Inactivation of the spxA1 or spxA2 gene of Streptococcus mutans decreases virulence in the rat caries model

SUMMARY

In oral biofilms, the major environmental challenges encountered by Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the transcriptional regulators SpxA1 and SpxA2 are involved in general stress survival of S. mutans with SpxA1 playing a primary role in activation of antioxidant and detoxification strategies whereas SpxA2 serves as a back up activator of oxidative stress genes. We have also found that spxA1 mutant strains (ΔspxA1 and ΔspxA1ΔspxA2) are outcompeted by peroxigenic oral streptococci in vitro and have impaired abilities to colonize the teeth of rats fed a highly cariogenic diet. Here, we show that the Spx proteins can also exert regulatory roles in the expression of additional virulence attributes of S. mutans. Competence activation is significantly impaired in Δspx strains and the production of mutacin IV and V is virtually abolished in ΔspxA1 strains. Unexpectedly, the ΔspxA2 strain showed increased production of glucans from sucrose, without affecting the total amount of bacteria within biofilms when compared to the parent strain. By using the rat caries model, we showed that the capacity of the ΔspxA1 and ΔspxA2 strains to cause caries on smooth tooth surfaces is significantly impaired. The ΔspxA2 strain also formed fewer lesions on sulcal surfaces. This report reveals that global regulation via Spx contributes to the cariogenic potential of S. mutans and highlights the essentiality of animal models in the characterization of bacterial traits implicated in virulence.

INTRODUCTION

Dental caries is one of the most prevalent infectious diseases worldwide (Marcenes et al., 2013; Selwitz et al., 2007). Even though the participation of other oral bacteria in the etiology of caries cannot be overlooked (Aas et al., 2005; Jenkinson, 2011), clinical and laboratory studies clearly implicate the oral pathogen Streptococcus mutans in the initiation and development of this disease (Lemos et al., 2005; Takahashi and Nyvad, 2011). The virulence of S. mutans is directly associated with its ability to form biofilms on tooth surfaces, to produce extracellular polysaccharides (EPS) and weak acid from sugars, and to efficiently adapt to large fluctuations in pH, oxygen tension and nutrient availability (Lemos et al., 2005).

Work from a number of laboratories demonstrated that S. mutans is well equipped to adapt to low pH values by activation of a robust physiological response to acidification referred to as the acid tolerance response (ATR) (Lemos et al., 2005). The ATR is accomplished by upregulation of the membrane-associated F-ATPase, induction of pathways that contribute to cytoplasm buffering and changes in membrane fatty acid composition, among other processes. While the S. mutans ATR has been studied in some detail (Lemos et al., 2005), the importance of O₂ metabolism and the mechanisms to cope with reactive oxygen species (ROS) in S. mutans have received limited attention. The relevance of oxidative stress survival to the pathophysiology of S. mutans is supported by studies that show an inverse correlation between the total numbers of S. mutans and peroxigenic Streptococci (e.g. S. sanguinis and S. gordonii) in dental plaque (Becker et al., 2002; Kreth et al., 2008; Mikx et al., 1972). Specifically, members of the mitis group are often associated with oral health; a series of elegant in vitro studies showed that H₂O₂ produced by S. gordonii or S. sanguinis via a pyruvate oxidase serves as a “chemical weapon” antagonizing the growth of S. mutans (Kreth et al., 2008). In addition, H₂O₂ present in certain oral hygiene and tooth bleaching products may represent another source of peroxide stress for oral bacteria (Marquis, 1995).

