Characterization of LrgAB as a stationary phase-specific pyruvate uptake system in Streptococcus mutans
Our recent ‘-omics’ comparisons of Streptococcus mutans wild-type and lrgAB-mutant revealed that this organism undergoes dynamic cellular changes in the face of multiple exogenous stresses, consequently affecting its comprehensive virulence traits. In this current study, we further demonstrate that LrgAB functions as a S. mutans pyruvate uptake system.
S. mutans excretes pyruvate during growth as an overflow metabolite, and appears to uptake this excreted pyruvate via LrgAB once the primary carbon source is exhausted. This utilization of excreted pyruvate was tightly regulated by glucose levels and stationary growth phase lrgAB induction. The degree of lrgAB induction was reduced by high extracellular levels of pyruvate, suggesting that lrgAB induction is subject to negative feedback regulation, likely through the LytST TCS, which is required for expression of lrgAB. Stationary phase lrgAB induction was efficiently inhibited by low concentrations of 3FP, a toxic pyruvate analogue, without affecting cell growth, suggesting that accumulated pyruvate is sensed either directly or indirectly by LytS, subsequently triggering lrgAB expression. S. mutans growth was inhibited by high concentrations of 3FP, implying that pyruvate uptake is necessary for S. mutans exponential phase growth and occurs in a Lrg-independent manner. Finally, we found that stationary phase lrgAB induction is modulated by hydrogen peroxide (H2O2) and by co-cultivation with H2O2-producing S. gordonii.
Pyruvate may provide S. mutans with an alternative carbon source under limited growth conditions, as well as serving as a buffer against exogenous oxidative stress. Given the hypothesized role of LrgAB in cell death and lysis, these data also provide an important basis for how these processes are functionally and mechanically connected to key metabolic pathways such as pyruvate metabolism.
KeywordsStreptococcus mutans Oxidative stress Pyruvate Glucose metabolism LrgAB
Development of a mature biofilm on the tooth surface is the central event in the pathogenesis of dental caries . This process primarily requires that cariogenic organisms, including Streptococcus mutans, withstand the limited resources or environmental fluctuations experienced in the oral cavity [2, 3]. An emerging concept for biofilm maturation is that the survival and persistence of these organisms during biofilm development may be mediated by regulated cell death and lysis processes, consequently eliminating bacterial cells damaged by adverse environments and benefiting the rest of the population within the biofilm [4, 5]. Toward this end, we have studied the S. mutans Cid/Lrg system, consisting of two dicistronic operons lrgAB (SMU.575c/574c) and cidAB (SMU.1701c/1700c) [6, 7, 8, 9, 10, 11]. These operons are currently annotated as encoding holin- and antiholin-like proteins. The primary basis for these annotations came from the predicted structural similarities between CidA/LrgA and the bacteriophage-encoded holin family of proteins [4, 5, 6]. Bacteriophage holins are small membrane proteins, regulating the timing and lysis of the host cell during lytic infection with inhibitor holins (antiholins) . Further support was provided by previously-observed phenotypes of Staphylococcus aureus cid and lrg mutants [13, 14, 15, 16]. Notably, in S. mutans, the Cid/Lrg system is also involved in comprehensive virulence traits, including antibiotic resistance, autolysis, biofilm development, genetic competence, oxidative and heat stress responses [6, 7, 9, 11], all of which are essential for successful colonization and persistence in the oral cavity. Nevertheless, the molecular details of how Cid and Lrg function to control cell death and lysis have not yet been completely elucidated in S. mutans and S. aureus. Direct evidence of their specific cellular functionality is still scarce.
A hallmark finding for the S. mutans Cid/Lrg system is that expression levels of lrg and cid are counterbalanced throughout the growth cycle and in response to the availability of oxygen and glucose [6, 8]. In fact, the lrg and cid operons were originally identified to be up- and down-regulated, respectively, in aerobically grown cells . The response of lrg and cid to glucose levels is particularly remarkable. The lrg genes are highly induced in cultures containing lower levels of glucose (≤ 15 mM) but almost completely repressed in cultures containing glucose at concentrations of 20 mM and higher . In contrast, cid expression is negligible when cells are cultured in the presence of lower glucose concentrations (≤20 mM) but increases at higher glucose concentrations (> 20 mM) . Our recent study further demonstrated that CcpA is a direct regulator of expression of cid and lrg , and lrgAB expression is also governed by the LytST two-component regulatory system (TCS), located immediately upstream of the lrgAB genes [6, 8]. These data suggest a functional linkage between the Cid/Lrg system and metabolic pathways. This idea was also reinforced by our recent omics studies of S. mutans wild-type and isogenic ΔlrgAB mutant strains, using RNA-seq and label-free quantitative mass spectrometry, showing that a large number of genes and/or proteins involved in carbohydrate metabolism, ABC transporters, and oxidative stress adaptation were significantly altered in the lrgAB mutant under stress inducing culture conditions (aerobic, heat and vancomycin treatment) [7, 11].
