Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA, USA
Correspondence: Patrick J. Piggot, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA. Tel.: +1 215 707 7927; fax: +1 215 707 7788; e-mail: firstname.lastname@example.org
Streptococcus mutans is a member of the dental plaque and is the primary causative agent of dental caries. It can survive extended periods of starvation, which may occur in different niches within the oral cavity. We have found that mucin compensated for the absence of amino acids to promote exponential growth and biofilm formation of S. mutans in minimal medium supplemented with glucose and sucrose, respectively. Mucin extended survival in conditions where there was no net growth provided the operon encoding the pyruvate dehydrogenase complex was intact. Mucin extended survival in conditions of amino acid sufficiency provided the tagatose pathway for galactose utilization was intact, suggesting that S. mutans can scavenge sufficient galactose from mucin to enhance survival, although not to serve as a primary carbon and energy source. The results suggest that mucin has a metabolic role in promoting survival of S. mutans.
The natural habitat of Streptococcus mutans is the human oral cavity, where it is generally considered a ubiquitous indigenous microorganism (Liljemark & Bloomquist, 1996). In the oral cavity, S. mutans is exposed to short periods of nutrient excess during meals, followed by longer periods of starvation between meals. It may be subjected to more prolonged starvation in particular niches. Colonization by S. mutans is often localized to a few surfaces, where it may be present in low numbers for extended periods without causing overt disease (Liljemark & Bloomquist, 1996). However, with a sucrose-rich diet and poor oral hygiene, S. mutans can become a major constituent of the dental plaque and cause dental caries (Loesche, 1986).
Streptococcus mutans grows when sucrose, glucose, or a range of other sugars and related compounds are available. Growth stops in their absence, and S. mutans cannot grow with amino acids as the sole carbon and energy source (Terleckyj & Shockman, 1975). We have been studying how S. mutans survives periods of sugar starvation. We have found that it can survive for extended periods in both batch cultures and biofilms provided the pH remains near neutral (Renye et al., 2004; Busuioc et al., 2010). Mucin can enhance survival (Renye et al., 2004). Mucins make up about 25% of salivary proteins and so constitute a major component of the oral cavity, the natural environment for S. mutans. Mucins contain abundant carbohydrate side chains (Derrien et al., 2010), and three of the common carbohydrate residues, galactose, mannose, and N-acetylglucosamine, are metabolizable by S. mutans (Homer et al., 1993; Ajdic et al., 2002; Abranches et al., 2003; Zeng et al., 2010). However, mucin does not serve as a primary carbon and energy source for S. mutans (van der Hoeven et al., 1990), so that those sugar residues are not readily accessible to S. mutans. Nevertheless, the observation that mucin can prolong survival led us to revisit the possibility that mucin may provide nutrients to enhance S. mutans growth and survival.
We show that mucin compensated for the absence of amino acids to promote exponential growth and biofilm formation in a minimal medium supplemented with sugar; that mucin extended survival in conditions where there was no net growth provided the operon encoding the pyruvate dehydrogenase (PDH) complex was intact; that mucin extended survival in conditions of amino acid sufficiency provided the tagatose pathway for galactose utilization was intact.
Materials and methods
Experiments were with S. mutans strain UA159 (Ajdic et al., 2002) and its derivatives. The pdh::kan mutant, SL14043, was described previously (Busuioc et al., 2010). The lacAB::erm mutant (Abranches et al., 2004) was kindly provided by Dr Robert Burne.
Chemically defined medium (CDM) was FMC of Terleckyj et al. (1975). It was supplemented with 6 mM glucose for batch cultures and 3 mM sucrose for biofilms. Minimal chemically defined medium (MCDM) was the same as CDM except that all amino acids were omitted. Todd Hewitt agar (THA) was used as solid medium. Type III porcine gastric mucin (Sigma) was used as a substitute for salivary mucin (van der Hoeven et al., 1990; Bradshaw et al., 1994; Kolenbrander, 2011). A 5% stock solution was prepared by dissolving mucin powder in 0.01 M phosphate buffer (6.5 mM K2HPO4, 3.5 mM, KH2PO4, pH 7) at room temperature. The dissolved mucin was dialyzed overnight against three changes of 5 L phosphate buffer using dialysis membrane with a 2300 molecular weight cutoff (Spectra/Por 7 Dialysis Membrane; Spectrapor). Mucin was added to media to a final concentration of 0.5% and filter-sterilized (Stericup 45 μm). When required, S. mutans was grown in the presence of kanamycin at 300 μg mL−1 or erythromycin at 25 μg mL−1.