Previously, we identified and characterized two genes, named spxA1 (formerly spxA) and spxA2 (formerly spxB) in S. mutans, which serve as global transcriptional regulators (Kajfasz et al., 2009; Kajfasz et al., 2010b). Spx regulators are highly conserved among Firmicutes and are directly involved in oxidative stress responses by positively affecting the transcription of genes involved in thiol homeostasis and detoxification (Kajfasz et al., 2010b; Nakano et al., 2003a; Nakano et al., 2003b; Zuber, 2004). Physiologic characterizations of ΔspxA1, ΔspxA2 and ΔspxA1/ΔspxA2 strains revealed that SpxA1 plays a major role in survival under acid and oxidative stress conditions (Kajfasz et al., 2010b). While stress tolerances were generally not impaired in the ΔspxA2 strain, the stress sensitivities of the double ΔspxA1/ΔspxA2 strain were more pronounced than in the single ΔspxA1 strain (Kajfasz et al., 2010b). In addition to activation of genes involved in thiol homeostasis and ROS scavenging (Kajfasz et al., 2010b; Kajfasz et al., 2015), we have recently shown that Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron (Galvao et al., 2015). Transcriptome and in vitro transcription analyses further supported that SpxA1 functions as the primary transcriptional activator of oxidative stress genes whereas SpxA2 appears to have a secondary but supportive role in the activation of oxidative stress responses (Kajfasz et al., 2010b; Kajfasz et al., 2015). Finally, inactivation of spxA1, spxA2 or both attenuated the virulence of S. mutans in the G. mellonella invertebrate model but only strains lacking the spxA1 gene (ΔspxA1 and ΔspxA1/ΔspxA2) showed a reduced ability to colonize the teeth of rats fed a highly cariogenic diet (Kajfasz et al., 2010b).

In this manuscript, we showed that loss of one or both spx genes affects the expression of important virulence attributes of S. mutans. Specifically, both Spx proteins were shown to mediate competence development and SpxA1 appears to be essential for mutacin production. While SpxA2 was not seemingly involved in mutacin production, inactivation of spxA2 resulted in enhanced biomass without affecting bacterial amount. We also assessed the virulence potential of the ΔspxA1 and ΔspxA2 strains in the rat caries model to show that both mutants were less cariogenic than the parental strain.

METHODS

Bacterial strains and growth conditions

The strains used in this study are listed in Table 1. The S. mutans UA159 (wild-type) and its Δspx derivatives (ΔspxA1, ΔspxA2 and ΔspxA1/ΔspxA2) were routinely grown in Brain Heart Infusion (BHI) at 37°C under anaerobic conditions (BBL Gaspack system, BD, Franklin Lakes, NJ). For overexpression of SpxA1 or SpxA2, strains harboring the pMSP3535 empty plasmid (Bryan et al., 2000) or pMSP3535 expressing spxA1 or spxA2 were grown in BHI broth containing 10 μg ml⁻¹ erythromycin. Overproduction of SpxA1 and SpxA2 was achieved by adding 16 ng ml⁻¹ nisin to the growth media.

Table 1.

Bacterial strains used in this study.

Strains	Relevant genotype	Source or reference
S. mutans
UA159	Wild-type	Laboratory stock
JL12 (ΔspxA1)	spxA1:: SpR	Kajfasz et al., 2009
JL13 (ΔspxA2)	spxA2:: ErmR	Kajfasz et al., 2009
JL21 (ΔspxA1/A2)	spxA1:: SpR, spxA2:: ErmR	Kajfasz et al., 2009
UA159+pMSP3535	ErmR	Kajfasz et al., 2015
UA159+spxA1	Wild-type harboring pMSP3535-spxA1, ErmR	This study
UA159+spxA2	Wild-type harboring pMSP3535-spxA2, ErmR	This study
Other species
L. lactis ATCC 11454	Wild-type	Laboratory stock
S. gordonii DL-1	Wild-type	Laboratory stock

Biofilm Assays

Biofilm development was measured in polystyrene 96-well (flat-bottom) microtiter plates (Costar .595; Corning Inc., Corning, NY). Briefly, cultures were grown in BHI to OD₆₀₀ of 0.5 and used to inoculate (1:100) the wells of a microtiter plate containing 200 μl of low molecular weight medium (LMW) (Koo et al., 2005) containing 1% sucrose. Following incubation for 24 h at 37°C in a 5% CO₂ aerobic atmosphere, culture medium was removed by aspiration and wells were gently washed with 200 μl sterile deionized water. Subsequently, 50 μl of a 0.1% solution of crystal violet dissolved in 99% ethanol was applied to each well and incubated at room temperature for 15 min, followed by removal of the fluid by aspiration. Wells were washed twice with water as before and allowed to air dry. The plates were de-stained with 200 μl of an acetone:ethanol solution (2:8) for 30 min at room temperature. The de-staining procedure was repeated and the OD₅₇₅ of the pooled de-staining solution was measured. Background was determined from staining non-inoculated wells with crystal violet. Significance was established as a P value ≤ 0.05. Experiments were conducted at least in triplicates.