The fact that the lrgAB promoter is highly active only in cells entering stationary phase, but not in cells growing exponentially, in the low-glucose condition , implies that LrgAB may be important for survival of cells when exogenous carbohydrate has been depleted. This lrgAB induction pattern was also shown in our previous microarray data, comparing RNA expression profiles of wild-type and lytS-deficient strains between early- and late-exponential growth phases in BHI medium . When this expression data was reconstituted for comparison of early- vs. late-exponential growth phases in the wild type, it was shown that lrgAB (SMU.575c-574c) was dramatically upregulated (about > 900-fold) at late-exponential phase, compared to early-exponential growth phase . A four-gene operon (SMU.1421 to SMU.1424), encoding the components of the pyruvate dehydrogenase complex (PDH), was also remarkably upregulated (by > 284-fold) at late-exponential phase, compared to that of early-exponential phase , suggesting that LrgAB may be related to pyruvate metabolism. Coincidentally, PftAB (YsbAB), homologous to LrgAB, was recently reported to function as a pyruvate transporter in Bacillus subtilis [18, 19]. Similarly to the observations for the lrgAB genes in S. mutans [6, 9], expression of the ysbAB genes was regulated by LytSR/LytST, located upstream of ysbAB, as well as by CcpA [20, 21]. Expression of ysbAB was also maximal at stationary phase of growth [18, 19] and was reduced by glucose addition [20, 21]. Esherichia coli also has two TCS (BtsSR and YpdAB) homologous to LytST that have been shown to regulate expression of pyruvate transporters in response to extracellular pyruvate [22, 23, 24]. Interestingly, these TCS and BtsT (high-affinity pyruvate transporter regulated by BtsSR and YpdAB) have also been recently implicated in the ability of pyruvate to rescue E. coli from the viable but non-culturable (VBNC) state . Collectively these studies, combined with the functional and genetic similarities of LrgAB to YsbAB in B. subtilis, suggest that LrgAB may also function as a pyruvate transporter in S. mutans.
To date little is known about the role and regulation of pyruvate in S. mutans, as well as other oral streptococci. Pyruvate is the final product of glycolysis, as well as a major substrate for oxidative metabolism. Pyruvate is converted to acetyl-coenzyme (acetyl-CoA) by the Pdh complex or Pfl (pyruvate formate lyase), depending on the presence or absence of oxygen, or the limitation or excess of a preferred sugar (e.g. glucose) [25, 26]. Acetyl-CoA is subsequently converted to end-products of fermentative metabolism, such as lactate, acetate, acetoin and formate. By ultilizing these pathways, cells maintain redox balance (NAD+/NADH) and generate ATP, which promotes cell homeostasis. In this present study, we reveal that pyruvate is excreted during growth of S. mutans as an overflow metabolite, and is reimported into cells via LrgAB when the primary carbon source (i.e. glucose) becomes exhausted. We performed a series of pyruvate quantification and lrg promoter reporter assays in order to characterize the role and regulation LrgAB as a pyruvate uptake system. These experiments demonstrate that LrgAB expression and activity is tightly regulated at the transcriptional level and modulated by both external and internal metabolic conditions. We also show that due to the H2O2- scavenging activity of pyruvate, re-uptake of pyruvate by S. mutans may be influenced by interactions with H2O2-producing oral commensals, such as S. gordonii. Given the possible involvement of LrgAB in inducing cell death and lysis in a programed manner, the presented data provide a new basis for how cell death and lysis mechanisms in S. mutans may be functionally and mechanically connected to pyruvate, a metabolic signal which may modulate homeostasis and virulence of this organism.