Growth and survival of batch cultures
Streptococcus mutans was grown at 37 °C in 5% CO2. Overnight cultures grown in CDM with 24 mM glucose were washed twice with 1× PBS and diluted 25-fold into fresh CDM, or MCDM, +/− glucose +/− mucin. Streptococcus mutans was grown in static culture tubes. Deletion mutants were grown in the presence of the appropriate antibiotic. Survival studies were conducted in the absence of antibiotic; the mutants were stable without selection. To monitor growth, optical density was measured at 675 nm. For determination of survival, samples were removed, serial dilutions made in 1× PBS, and samples plated onto THA.
Biofilms were set up in flow cell chambers at 37 °C. MCDM or CDM with 3 mM sucrose, and mucin where indicated, was supplied at a flow rate of 200 μL min−1 (Renye et al., 2004). To inoculate the chambers, bacteria were grown overnight in batch cultures in CDM containing 24 mM glucose in 5% CO2 at 37 °C. The bacteria were diluted 25-fold into fresh CDM containing 24 mM glucose and were incubated for 4–5 h. The bacteria were washed twice with 1× PBS and diluted to an OD675 of 0.1. Five hundred microliters of the diluted culture were injected directly into the flow chamber tubing with a syringe. After all chambers were inoculated, the flow (200 μL min−1) of medium was started using a digital pump. The chambers were initially inverted for 20 min to allow the bacteria to adhere to the glass coverslip and then returned to an upright position. After the growth period (18 h), biofilms were starved by supplying the growth medium without sugar, but with mucin where indicated.
Biofilms were monitored by use of the BacLight stain (Molecular Probes, Eugene Oregon). BacLight solution (3 μL) was diluted into 1 mL of PBS. A 500 μL volume of the diluted stain was injected directly into the flow cell tubing (Renye et al., 2004). The flow cell chamber was incubated at room temperature for 15 min, and the biofilm was viewed using a Leica DM IRE2 confocal microscope with a TCS SL system. The excitation wavelength was 488 nm; emission for live (green) cells was captured between 500 and 550 nm, and for dead (red) cells between 600 and 730 nm.
Mucin can substitute for amino acids to promote planktonic growth and biofilm formation in the presence of a sugar
Previous reports indicate that mucin cannot be used as a primary carbon and energy source to support exponential growth of S. mutans, but can modestly increase growth rate and growth yield in the presence of glucose (van der Hoeven et al., 1990; Renye et al., 2004). To examine potential roles of mucin, experiments were performed using a chemically defined medium (CDM) that contains the 20 common amino acids and a minimal chemically defined medium (MCDM) that lacks all amino acids. The mucin was dialyzed prior to use to remove free amino acids and fragments of < 2300 mw.
Streptococcus mutans could grow in MCDM supplemented with mucin and glucose (filled squares, Fig. 1), but not in MCDM supplemented only with glucose (filled circles, Fig. 1). In CDM + glucose, S. mutans could grow without mucin (filled circles, Fig. 2; Renye et al., 2004), suggesting that mucin enabled S. mutans to grow in MCDM containing glucose by providing amino acids. To determine whether the effect of mucin was because the amino acids served as sulfur and/or nitrogen source, MCDM + glucose was supplemented with cysteine and/or combinations of arginine, glutamine, and glutamic acid; no combination of these amino acids supported growth (data not shown). Thus, the mucin was not simply providing a sulfur or nitrogen source. Consistent with previous observations, mucin could not act as a primary carbon and energy source and did not support growth in CDM in the absence of glucose. However, mucin did increase growth rate and slightly increased growth yield in CDM in the presence of glucose (Fig. 2), as reported previously (Renye et al., 2004).
In the absence of glucose, no growth was observed in CDM (Fig. 2). Thus, there was no indication of any reserve of intracellular polysaccharide (IPS) in the inoculum contributing to growth after inoculation. Streptococcus mutans can accumulate IPS reserves when it is grown in media containing excess glucose (Busuioc et al., 2009). However, glucose was limiting in the medium used here to prepare the inoculum so that little if any IPS was anticipated (Renye et al., 2004; Busuioc et al., 2009).