Confocal microscopy analysis of biofilms was performed on saliva-coated hydroxyapatite discs (sHA) (diameter, 1.25 cm; Clarkson Chromatography Products, Inc., South Williamsport, PA). Stimulated human saliva obtained from healthy subjects (RSRB 00030432, University of Rochester) was pooled, clarified by centrifugation and filter-sterilized prior to use. Cells of S. mutans UA159 or ΔspxA2 were grown in ultrafiltered (10-kDa-cutoff membrane; Prep/Scale; Millipore, MA) buffered tryptone-yeast extract broth (UFTYE) containing 2.5% tryptone and 1.5% yeast extract (pH 7.0) with 1% glucose to OD₆₀₀ of 0.5. Biofilms were formed on sHA discs placed vertically using a disc holder in a 24-well plate containing batch (static) cultures (Falsetta et al., 2012; Koo et al., 2005). The bacterial cells were grown in 2.8 ml (per well) of UFTYE containing 1% sucrose and an sHA disc at 37°C in 5% CO₂ for 24 h. At the end of the experimental period, the biofilms were dip-washed three times and then gently swirled in physiological saline to remove loosely adherent material. The biofilms were subjected to confocal imaging analysis as described elsewhere (Falsetta et al., 2012; Xiao and Koo, 2010). Briefly, Alexa Fluor 647-dextran conjugate (10 kDa; absorbance/fluorescence emission maxima, 647/668 nm; Molecular Probes, Invitrogen Corp., Carlsbad, CA) was used to label glucans in the EPS matrix, while total microbial biomass was stained with Syto 9 (485/498 nm; Molecular Probes). Imaging was performed using an Olympus FV 1000 two-photon laser scanning microscope (Olympus, Tokyo, Japan). Each biofilm was scanned at five positions randomly selected at the microscope stage for a total of 15 stacks per sample from 3 independent experiments. The confocal images were analyzed using COMSTAT software for the quantitation of EPS and microbial cells biomass within intact biofilms (Falsetta et al., 2012; Xiao and Koo, 2010).

Deferred antagonism assay

Cultures were grown in BHI to an OD₆₀₀ of 0.3 when a 15 μl aliquot was spotted onto BHI agar and incubated for 24 h. Following incubation, plates were exposed to UV light for 20 min to ensure that subsequent antagonism was not due to actively growing S. mutans cells. Then, 500 μl of an overnight culture of S. gordonii DL-1 (mutacin IV sensitive) or Lactococus lactis ATCC 11454 (mutacin V sensitive) was added to 5 ml soft (0.75 %) BHI agar, spread as an overlay and incubated for another 24 h before zones of growth inhibition around the S. mutans spots were measured (Hossain and Biswas, 2011).

Genetic competence assay

Overnight cultures were sub-cultured 1:20 into fresh BHI supplemented with 10% horse serum and grown to OD₆₀₀ of 0.125. At this point, cultures were split in two and incubated for an additional 15 min with or without 5 μM of the synthetic competence stimulating peptide (CSP) (Li et al., 2002) followed by addition of 0.2 μg of the shuttle plasmid pMC340B that confers resistance to kanamycin. Cells were then incubated until stationary phase was reached and plated in duplicate on BHI agar plates with or without 1 mg ml⁻¹ kanamycin. Transformation efficiency was determined after 48 h incubation at 37°C in a 5 % CO₂ atmosphere and was expressed as the percentage of transformants (kanamycin plates) among the total viable recipient cells (antibiotic-free plates).