Bacterial strains, plasmids, and growth conditions
Streptococcus mutans UA159 and its previously-constructed mutant derivatives [6, 9] were cultured in brain heart infusion (BHI) medium (Difco Laboratories, Detroit, MI) or chemically defined medium FMC  containing 11 mM (or 45 mM) glucose. The medium was supplemented by sodium pyruvate (Fisher Scientific), β-fluoropyruvic acid sodium salt monohydrate (3FP, Sigma-Aldrich), pyruvic acid (Sigma-Aldrich), carbonyl cyanide m-chlorophenyl hydrazine (CCCP, Sigma-Aldrich) and 2,4-dinitrophenol (DNP, Sigma-Aldrich) or hydrogen peroxide (H2O2, Fisher Scientific), as necessary. Antibiotics were used to supplement growth media in the following concentrations: spectinomycin (1 mg/ml), kanamycin (1 mg/ml), and erythromycin (10 μg/ml). Unless otherwise noted, cultures were grown at 37 °C in a 5% CO2, aerobic atmosphere. For aerobic growh, cultures were grown in an aerobic incubator. To achieve anaerobic conditions, sterile mineral oil was placed on top of the cultures [6, 17, 28]. For growth measurements, fresh medium was inoculated with 1 : 100 dilutions of overnight cultures of S. mutans. The optical density at 600 nm (OD 600) was measured at 37 °C at 30 min-intervals using a Bioscreen C growth curve analysis system.
Sequence analysis and alignments
Amino acid sequences for S. mutans UA159 LrgA/B (SMU.574/575c), B. subtilis 168 YsbA/B (BSU_28900/28910), S. aureus MRSA252 LrgA/B (SAR_0259/0260), S. gordonii DL1 LrgA/B (SGO_1268/1269), and E. coli K12 LrgA/B (YohJ/K, JW2129/2130) were retrieved from the National Center of Biotechnology Information (NCBI) Protein database or from Uniprot (www.uniprot.org). Alignments were then generated utilizing the T-COFFEE M-coffee protein alignment tool  with shading completed via BoxShade version 3.21 (https://embnet.vital-it.ch/software/BOX_form.html). Percent identity matrices were then produced using the same sequences using Clustal Omega .
Microplate reporter assay
GFP activities of the S. mutans strains harboring Plrg-gfp gene fusion, previously constructed , were observed using a Synergy microplate reader (BioTek) controlled by Gen5 software [10, 31, 32]. Overnight cultures were diluted 1:50 into 2 ml of FMC medium and grown to an OD600 = 0.5. At this point, these cultures were diluted 1:50 into 175 μl FMC in individual wells of a 96-well plate (black walls, clear bottoms; Corning). To evaluate the possible role of pyruvate as an antioxidant against H2O2 in the oral cavity, the reporter strain was also cultivated with Streptococcus gordonii DL1, a H2O2-producing oral commensal, at a ratio of 1:1. The optical density at 600 nm (OD600) and green fluorescence were monitored (sensitivity = 45; excitation = 485 nm; emission = 520 nm) at 30 min intervals. The fluorescence of wild-type harboring plasmid without the reporter gene fusion was subtracted from fluorescence readings of the S. mutans strains harboring Plrg-gfp gene fusion. The results are representative of at least three independent replicates, each performed in triplicate.
Measurement of extracellular pyruvate and glucose levels
S. mutans UA159 wild-type and isogenic mutants (ΔlrgAB, ΔlytS, ΔcidB, or UA159/184-cidAB) strains were grown in chemically defined FMC medium, supplemented with either 11 mM or 45 mM glucose. For time course measurements of extracellular pyruvate during growth, samples (200 μl) were taken at 1–2 h intervals and half of this volume (100 μl) was used to measure the OD600 in a spectrophotometer for monitoring growth. The other half (100 μl) was centrifuged for 2 min at 18,000 xg to remove the cells, and pyruvate and glucose concentrations of the supernatant were quantified with an EnzyChrom™ pyruvate assay kit (BioAssay Systems, Hayward, CA) or glucose (HK) assay kit (Sigma-Aldrich), respectively, according to the manufacturer’s instructions. The results are average or representative of at least two independent replicates, each performed in duplicate.
Sequence analysis of LrgA and LrgB homologues
lrgAB encodes a pyruvate uptake system in S. mutans
Glucose and oxygen levels are important for stationary-phase pyruvate uptake of S. mutans
S. mutans metabolizes a large proportion of glucose only as far as pyruvate and acetyl CoA, and their subsequent metabolism depends on the availability of oxygen, particularly when the glucose concentration is limited [26, 34, 35]. Our previous data showed that expression of lrgAB was highly upregulated in response to oxygen, and its deficiency rendered the organism super-sensitive to oxygen [6, 11, 17], suggesting that oxygen availability may influence the efficacy of LrgAB to function as a pyruvate uptake system. To test this, we quantified extracellular pyruvate in both wild type and ΔlrgAB mutant cells, cultivated aerobically or anaerobically. To achieve anaerobic conditions, sterile mineral oil was placed on top of the cultures. All cultures were incubated in an aerobic incubator. The overall extracellular pyruvate profile during growth of both wild type (Additional file 5: Figure S5A and S5C) and ΔlrgAB (Additional file 5: Figure S5B and S5D) cultures were similar between the cells, grown aerobically and anaerobically. However, we found that pyruvate was about 50% more excreted during aerobic growth (Additional file 5: Figure S5A and S5B), relative to that observed during anaerobic growth (Additional file 5: Figure S5C and S5D) in both strains, respectively. We also found that the re-uptake of excreted pyruvate was somewhat inhibited during aerobic growth in both strains, relative to anaerobic growth, suggesting that pyruvate consumption may be decelerated during aerobic growth. Overall, these results suggest that LrgAB activity may be modulated by internal and external metabolic conditions of the cell.
