilm formation (Gregoire et al. 2011; Falsetta et al. 2014; Koo and Bowen 2014). The properties of S. mutans in mixed-species biofilms with C. albicans and the influences of fungal presence on bacterial biology have been explored. In contrast, understanding the role of S. mutans on the C. albicans counterpart remains incomplete. Taking this research gap into consideration, the present study aimed to characterize the effect of S. mutans on C. albicans accumulation in mixed-species biofilm. For this purpose, we took advantage of the recently developed haploid model of C. albicans biofilms (Seneviratne et al. 2015). The diploid genetic nature of C. albicans makes targeted gene deletion challenging (Noble and Johnson 2007). Discovery of the haploid nature of C. albicans opened a new avenue for simpler genetic manipulation where 1-step targeted gene mutations can be performed for constructing mutant libraries (Zeng et al. 2014). We screened a haploid C. albicans mutant library and found that bcr1 mutant (known to defective in single-species biofilms) formed comparable biofilms to its parent strain in the presence of S. mutans. These findings were confirmed using diploid C. albicans strains (predominant form in nature), and further experiments demonstrated that GtfB from S. mutans plays a key role in the fungal accumulation within mixed-species biofilm. Altogether, it appears that S. mutans–derived GtfB augments the ability of C. albicans to form biofilms through a bcr1-independent mechanism, providing further insights into this unique and virulent bacterial-fungal association.

Materials and Methods

Microbial Strains and Culture Conditions

The haploid and diploid C. albicans strains used in the study are described in Appendix Table 1. S. mutans UA159 strain was taken from the archival collection of the oral sciences laboratory (National University of Singapore). C. albicans and S. mutans cells were grown to mid-exponential phase in ultra-filtered tryptone yeast extract medium (UFTYE; pH 5.5 and 7.0 for C. albicans and S. mutans, respectively) containing 1% (w/v) glucose and harvested by centrifugation (6,000 g, 10 min, 4°C) as described previously (Gregoire et al. 2011). The cells were then washed in phosphate-buffered saline (PBS) prior to being used for biofilm formation.

In Vitro Biofilm Development

For the C. albicans single-species biofilm formation, approximately 1 × 106 colony-forming units (CFU)/mL yeast cells were inoculated in UFTYE broth containing 1% (w/v) sucrose. For mixed-species biofilm formation, approximately 1 × 106 CFU/mL of C. albicans yeast cells and 2 × 106 CFU/mL of S. mutans cells were diluted in UFTYE broth containing 1% (w/v) sucrose. The biofilms were formed in presterilized flat-bottomed 96-well microtiter plates (Greiner Bio-One) at 37°C for 24 h under aerobic conditions.

Biofilm Quantification

Three different methods were used for the biofilm quantification:

  1. XTT reduction assay: The XTT assay was performed according to a previously optimized protocol (Seneviratne et al. 2008). The colorimetric changes of XTT were measured spectrophotometrically at 490 nm (Multiskan GO; Thermo Fisher Scientific).

  2. Crystal violet assay: Biofilms were quantified using crystal violet (CV) assay (O’Toole 2011). Briefly, the 2% formalin fixed biofilms were stained with 1% (w/v) CV and subsequently filled with 95% ethanol. The optical density of 95% ethanol was measured at 570 nm (Multiskan GO; Thermo Fisher Scientific). XTT or CV assay provides total biomass without distinguishing C. albicans from S. mutans in mixed-species biofilms. CFU counting was performed to enumerate each species despite limitations due to the C. albicans yeast-hyphae transition.

  3. CFU counting method: A dilution series of the cell suspensions from biofilms was prepared and spread plated on glucose minimal medium agar plates supplemented with suitable amino acids and 8 µg/mL gentamicin sulfate salt (Sigma-Aldrich) to prevent bacterial growth. The plates were incubated at 30°C for 48 h and the fungal colonies were counted.