Rat caries model

The animal experiment protocol was reviewed and approved by the Ethical Committee on Animal Research at the University of Campinas, SP, Brazil (Protocol # 2637-1) and was performed according to methods previously described (Falsetta et al., 2012). Female pups free of S. mutans and SDA virus from 10 litters of SPF Wistar rats were provided by CEMIB (UNICAMP). At the age of 19 days, pups were weaned and randomly divided into three groups of 10 animals. Animals were orally infected for three successive days by means of a cotton swab containing mid-exponential cultures of S. mutans strains UA159, ΔspxA1 or ΔspxA2. The oral infection of the pups was confirmed 1 week later by plating on Mitis-Salivarius (MS) agar plus bacitracin (MSB; Sigma-Aldrich, St Louis, MO). Each group received highly cariogenic diet 2000 and 5% sucrose water ad libitum (Bowen et al. 1988). The animals were weighed weekly and their behavior and physical appearance noted on a daily basis. After five weeks, animals were killed by CO₂ asphyxiation. The lower left jaw was aseptically dissected, suspended into 5.0 ml of sterile saline solution (0.9%, w/v), and sonicated (three 10 s pulses at 5 s intervals, at 30 W; Vibracell, Sonics and Material Inc). The suspension was plated on MSB to estimate the populations of S. mutans UA159, ΔspxA1 and ΔspxA2 and on blood agar to determine the total cultivable microorganisms. Smooth-surface and sulcal caries and their severities were evaluated according to Larson’s modification of Keyes’ system (Keyes, 1958; Larson, 1981) by a single calibrated examiner blinded to the study. Statistical significance was determined by ANOVA in the Tukey-Kramer HSD test for all pairs. The statistical software GraphPad PRISM version 5.0 was used to perform the analyses. The level of significance was set at 5%.

RESULTS

Inactivation of either SpxA1 or SpxA2 impairs competence development and loss of SpxA1 abolishes mutacin production

Previously, we showed that competence and production of at least two mutacins (mutacin IV and mutacin V) were significantly impaired in ΔclpP and ΔclpX strains (Kajfasz et al., 2011). As Spx proteins are targeted for degradation by the ClpXP proteolytic system (Kajfasz et al., 2009), this result indirectly suggests that the Spx proteins negatively regulate competence and mutacin production in S. mutans. Unexpectedly, transformation efficiency was also lower in strains lacking one or both spxA genes, regardless of the presence of exogenously added CSP, which boosted the number of transformants in all strains (Fig. 1A-B). In an attempt to understand the conflicting results involving Spx levels and competence activation, we used a nisin-inducible plasmid to overexpress SpxA1 or SpxA2 in the parental strain thereby mimicking the high Spx levels observed in ΔclpP and ΔclpX strains. Then, we tested the transformation efficiency of these strains after induction with a sub-inhibitory concentration of nisin that does not affect growth rates of any of the strains. As observed in both Δclp and Δspx strains, overexpression of SpxA1 or SpxA2 also resulted in competence deficiency as compared to the parent strain (Fig. 1C-D).

Fig. 1.

Competence of S. mutans UA159 and its derivatives. Transformation efficiency of (A, B) parent UA159 and ΔspxA strains and (C, D) UA159 harboring the nisin-inducible pMSP3535 (control) or pMSP3535-Spx plasmids (Spx-overexpressing strains). The integration plasmid pMC340A (donor DNA) was added to cells in early-logarithmic phase (OD₆₀₀ of 0.15) with or without the addition of CSP or nisin. Cultures were incubated at 37°C in 5% CO₂ until entering stationary phase, serially diluted and plated in BHI for total CFU and in BHI containing kanamycin for transformants. (A) pMC340A only (B) pMC340A + CSP, (C) pMC340A only, and (D) pMC340A + nisin. The results represent the mean and standard deviations of at least three independent experiments. Asterisks indicate differences were statistically significant (P ≤0.05) when compared to UA159 (A, B), or UA159 harboring the empty pMSP3535 vector (C, D).

Next, we measured production of two competence-coordinated bacteriocins by the Δspx strains using a deferred antagonism assay (Fig. 2). Inhibition of the growth of S. gordonii was used to assess the ability of the wild-type and ΔspxA strains to produce mutacin IV, which is active against the mitis group of streptococci (Qi et al., 2001). Inhibition of L. lactis was used to assess the ability of the strains to produce mutacin V, which has broader antimicrobial activity, ranging from mitis streptococci to lactococci and micrococci (Hale et al., 2005). Based on our results, mutacin production by the ΔspxA1 and ΔspxA1/ΔspxA2 strains was diminished to the extent that both strains were completely unable to antagonize the growth of S. gordonii or L. lactis. The link to mutacin production appears to be limited to spxA1, as the ΔspxA2 strain showed antagonism similar to that of UA159, generating average zone diameters of 12.3mm (UA159) to 14.3mm (ΔspxA2) against S. gordonii.and 19.7mm (UA159) to 20.7mM (ΔspxA2) against L. lactis.