Characterization of lrgAB expression and pyruvate uptake in response to extracellular pyruvate
The toxic pyruvate analogue 3FP interferes with the response of lrgAB to extracellular pyruvate
High concentrations of extracellular pyruvate can delay the stationary phase, regardless of LrgAB
Overflowed pyruvate can modulate the level of environmental hydrogen peroxide
The S. mutans Cid/Lrg system represents an excellent model to study how this organism withstands various stressors encountered in the oral cavity. Our previously-published “-omics” data suggested that the adaptation process to adverse environments requires metabolic remodeling [7, 8, 9, 11], which is in accordance with the fact that (i) lrgAB is specifically induced at stationary phase and (ii) is tightly regulated by glucose and oxygen levels [6, 8, 17]. The primary finding of this current study is that LrgAB is important to initiate the rapid re-uptake of pyruvate, excreted during growth, when cells confront carbon starvation/nutrient limitation in stationary growth phase. The excretion (overflow) of pyruvate is a common feature of many bacterial species when cultivated under a carbon-excess condition, contributing to metabolic balancing between carbon uptake and consumption [33, 43, 44, 45]. Because of its central role in metabolism, it is not surprising that excretion and re-uptake of pyruvate are tightly regulated at multiple levels, and the three main enzymes that utilize pyruvate as a substrate, Pdh, Pfl and Ldh, are accordingly modulated to the cell’s metabolic status and environmental condition [25, 46]. We have previously shown that the pdh and pfl genes, responsible for the conversion of pyruvate to acetyl-CoA, were highly upregulated at late-exponential phase , suggesting that their gene expression is linked with the uptake of pyruvate, functionally and metabolically. Given that the pdh genes were commonly upregulated in response to three stress conditions (aerobic, heat and vancomycin challenge) previously tested , pyruvate uptake and metabolism should affect the ability of S. mutans to adapt to suboptimal conditions. Pyruvate is likely a common nutrient in the microbiome environment, such as oral cavity. But its utilization and regulation in S. mutans have been poorly characterized, especially with respect to how they are connected to the virulence network of this organism.
It seems that the re-uptake of pyruvate is tightly regulated by glucose levels and stationary growth phase lrgAB induction. Growth in high levels of glucose abrogates stationary-phase response of lrgAB to extracellular pyruvate, suggesting that the response of lrgAB to extracellular pyruvate is subject to carbon catabolite repression. Supplementation of exogenous pyruvate was unable to induce earlier induction of lrgAB (normally occurring at stationary phase), reinforcing that uptake of pyruvate occurs only under limited growth, and is subject to tight metabolic control. Given that lrgAB expression requires activation by LytST TCS , repression of lrgAB may be primarily due to inaccessibility of LytT to the promoter region of lrgAB. In our recent study, we found that the cre site for CcpA binding overlapped, in part, with a potential LytT binding site in the promoter region of lrgAB , suggesting that activation of lrgAB by LytT may be prevented by CcpA binding to the promoter region of lrgAB. However, repression of lrgAB was not released even in the absence of CcpA when the cell was cultivated in high-glucose medium , suggesting that additional regulation(s) may be involved in the expression of lrgAB. In fact, CcpA often works together with CodY to sense changes in nutrient availability and coordinate, directly and indirectly, the expression of hundreds of genes involved in carbon and nitrogen metabolism of Gram-positive bacteria [45, 47, 48, 49]. CodY acts mainly as a repressor, and many genes encoding metabolic pathway components are repressed during growth in the presence of excess nutrients and involved in adaptation to poor growth conditions . Accordingly, we also observed that the codY gene was several-fold upregulated at early-exponential phase, compared to late-exponential growth phase . Thus, it is possible that these two global regulators (CcpA and CodY) interact and modulate lrgAB expression in response to environmental conditions and nutritional needs, which is currently under investigation.