Growth Kinetics of C. albicans Strains

Cell concentrations of 1 × 106 CFU/mL of C. albicans strains were diluted in 200 µL UFTYE broth containing 1% (w/v) sucrose in 96-well microtiter plates (Greiner Bio-One). The optical density of the microbial cultures was measured at a wavelength of 600 nm (Multiskan GO; Thermo Fisher Scientific) at 30-min intervals up to 72 h at 37°C under aerobic conditions.

Confocal Laser Scanning Microscopy

Single-species and mixed-species biofilms were developed on Thermanox (Nunc) 8-well plates. Biofilms were fixed with 4% (v/v) paraformaldehyde for 24 h at 4°C and stained using propidium iodide (Invitrogen) and 0.001% (w/v) calcofluor white (Sigma-Aldrich) with modifications to a previous protocol (Cavalcanti et al. 2016). Propidium iodide (excitation/emission 535/617 nm) stained the bacteria while calcofluor white (excitation/emission 365/435 nm) stained the C. albicans. The biofilms were observed using an Olympus-Fluoview FV1000 TIRF confocal microscope. Z sections were collected from 4 different fields of 3 biological samples, and biofilm biovolumes were determined using the Imaris software. Glucan formation by GtfB was visualized using Alexa Fluor 647–labeled dextran conjugate (10,000 molecular weight; absorbance/ fluorescence emission maxima, 647/ 668 nm; Molecular Probes) as detailed previously (Klein et al. 2009).

Analysis of C. albicans Adhesins by Quantitative Reverse Transcription Polymerase Chain Reaction

Cells recovered from biofilms were centrifuged (10,000 g for 10 min) and cell pellets were used for RNA extraction using TRIzol (Thermo Fisher Scientific) reagent according to an established method (Rio et al. 2010). The integrity and purity of RNA were spectrophotometrically determined by the absorbance ratio at 260/280 nm (NanoDrop ND-1000; Thermo Fisher Scientific). Reverse transcription of RNA was performed using the M-MLV Reverse Transcriptase system (Promega). Primers used for quantitative polymerase chain reaction (qPCR) analysis are presented in Appendix Table 2. Amplification and quantification of target RNA by quantitative polymerase chain reaction were performed in MicroAmp optical 96-well plates (Applied Biosystems) using a KAPA SYBR FAST qPCR Kit (Kapa Biosystems) as described previously (Truong et al. 2016). The expression of targeted genes was normalized according to the housekeeping reference gene PMA1 (Chau et al. 2004). Each C. albicans strain forming single-species biofilms was used as the reference group for gene expression analysis. Analysis of relative gene expression was achieved according to the ΔΔCt method (Schmittgen and Livak 2008).

Statistical Analysis

Each error bar indicates mean ± standard deviation (SD) calculated from 3 biological replicates and 4 technical replicates. For any pairwise comparison, Student’s t test or Mann-Whitney U test was performed. Kruskal-Wallis test was used for multiple comparisons. P < 0.05 was considered statistically significant (*).

Results

S. mutans Augments the Accumulation of Haploid and Diploid C. albicans in Mixed-Species Biofilms

The XTT reduction assay reading represents total biomass, including both S. mutans (Sm) and C. albicans (Ca). The XTT assay showed an increase in the total biofilm-biomass of the mixed-species biofilm in comparison to the respective single-species biofilms of C. albicans (Fig. 1A; P < 0.05). Similar to the XTT assay, the CV assay also showed a similar pattern in the biofilm formation (Fig. 1B; P < 0.05). On the other hand, the haploid bcr1Δ and the diploid bcr1Δ/Δ mutants formed comparable biofilms to that of parent strains in Sm-Ca mixed-species biofilm conditions (Fig. 1A, B).

Figure 1.

Figure 1.

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Streptococcus mutans augmented the biofilm formation of haploid and diploid Candida albicans strains in S. mutans–C. albicans mixed-species biofilms. Biofilm formation was evaluated using (A) XTT reduction assay, (B) Crystal violet assay, and (C) colony-forming unit (CFU) counting. The presence of S. mutans UA159 rescued the inability of haploid bcr1Δ and diploid bcr1Δ/Δ mutants to form biofilms, showing comparable biofilm-forming ability to their respective haploid and diploid parent strains, GZY803 and SC5314. (D) Growth kinetics demonstrated that biofilm formation is not dependent on growth as bcr1Δ and diploid bcr1Δ/Δ mutants have a similar growth rate to their parent strains, GZY803 and SC5314.