Fig. 2.

Mutacin production by S. mutans UA159 and its derivatives. Cultures of S. mutans were grown in BHI to OD₆₀₀ of 0.3 and 15 μl of each culture spotted on BHI plates followed by incubation at 37°C in 5% CO₂. After 24 h incubation, plates were exposed to UV light for 20 min and then overlayed with 5 ml soft BHI agar containing 500 μl of overnight cultures of S. gordonii or L. lactis. Plates were incubated for additional 48 h and the zone of inhibition recorded.

The ΔspxA2 strain shows enhanced glucan production

The ability to form biofilms via production of EPS such as glucans in the presence of sucrose is a major virulence factor of S. mutans (Koo et al., 2005; Xiao et al., 2012; Yamashita et al., 1992). Because spx genes have been linked to biofilm formation in Staphylococci (Pamp et al., 2006; Wang et al., 2010), we used in vitro assays to determine the ability of the single Δspx strains to form biofilms in media containing sucrose as the sole carbohydrate source. In saliva-coated microtiter plates, biofilm formation by ΔspxA2, but not the ΔspxA1 strain, was significantly enhanced (Fig. 3A). We also tried to measure the biofilm-forming capacity of the double ΔspxA1/ΔspxA2 strain. However, growth of this strain in biofilms was dramatically impaired and the biofilm quantifications were not reproducible. To exclude any possibility that the differences observed between the parent and single Δspx strains were due to small variations in the growth rates of the strains, the biofilm data were normalized by the total final OD₆₀₀ comprised of both planktonic and biofilm cells. To confirm the enhanced sucrose-dependent biofilm phenotype of the ΔspxA2 strain, a saliva-coated HA disc assay (a tooth enamel surrogate) was used to measure total biomass of bacteria and EPS glucans as well as EPS/bacteria ratio via confocal microscopy. In agreement with the results using microtiter plates, the ΔspxA2 strain showed total biomass by increasing production of EPS glucans (Fig. 3B) without, however, affecting the amount of bacterial biomass. COMSTAT analysis revealed that the glucan/ bacteria ratio of the ΔspxA2 strain (3.71± 0.98) was significantly higher (P ≤ 0.05) than the ratios observed for the parent strain (2.56± 0.68).

Fig. 3.

Biofilm fomation and biomass by S. mutans UA159 and its derivatives. (A) Biofilm formation on microtiter plates. Cultures were grown in LMW containing 1% sucrose for 24 h. Biofilm formation was normalized by total growth to exclude apparent differences due to the growth abilities of each strain (*, P ≤ 0.05). (B) The imaging of EPS and bacteria within intact 3D biofilms was conducted using a method based on incorporation of fluorescently labeled dextran (Alexa Fluor 647, red) by Gtfs during glucan synthesis. Bacterial cells were labeled with SYTO9 (green). The images shown are representative of n = 15. The confocal images were analyzed by AMIRA and COMSTAT (*, P ≤ 0.05).

Inactivation of spxA1 or spxA2 decreases the colonization and cariogenic potentials of UA159

Previously, we showed that the ability of the ΔspxA1 and Δspx1/ΔspxA2 strains to colonize the teeth of rats was significantly impaired (Kajfasz et al., 2010b). As stated above, the Δspx1/ΔspxA2 strain shows poor growth in biofilms, making it virtually impossible to separate a low cariogenic potential from an overall loss of bacterial fitness. As in the case of the biofilm analysis, an animal caries study was conducted only with the wild-type and Δspx single mutant strains. During the course of the study, no differences were observed in the pattern of meals consumed by the animals and consistent with this was the finding that there were no significant differences observed in the weight gains of animals. Counts of total cultivable microflora revealed no significant differences among the groups infected with the wild-type, ΔspxA1 or ΔspxA2 strains (Fig. 4A). However, when focusing on S. mutans, the numbers of ΔspxA1 and ΔspxA2 colonies recovered from the animals were lower when compared to the parent strain (Fig. 4B) but only the differences between parent and ΔspxA1 strains were statistically significant (P ≤ 0.05). Likewise, the proportions of S. mutans to total flora were significantly lower (P ≤ 0.05) for the ΔspxA1 strain (29.08 %± 17.61) but not for ΔspxA2 (59.79 % ± 21.9) when compared to the proportions of the wild-type to total flora (53.35 % ± 23.42) (Fig. 4C).