Streptococcus mutans could not form biofilms in MCDM supplemented only with sucrose; however, it did form biofilms in MCDM containing mucin as well as sucrose. Lived/dead stained images of biofilms formed in flow cell chambers are shown in Fig 3. About 3.5 × 109 bacteria per sq cm were recovered from a similar biofilm formed with mucin, whereas only 1.5 × 104 bacteria per sq cm were recovered from a parallel chamber lacking mucin. A major difference between biofilms formed with and without mucin was also observed for static biofilms (Busuioc et al., 2010) grown in MCDM with 3 mM sucrose: 108 bacteria per sq cm with mucin, and 2 × 104 per sq cm without mucin; biofilms in flow cell were consistently thicker than static biofilms (Busuioc et al., 2010) because of the continuous supply of sucrose during their formation. In CDM containing sucrose, S. mutans formed biofilms without mucin (Renye et al., 2004; results not shown), so that, as for planktonic growth, mucin appeared to be providing amino acids to enable S. mutans to form biofilms in MCDM.
Mucin can extend survival in stationary phase
It was previously shown that in both batch cultures and biofilms, mucin prolonged the survival of S. mutans grown in CDM containing 6 mM glucose or 3 mM sucrose. Under these conditions, sugar was exhausted after exponential growth, so that the surviving cultures were starved for sugar (Renye et al., 2004). Amino acid analysis of the spent medium from batch cultures established in CDM + 6 mM glucose (and no mucin) indicated that none of the 20 amino acids in CDM was substantially depleted even 30 days after the glucose was used up (data not shown). Thus, the effects of mucin on survival in CDM were presumably not mediated by amino acids.
Streptococcus mutans survived for at least 70 days after inoculation into MCDM containing both mucin and glucose, where there was growth prior to entering stationary phase (open squares, Fig. 4). It survived for < 15 days after inoculation into MCDM containing glucose, but no mucin, where there was no growth following inoculation (data not shown). The interpretation of these data was complicated by growth prior to entry into stationary phase in the presence of glucose and no growth in its absence.
To explore the role of mucin further, we studied the effect of mucin when it was only present in conditions in which there was no net growth. Bacteria were grown in CDM with 24 mM glucose (with no mucin), washed twice to remove spent medium and residual sugar, and diluted 25-fold into CDM, or MCDM, ± mucin. Cell numbers decreased substantially following inoculation and then stabilized at about 104–105 CFU mL−1 for cultures containing mucin (CDM, Fig. 5a; MCDM, Fig. 5b). Mucin (filled squares, Fig. 5a and b) prolonged survival in both CDM and MCDM to > 70 days, compared to < 7 days in its absence (open squares, Fig. 5a and b). Thus, it seemed possible that in these conditions, mucin enhanced survival by providing amino acids and/or by the slow release of sugar residues, even though it did not serve as primary carbon/energy source.
We had previously found that in stationary phase, S. mutans upregulates the pdh operon, encoding the PDH complex, and that the operon is important for survival (Busuioc et al., 2010). The PDH complex occupies an important junction in carbon metabolism and can have a key role in environments where the concentration of sugar is low (Carlsson et al., 1985). We examined the role of the PDH complex in prolonging survival in the presence of mucin. We found that the extension of survival resulting from the addition of mucin to either CDM or MCDM (with no added sugar) depended on having a functional pdh operon; the pdh mutant (filled circles, Fig. 5a and b) survived for < 9 days compared to > 70 days when pdh was intact (filled squares, Fig. 5a and b).
In conditions in which the bacteria grew in the presence of mucin, prolonged survival also depended on the pdh operon. In CDM containing glucose and mucin, S. mutans UA159 survived for > 70 days (filled squares, Fig. 4), whereas the pdh mutant survived for < 16 days (open squares, Fig. 4). In MCDM with glucose and mucin, the parental strain survived for over 70 days (open squares, Fig. 4), whereas the pdh mutant survived for < 10 days (open circles, Fig. 4). The dependence on pdh suggested that mucin might have a metabolic role in prolonging survival of S. mutans.