CFU counting method demonstrated that GZY803 and SC5314 had significantly higher C. albicans cells compared to the bcr1Δ and bcr1Δ/Δ mutants in single-species biofilms (Fig. 1C). There was no significant difference in the growth kinetics of the C. albicans bcr1Δ and bcr1Δ/Δ mutant strains with their respective parent strains (Fig. 1D). Therefore, the difference in biofilm formation between C. albicans parent and bcr1 mutants was not due to the growth kinetics. Rather, the presence of S. mutans significantly augmented the accumulation of the C. albicans counterpart in Sm-Ca mixed-species biofilm (Fig. 1C). Surprisingly, C. albicans bcr1Δ and bcr1Δ/Δ mutants, which were defective in single-species biofilm formation, showed an increase in biomass (Fig. 1A, B) and viable cell numbers in Sm-Ca mixed-species biofilms (Fig. 1C; P < 0.05). CFU counting showed that S. mutans cells in the Sm-Ca mixed-species biofilm were significantly higher compared to the S. mutans single-species biofilm (Appendix Fig. 3; P < 0.05). There was no statistical significance between the number of S. mutans cells in the Sm-bcr1Δ/Δ and Sm-SC5314 mixed-species biofilms.

Microscopic Comparison of Haploid and Diploid C. albicans Biofilms in the Presence of S. mutans

Confocal laser scanning microscopy (CLSM) imaging corroborated the foregoing findings, providing evidence on the increased biofilm formation of C. albicans in the presence of S. mutans (Fig. 2A). C. albicans bcr1Δ (Fig. 2A.II, A.VI) and bcr1Δ/Δ (Fig. 2A.X, A.XIV) formed sparse biofilms compared to the haploid-parent GZY803 (Fig. 2A.I, A.V) and diploid-parent SC5314 (Fig. 2A.IX, A.XIII), respectively. Consistent with colorimetric and microbiological data, the presence of S. mutans enhanced the biofilm formation of C. albicans haploid-parent (Fig. 2A.III, A.VII) and haploid-bcr1Δ (Fig. 2A.IV, A.VIII) as well as diploid-parent (Fig. 2A.XI, A.XV) and diploid-bcr1Δ/Δ (Fig. 2A.XII, A.XVI). It was noteworthy that there was a drastic increase in the biofilm formation of bcr1Δ (Fig. 2A.IV, A.VIII) and bcr1Δ/Δ (Fig. 2A.XII, A.XVI) in the presence of S. mutans compared to single-species biofilms of bcr1Δ (Fig. 2A.II, A.VI) and bcr1Δ/Δ (Fig. 2A.X, A.XIV). The visual observations were supported by the quantitative analysis of biovolumes of C. albicans cells using the Imaris software in both single-and Sm-Ca mixed-species biofilms (Fig. 2B; P < 0.05). Taken together, S. mutans appears to augment the biofilm formation of haploid and diploid C. albicans in Sm-Ca mixed-species biofilms, including bcr1Δ and bcr1Δ/Δ mutants, suggesting that the enhancement of the C. albicans counterpart is independent of BCR1.

Figure 2.

Figure 2.

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Microscopic comparison of haploid and diploid Candida albicans biofilms in the presence and absence of Streptococcus mutans. C. albicans in the single-and mixed-species biofilms were fixed and stained with calcofluor white (in blue), and the S. mutans in mixed-species biofilms were stained with propidium iodide (in red) for confocal laser scanning microscopy (CLSM) visualization (images I-IV and IX-XII). Images V-VIII and XIII-XVI represent only the C. albicans cells in the single-or mixed-species biofilms. (A) The presence of S. mutans notably enhanced the biofilm formation of bcr1Δ (IV and VIII) and bcr1Δ/Δ (XII and XVI) mutants in comparison to the absence of S. mutans (II, VI and X, XIV). Haploid and diploid parent strains, GZY803 (III and VII) and SC5314 (XI and XV), also showed a slight increase in biofilm formation in the presence of S. mutans. (B) The biovolumes of C. albicans single-and mixed-species biofilms were compared using the Imaris software.