Fig. 4.

Total cultivable microflora (A) and S. mutans (B) recovered from left jaws of animals five weeks after infection with UA159, ΔspxA1 or ΔspxA2 strains. (C) Proportion (%) of S. mutans per total flora. The results represent the mean and standard deviations of one experiment (n=10). Asterisks indicate differences were statistically significant (P ≤0.05) when compared to UA159 * P ≤ 0.05 (ANOVA).

The incidence and severity of smooth surface and sulcal surface caries in animals infected with the parent or Δspx strains are shown in Table 2. Scoring of carious lesions on smooth and sulcal surfaces was divided into parameters evaluating either lesion extension (enamel [E]), or lesion severity (slight [Ds], moderate [Dm], or severe [Dx]) as defined by Larson (Larson, 1981). Rats infected with ΔspxA1 or ΔspxA2 had reduced dental caries based on lower levels of average lesion extension [E] on smooth surfaces. The ΔspxA2 strain also caused fewer sulcal carious lesions when compared to animals infected with the UA159 or ΔspxA1 strains (P ≤ 0.05). Despite the differences in lesion extension, the differences in lesion severity among strains were not significant at any level of dentin damage.

Table 2.

Influence of different strains on development of dental caries in Wistar rats (Keyes’ score).

	Smooth surface				Sulcal surface
Strains	Lesion extension (E)	Lesion severity			Lesion extension (E)	Lesion severity
		Ds	Dm	Dx		Ds	Dm	Dx
UA159	40.8 (±9.05)	16.5 (±6.58)	4.8 (±4.75)	0.7 (±1.49)	47.2 (±3.04)	41 (±2.82)	11.6 (±6.73)	1.2 (±1.47)
Δ spxA1	30.3* (±12.01)	16.8 (±9.18)	6.9 (±5.98)	0.1 (±0.31)	45.8 (±4.8)	40.1 (±3.34)	11.3 (±6.58)	4 (±5.07)
Δ spxA2	25.2* (±8.16)	14.3 (±6.75)	4.7 (±3.88)	0	40.4* (±9.41)	35 (±8.65)	6.6 (±7.9)	1 (±1.15)

Asterisks denote statistically significant differences when compared to the wild-type (UA159) values in the same column, n=10. ANOVA, using Tukey-Kramer HSD test for all pairs, was used to determine statistical significance.. Notes: E – enamel; Ds – slight dentinal caries; Dm – moderate dentinal caries; Dx – extensive dentinal caries.

DISCUSSION

While the majority of work that has contributed to the understanding of Spx function has come from the model Gram-positive organism Bacillus subtilis, evidence is now accumulating that Spx proteins have similar regulatory functions in other bacteria (Chen et al., 2012; Kajfasz et al., 2010a; Kajfasz et al., 2012; Pamp et al., 2006b; Turlan et al., 2009; Turner et al., 2007; Zheng et al., 2014). After our initial discovery of the existence of two Spx proteins in S. mutans, evidence that two Spx paralogs, with overlapping but also unique regulatory functions, contribute to adaptive stress responses of other Streptococci and, more recently, Bacillus anthracis has emerged (Barendt et al., 2013; Chen et al., 2012; Kajfasz et al., 2009; Kajfasz et al., 2010b; Turlan et al., 2009; Zheng et al., 2014). From our previous studies, it becomes clear that SpxA1 and SpxA2 are global regulators and, while SpxA1 has a primary role in the activation of oxidative stress genes, both Spx proteins appear to modulate the expression of a variety of other cellular traits, including cell envelope homeostasis by SpxA2 (Kajfasz et al., 2010b) and, as shown here, competence development (SpxA1 and SpxA2), mutacin production (SpxA1) and biofilm formation (SpxA2).