The experiments described above suggested that survival in CDM might be enhanced through the slow release of sugars from mucin. Of the sugar residues present in mucin, galactose is the most abundant that is utilizable by S. mutans. To explore whether release of galactose from mucin was prolonging survival, we tested a ΔlacAB mutant. Galactose can be utilized by S. mutans by two pathways, the tagatose and the Leloir pathways, with the tagatose pathway having the major role (Zeng et al., 2010). A ΔlacAB mutant is impaired in the conversion of galactose-6-phosphate to tagatose-6-phosphate and hence is blocked in the tagatose pathway (Zeng et al., 2010). The lacAB mutation is lethal when S. mutans is provided with galactose as sole carbon source because of the accumulation of toxic levels of galactose-6-phosphate (Zeng et al., 2010), but plausibly slow release of galactose from mucin would not be lethal. The ΔlacAB mutant survived for < 18 days in batch cultures established in CDM supplemented with mucin (filled triangles, Fig. 5a), compared to > 70 days for the parental strain (filled squares, Fig 5a). These results suggest that in CDM, the mucin might be providing very low amounts of galactose that were sufficient to enhance survival, although not to produce net growth after inoculation nor to rapidly accumulate toxic levels of galactose-6-phosphate in the ΔlacAB mutant. In contrast, mucin-enhanced survival in MCDM was unaffected by the ΔlacAB mutation (filled triangles, Fig. 5b), suggesting a different role for the mucin than providing galactose; it remains unclear why galactose would not be needed for survival in MCDM, but would be needed in CDM.
Mucin has been shown to provide nutrients for a variety of bacterial species that inhabit mucosal surfaces (Derrien et al., 2010). Mucin is a complex glycoprotein, with multiple carbohydrate side chains (Derrien et al., 2010). The ability of bacteria to utilize mucin as a nutrient depends on their ability to cleave the peptide backbone by proteases, cleave O-linked glycosidic bonds to liberate sugars (predominantly fucose, galactose, and N-acetyl-glucosamine) or N-linked glycosidic bonds for mannose, and remove the sulfate and sialic acid modifications from the terminal sugars (Derrien et al., 2010). There are only rare examples of bacteria, such as Bacteroides thetaiotaomicron and Akkermansia muciniphila, that are capable of using mucin as sole carbon and nitrogen source (Salyers et al., 1977; Derrien et al., 2004; Sonnenburg et al., 2005). In the oral cavity, a consortium of bacteria acts in concert to degrade mucin and release its nutrients (de Jong et al., 1984; van der Hoeven et al., 1990). Streptococcus mutans cannot use mucin as a sole carbon and energy source (van der Hoeven et al., 1990); however, mucin prolonged the survival of S. mutans (Renye et al., 2004). The purpose of the present study was to determine whether S. mutans in monospecies cultures was capable of scavenging nutrients from mucin to enhance its growth and survival.
We found that mucin can provide amino acids to support growth in the absence of free amino acids, when a primary energy source such as glucose is provided. Consistent with this conclusion, S. mutans encodes multiple proteases and transporters for free amino acids and oligopeptides (Ajdic et al., 2002). As the mucin was dialyzed extensively before use, it is likely that the amino acids were scavenged by the cleavage of the peptide backbone and were not a result of free amino acids in the mucin preparation.
Our results also indicate that, at least in the conditions tested, S. mutans can scavenge sufficient galactose from mucin to enhance long-term survival, although not sufficient to serve as primary carbon and energy source. This conclusion is supported by the extended survival in CDM containing mucin depending on having an intact tagatose pathway (Fig. 5a); it is consistent with sequence analysis indicating that S. mutans putatively encodes α- and β-d-galactosidase, which might release galactose from mucin (Derrien et al., 2010). Although amino acids can support growth, the dependence on the tagatose pathway for survival in the amino acid replete CDM makes it unlikely that amino acids are the primary part of mucin being used to support survival. Streptococcus mutans can grow on galactose as a primary carbon and energy source (Zeng et al., 2010). Therefore, the inability of mucin to act as primary carbon and energy source may be because the organism has a limited ability to scavenge galactose from mucin; the putative galactosidases may be poorly expressed or may have very low activity against mucin, possibly because of extensive modification of the terminal sugars on mucin by sulfate and sialic acid residues (Roberton et al., 2000; Brockhausen, 2003). The dependence of survival on pdh may be metabolic, as an end product of the tagatose pathway is d-glyceraldehyde-3-P, which could be converted to pyruvate and serve as a substrate for PDH. However, a role for PDH as a regulator responding to pyruvate availability cannot be ruled out.
Taken together, the results suggest that, in a monospecies culture or biofilm, S. mutans can scavenge amino acids and a small amount of galactose from mucin. The galactose is sufficient to enhance the survival of the population dependent on the tagatose pathway and the presence of PDH. The results support the notion that mucin is an important nutrient for the survival of oral bacteria.
This work was supported in part by Public Health Service grant DE-014604 from the National Institutes of Health to PJP and BAB. We thank Robert Burne for the lacAB mutant. We thank Monica Busuioc and April Suriano for helpful discussions.