S. mutans Enhances the Expression of C. albicans HWP1, ALS1, and ALS3 in Mixed-Species Biofilms

qPCR analysis showed that the presence of S. mutans enhanced the expression of C. albicans HWP1 (Fig. 3A), ALS1 (Fig. 3B), and ALS3 (Fig. 3C) genes in Sm-Ca mixed-species biofilms compared to the single-species biofilms. In Sm-Ca mixed-species biofilms, HWP1 expression was significantly increased in both haploid bcr1Δ and diploid bcr1Δ/Δ mutants as well as in their respective parent strains, GZY803 and SC5314 (P < 0.05). ALS1 and ALS3 expressions were significantly increased in both haploid-parent strain GZY803 and diploid-parent strain SC5314 and in the bcr1Δ/Δ mutant (P < 0.05). However, there was no significant increase in ALS1 and ALS3 expression in the bcr1Δ mutant in the presence of S. mutans.

Figure 3.

Figure 3.

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Streptococcus mutans increase relative gene expression of Candida albicans hyphal wall protein (HWP1) and adhesins (ALS1, ALS3). Quantitative reverse transcription polymerase chain reaction analysis was performed for C. albicans genes (A) HWP1, (B) ALS1, and (C) ALS3. Target genes were normalized with the C. albicans housekeeping gene PMA1 using the ΔΔCt method. Single-species biofilms of each C. albicans strain were used as reference biofilm to compare the relative expression of above genes in the respective C. albicans strain in the C. albicans–S. mutans mixed-species biofilm. The fold change of the gene of interest for each strain forming mixed-species biofilms was compared to that of a fold change value of 1 of the gene of interest in the single-species biofilm of the same strain. Bars show the average and the standard deviation (SD) within each group. The presence of S. mutans UA159 helped haploid bcr1Δ and diploid bcr1Δ/Δ mutants and their respective parent strain GZY803 and diploid SC5314 to increase the expression of (A) HWP1, (B) ALS1, and (C) ALS3 genes, which are under the direct regulation of bcr1 transcription factor.

GtfB Enhances C. albicans Biofilm Formation

From the above observations, S. mutans appeared to produce an “effector molecule” that promoted C. albicans accumulation in mixed-species biofilms. Previous studies have demonstrated that S. mutans–derived GtfB is a key mediator associated with C. albicans and development of mixed-species biofilm (Gregoire et al. 2011; Falsetta et al. 2014). Gtfs can use sucrose to synthesize glucans, which are the main components of extracellular matrix in cariogenic biofilms (Kuramitsu 1993). Therefore, we examined the formation of glucans in biofilms using an established fluorescence-labeling method detailed previously (Xiao et al. 2012) (Appendix Fig. 1). As expected, Sm-Ca biofilms showed the presence of abundant glucans (Appendix Fig. 1A.I –A.IV), whereas the polysaccharides were absent in all C. albicans single-species biofilms (Appendix Fig. 1B.I–B.IV). Hence, we postulated that GtfB produced by S. mutans can influence the ability of C. albicans to form biofilms. We used purified, cell-free GtfB enzyme preparation in sterile buffer as described previously (Koo et al. 2002). C. albicans single-species biofilms were formed in the presence of 5 µg/mL of enzymatically active GtfB. This GtfB amount was selected following a dose-dependent study (Appendix Fig. 2). Interestingly, GtfB enhanced the biofilm formation by C. albicans parental strain as well as bcr1Δ/Δ mutant (Fig. 4A–C; P < 0.05) and their haploid counterparts (Appendix Fig. 5). Notably, extracellular glucans (in red) were detected in C. albicans biofilms formed in the presence of GtfB, as shown by confocal imaging (Fig. 4D). In contrast, S. mutans strain defective in gtfB expression is incapable of enhancing C. albicans biofilm formation (Appendix Fig. 6). Previous studies have shown that GtfB can bind to both yeast and hyphal cells of C. albicans (Gregoire et al. 2011; Hwang et al. 2015). Therefore, it is possible that GtfB bound to C. albicans cells may modulate the enhanced fungal biofilm formation when grown together with S. mutans. To further confirm this modulating effect of GtfB on C. albicans biofilm formation, an ira2Δ/Δ mutant previously shown to be defective in biofilm formation (Seneviratne et al. 2015) was used for similar experiments (Appendix Fig. 4). GtfB produced by S. mutans was able to stimulate the biofilm formation of the ira2Δ/Δ mutant, confirming our observation on bcr1Δ/Δ.