The association of Spx regulation with competence development was first demonstrated in B. subtilis. The B. subtilis Spx was shown to negatively affect competence by assisting the formation of a complex with the ClpC ATPase and the MecA adaptor protein that sequesters ComK, a transcriptional regulator required for late competence gene activation (Nakano et al., 2002). More recently, the S. pneumoniae SpxA1 was shown to repress transcription of the early competence operon comCDE thereby also acting as a negative regulator of competence (Turlan et al., 2009). In S. mutans UA159, competence development is controlled by quorum-sensing mechanisms involving the σ^X inducing peptide (XIP) and the competence stimulating peptide (CSP) (Mashburn-Warren et al., 2010; van der Ploeg, 2005). Competence activation has been shown to influence biofilm formation, stress tolerance, persistence and virulence (Kaspar et al., 2015; Leung et al., 2015; Li et al., 2008; Perry et al., 2009a; Perry et al., 2009b; Wenderska et al., 2012), and is intertwined with antagonism of other species, as bacteriocin (mutacin) production is controlled by the CSP quorum-sensing factor (Dufour et al., 2011; Kreth et al., 2005; Reck et al., 2015; van der Ploeg, 2005). Our previous observation that competence was impaired in S. mutans strains lacking clpP (ΔclpP) or clpX (ΔclpX) (Kajfasz et al., 2011), known to accumulate the Spx proteins (Kajfasz et al., 2010), was in line with the literature. Unexpectedly, deletion of either spxA1 or spxA2 negatively affected competence suggesting a much more complex relationship between Spx and competence development in S. mutans. Given these conflicting results, we took a further step and assessed competence efficiency in wild-type strains (ClpXP⁺) overproducing spxA1 or spxA2 through an inducible plasmid. Surprisingly, competence efficiency was impaired to similar levels as seen in the Δclp and Δspx strains. Thus, our results indicate that any fluctuations in Spx pools can have a detrimental effect in S. mutans competence. By perusing our previous microarrays comparing the transcriptome of the wild-type UA159 and ΔspxA strains grown in BHI medium to an OD₆₀₀ of 0.5 (Kajfasz et al., 2010), we found that expression of comR (smu61) and comE (smu1917), the transcriptional regulators involved in activation of the XIP competence and CSP competence/bacteriocin pathways, respectively, was downregulated (~ 2-fold each) in the ΔspxA1 strain. However, the transformation efficiency of all strains in this study was improved upon addition of CSP to the cultures, suggesting that cells lacking the SpxA proteins are able to respond to the presence of CSP. Additional studies to obtain mechanistic insight into the significance of Spx in competence development are underway.

The production of bacteriocins by S. mutans strains, the so-called mutacins, is thought to play an important role in oral colonization. Mutacin production helps S. mutans to compete with other oral bacteria enabling its establishment and persistence in dental plaque (Merritt and Qi, 2012). Mutacins are divided in two types: lantibiotic and non-lantibiotic, with lantibiotics showing a higher spectrum of activity.than non-lantibiotics (Merritt and Qi, 2012). The genome of S. mutans UA159 encodes at least three non-lantibiotic mutacins; mutacin IV, V and VI. These mutacins are regulated by the ComCDE quorum-sensing system, initially identified as a competence activator (Li et al., 2002). The observed defects in competence of strains with abnormal Spx levels and the intimate relationship between the regulatory networks controlling competence and mutacin production led us to assess mutacin production in the Δspx strains. Using S. gordonii (sensitive to mutacin IV) and L. lactis (sensitive to mutacin V) as indicators, we found that strains lacking spxA1 could no longer inhibit the growth of both indicator strains. Different from competence activation that is influenced by both SpxA1 and SpxA2, mutacin production was not affected in the ΔspxA2 single mutant. We also searched our previous transcriptome analysis (Kajfasz et al., 2010) for the differential expression of mutacin-coding genes in the Δspx strains. Transcription of the genes responsible for mutacin IV (nlmAB) or mutacin V (nlmC) was not significantly affected in the Δspx strains albeit this result remains to be validated using targeted approaches (e.g., qRT RT-PCR). While the specific association of Spx regulation with mutacin production is still unclear, it seems unlikely that it occurs through direct regulation of the mutacin-encoding genes by Spx.

It is well recognized that glucosyltransferases (Gtf) from S. mutans play critical roles in the development of highly cariogenic biofilms through formation of an insoluble polysaccharide matrix comprised primarily of glucans (Bowen and Koo, 2011). The genome of S. mutans UA159 encodes three Gtf genes; gtfB, gtfC and gtfD. The gtfB and gtfC genes are genetically linked and co-regulated by multiple transcriptional regulators (Bowen and Koo, 2011; Goodman and Gao, 2000; Yoshida and Kuramitsu, 2002). The gtfD gene is located elsewhere in the chromosome and also appears to be under complex transcriptional control (Bowen and Koo, 2011; Goodman and Gao, 2000). Our observations that glucan production and, as a result, biofilm biomass, is enhanced in the ΔspxA2 strain aligns well with previous microarray analysis showing that expression of gtfB and gtfD was enhanced in ΔspxA2 (Kajfasz et al., 2010). Additionally, we observed the upregulation of the lytTS genes that are involved in autolysis and biofilm formation through the release of eDNA which contributes to the structure of the extracellular matrix (Klein et al., 2010). When compared to the parent strain, lytS was found to be upregulated more than 2-fold in ΔspxA2 in our earlier microarray analysis, which may help to explain the enhanced biofilm formation seen for the ΔspxA2 strain. Aside from serving as a structural component, the presence of eDNA may further contribute to matrix formation by stimulating glucan synthesis by GtfB on the S. mutans cell surface (Klein et al., 2015).

In a previous oral colonization study, we showed that 15 days post-infection, the ΔspxA1 strain was recovered in significantly fewer numbers from the teeth of specific pathogen-free (SPF) rats (Kajfasz et al., 2010). However, when SPF rats were infected with the ΔspxA2 mutant, the CFU numbers recovered were similar to those obtained from the parental strain. Here, in a 5-week caries study, when compared to animals infected with the parent strain, we showed that SPF rats infected with either the ΔspxA1 or ΔspxA2 strain had fewer carious lesions on smooth surfaces whereas the ΔspxA2 strain also caused fewer sulcal caries. Interestingly, while the ΔspxA1 strain was recovered at fewer numbers upon termination of the study, the difference between the number of S. mutans recovered from animals infected with the parent or ΔspxA2 strain was not significant, consistent with in vitro data showing unchanged bacterial amounts. Thus, it appears that loss of spxA2 does not affect colonization of the tooth surface, as observed in our colonization study (Kajfasz et al., 2010), but rather affects the cariogenic potential of S. mutans in vivo. While the result of the caries study with the ΔspxA1 strain was a logical and expected extension of the oral colonization study and aligns well with the general stress sensitivity (Galvao et al., 2015; Kajfasz et al., 2010; Kajfasz et al., 2015) and defects in competence and mutacin production of the ΔspxA1 strain, the decreased cariogenicity of ΔspxA2 was somewhat surprising. With the exception of competence and biofilm formation, the in vitro expression of all other virulence attributes investigated in the ΔspxA2 strain were not affected. At first glance, the decrease in formation of carious lesions by ΔspxA2 may appear surprising considering the enhanced in vitro biofilm biomass due to increased EPS production by this strain. However, it is noteworthy that the bacterial accumulation within the biofilm was not significantly enhanced. Thus, it appears that the ability of S. mutans themselves, within the EPS-rich matrix milieu, to cause the disease was attenuated in ΔspxA2. Whether this aberrant glucan accumulation affects the mechanical stability of the biofilm, proton diffusion, or proton retention of S. mutans as suggested by others (Guo et al., 2015; Hata and Mayanagi, 2003), it still needs further evaluation.

We previously reported diminished virulence of ΔspxA2, as well as the ΔspxA1 strain, in the G. mellonella systemic infection model (Kajfasz et al., 2010). In addition, the fitness of the strain lacking both spx genes has consistently proven to be severely compromised in physiologic assays and animal models (Kajfasz et al., 2010; Kajfasz et al., 2015), which highlights the cooperative nature of these two Spx paralogs. Thus, it appears that SpxA2 performs important functions in S. mutans and that further characterization of the regulatory activity of SpxA2 during infection and the nature of its cooperative relationship with SpxA1 is warranted. For example, it will be interesting to obtain the global transcriptional profile of cells from Δspx strains isolated directly from dental plaque of infected SPF rats.