Figure 4.

Figure 4.

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Glucosyltransferase B (GtfB) increases biofilm formation in Candida albicans. The biofilm formation in the presence of GtfB was evaluated using (A) visual observation, (B) XTT reduction assay, (C) colony-forming unit (CFU) counting, and (D) confocal microscopy. Confocal laser scanning microscopy (CLSM) images show C. albicans cells stained with Calcofluor white (in blue) and GtfB-derived glucans labeled with Alexa Fluor 647–labeled dextran conjugate (in red). Adding cell-free, purified GtfB significantly increased biofilm formation in C. albicans. This figure is available in color online.

GtfB Increases the Expression of HWP1, ALS1, and ALS3 in C. albicans Biofilms

We postulated that GtfB is responsible for the increased expression of HWP1, ALS1, and ALS3 in C. albicans biofilms when grown as mixed-species biofilms. Parent C. albicans strain SC5314 and the bcr1Δ/Δ biofilms in the presence of GtfB exhibited a significant increase in the expressions of the HWP1 (Fig. 5A), ALS1 (Fig. 5B), and ALS3 (Fig. 5C) genes in the presence of GtfB (P < 0.05); similar gene expression enhancement was observed using the equivalent haploid C. albicans strains (Appendix Fig. 7). The ability of GtfB to enhance the expression of the aforementioned genes in biofilms of C. albicans SC5314 parent isolate as well as bcr1Δ/Δ mutant shows that the expression happens through a BCR1-independent mechanism.

Figure 5.

Figure 5.

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Glucosyltransferase B (GtfB) increases relative gene expression of Candida albicans hyphal wall protein (HWP1) and adhesins (ALS1, ALS3). Quantitative reverse transcription polymerase chain reaction analysis was performed to evaluate the relative expression of C. albicans (A) HWP1, (B) ALS1, and (C) ALS3 genes using the ΔΔCt method. Target genes were normalized using the C. albicans housekeeping gene (PMA1). Single-species biofilms of each C. albicans strain developed without the supplementation of GtfB were used as the reference biofilm to compare the expression of the above genes in C. albicans strains forming biofilms in the presence of GtfB. Bars show the average and the standard deviation (SD) within each group. Cell-free, purified GtfB increased the expression of (A) HWP1, (B) ALS1, and (C) ALS3 genes in haploid bcr1Δ and diploid bcr1Δ/Δ mutants and their respective parent strains GZY803 and diploid SC5314.

Discussion

Previously, we and others demonstrated that the presence of C. albicans promotes S. mutans cells accretion and GtfB production in mixed-species biofilm grown in sucrose (Pereira-Cenci et al. 2008; Falsetta et al. 2014). Here, we found that S. mutans also augments C. albicans accumulation in mixed-species biofilms, providing further evidence of a synergistic interaction between the 2 organisms. Strikingly, we also observed that C. albicans strains lacking the bcr1 transcription factor were capable of forming enhanced biofilm in the presence of S. mutans or by GtfB itself. This observation suggests that GtfB secreted by S. mutans is able to restore the biofilm-defective phenotype of C. albicans. These findings support the clinical and in vivo observations that C. albicans and S. mutans develop a symbiotic relationship allowing coexistence and mixed-biofilm formation on tooth surfaces, an atypical habitat for C. albicans (de Carvalho et al. 2006; Raja et al. 2010; Yang et al. 2012; Falsetta et al. 2014).

BCR1 is a key regulator for C. albicans biofilm development, and bcr1Δ/Δ mutant is biofilm defective (Nobile and Mitchell 2005; Nobile et al. 2006). Major functional downstream targets of bcr1 include HWP1, ALS1, and ALS3 genes that encode cell surface proteins. Overexpression of these genes in C. albicans bcr1Δ/Δ mutant restored the biofilm formation (Nobile et al. 2006; Nobile et al. 2012). Interestingly, C. albicans bcr1Δ and bcr1Δ/Δ mutants were able to form comparable biofilms to their respective parent strains GZY803 and SC5314 in the presence of S. mutans. Hence, it appears that the presence of S. mutans enables C. albicans to bypass the BCR1-dependent biofilm defect during the formation of Sm-Ca mixed-species biofilms. Moreover, S. mutans also increased the HWP1, ALS1, and ALS3 expressions of C. albicans. These findings suggest that S. mutans increases the expression of the above genes in a BCR1-independent mechanism, which may partly explain the comparable biofilm formation of BCR1 mutants in Sm-Ca mixed-species biofilms. A recent study is in agreement with our findings where S. mutans and Streptococcus sanguinis together were able to increase the ALS3 and HWP1 gene expressions in C. albicans during mixed-species biofilm formation (Cavalcanti et al. 2016).

Notably, GtfB by itself increased C. albicans biofilm formation. Introducing GtfB allowed C. albicans diploid bcr1Δ/Δ mutant to form comparable biofilms to wild-type SC5314. IRA2 is another hyphal-independent, biofilm-specific regulator upstream to BCR1 in C. albicans (Seneviratne et al. 2015). Interestingly, observations on ira2Δ/Δ mutant in the present study further confirmed the augmented effect of GtfB on C. albicans biofilm formation. Moreover, addition of GtfB increased the expression of HWP1, ALS1, and ALS3 in C. albicans SC5314 as well as bcr1Δ/Δ mutant. These findings provide supportive evidence that S. mutans–derived GtfB may be the reason for enhanced C. albicans accumulation in Sm-Ca mixed-species biofilms. Previous studies have demonstrated that GtfB can bind to both S. mutans and C. albicans (Vacca-Smith and Bowen 1998; Hwang et al. 2015) but with different avidity. Intriguingly, the binding strength and binding stability of GtfB to the C. albicans surface were several folds higher compared with the enzyme adhesion to S. mutans (Hwang et al. 2015). Hence, there may be a highly specific interaction between S. mutans GtfB and the cell-wall component in C. albicans, as suggested previously (Gregoire et al. 2011; Hwang et al. 2015), that may also induce the expression of HWP1, ALS1, and ALS3 genes. However, future studies are required to identify the binding sites of GtfB and the downstream regulation of genes activated upon binding. We are generating targeted gene mutations using the genetic flexibility of the haploid system, which will be examined in the haploid C. albicans–S. mutans mixed-species biofilm model established here. Inclusion of mutants affecting the bcr1 pathway and GtfB-defective S. mutans strains as well as nonmutans streptococci may help to further elucidate the mechanisms of this bacterial-fungal interaction.

These findings shed new light on the previously observed C. albicans and S. mutans synergism in biofilms associated with early childhood caries. Furthermore, the presence of C. albicans on the tooth surface may become a fungal reservoir for mucosal fungal infections and systemic mycoses in compromised host populations. Hence, targeting GtfB may be a promising strategy to counter the formation of pathogenic bacterial-fungal biofilms.

Author Contributions

K. Ellepola, contributed to conception, design, data acquisition, analysis, and interpretation, drafted the manuscript; Y. Liu, contributed to data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; T. Cao, contributed to data interpretation, drafted the manuscript; H. Koo, C.J. Seneviratne, contributed to conception, design, data analysis, and interpretation, drafted the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplementary Material

Appendix

Footnotes

A supplemental appendix to this article is available online.

This study was funded by NMRC grant NMRC/CNIG/111/2013 and NMRC/CIRG/1408/2014 to CJS and the National Institute for Dental and Craniofacial Research grant DE025220 to HK.

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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Appendix
DS_10.1177_0022034517714414.pdf (427.5KB, pdf)

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