The specific response of lrgAB to extracellular pyruvate was supported by the observation that supplementation of 10 μM 3FP had a profound effect on repressing the expression of lrgAB, likely by interfering with activation of the LytS sensor kinase by pyruvate. These results suggest that LrgAB may be responsible for facilitating the recovery from carbon starvation when concentrations of pyruvate are low. On the contrary, the finding that the stationary phase induction of lrgAB was alleviated by high concentrations of extracellular pyruvate (approx. > 20 mM), revealed the existence of a negative feedback regulation acting on LytST by the presence of high levels of extracellular pyruvate. This feedback regulation may contribute to balanced extracellular and intracellular pyruvate levels, although it remains to be elucidated. Given that the re-uptake of accumulated pyruvate is subject to a tight metabolic control, pyruvate may play a role as a potential metabolic signal to determine cellular fate under limited nutrient conditions. However, this regulatory model does not account for the observation that cells were still capable of utilizing extracellular pyruvate even in the absence of LrgAB, resulting in prolonged exponential growth, suggesting the existence of an additional pyruvate uptake system that operates in a LrgAB-independent manner and preferably when pyruvate concentrations are relatively high (at several mM levels). Indeed, it has been suggested that E. coli uses one exporter and two uptake transporters to modulate the level of intracellular pyruvate . However, uptake of pyruvate does not appear to be attributed to diffusion, because the cell displayed the same growth rate even in the presence of high concentrations of pyruvate. It is also supported by the observation that S. mutans was also unable to grow in a medium containing high concentrations of pyruvate (up to 80 mM) as sole carbon source (Additional file 2: Figure S2). These multiple pyruvate uptake systems and feedback regulations may ensure a tight management of pyruvate homeostasis which in turn may facilitate cellular adaptation.
Although the data herein suggest that LrgAB is primarily responsible for uptake of pyruvate in S. mutans, it remains to be elucidated how LrgAB mechanistically mediates the uptake (transport) of pyruvate. In a preliminary experiment, we found that stationary phase lrgAB induction was significantly inhibited by two hydrophobic protonophores, CCCP (carbonyl cyanide m-chlorophenyl hydrazine) and DNP (2,4-dinitrophenol) at 1 μM and 100 μM, respectively (Additional file 8: Figure S8), suggesting that a pH gradient between interior and exterior of cell may be involved in the activation of LrgAB. Accordingly, a recent study showed that BtsT (also named YjiY) functions as a specific pyruvate/H+ symporter in E. coli . This aspect is being further investigated in LrgAB. It is also noteworthy that lrgAB is induced by pyruvic acid, which still occurs at stationary growth phase (Additional file 9: Figure S9). Although pyruvate could freely diffuse through the lipid bilayer of the cell membrane in its protonated form , most of pyruvic acid should be in a dissociated, charged state (pyruvate), due to acidity of pyruvic acid (pKa = 2.5), suggesting that LrgAB may not be energized only by a proton gradient. Thus, in order to metabolize pyruvate, the uptake systems, including LrgAB, would be required for internalizing pyruvate. It also appears that other metabolic intermediates with structural similarity with pyruvate are transported by LrgAB. In our preliminary experiment using the PlrgA-gfp reporter strain, we observed that lrgAB did not repond to α-ketoglutarate, malate, oxaloacetate, and succinate, but citrate could trigger lrgAB induction (data not shown), suggesting that LrgAB may be specifically responsible for uptake of pyruvate. This theory is also supported by the observation that a synthetic analogue of pyruvate (3FP) effectively competed with pyruvate. This specificity also reinforces the role of pyruvate as a potential metabolic signal, because pyruvate is excreted together with other metabolic compounds. The detailed mechanism for how pyruvate is transported via LrgAB through the cell membrane is an important part of understanding the Cid/Lrg system and warrants further investigation.
Another important finding of this study was that pyruvate directly reacts with H2O2, suggesting that the re-uptake of pyruvate may be influenced by interspecies interactions, especially with H2O2-producing oral commensals, such as S. gordonii and S. sanguinis [53, 54]. The reaction of H2O2 with pyruvate is presented as CH3-CO-COOH+H2O2 → CH3-COOH+H2O + CO2 . Indeed, acetate and CO2 are the major by-products of pyruvate reaction with H2O2, with pyruvate being produced and excreted during exponential growth while glucose is converted to biomass. Acetate would be presumably also taken up into the cell in parallel with pyruvate under nutrient limited growth condition [33, 55]. Intriguingly, the pta-ackA pathway which generates acetate and ATP was reported to cause cell death in S. aureus , and to be regulated by CcpA and CodY in S. mutans  and B. subtilis . Therefore, these observations further suggest a potential connection between pyruvate metabolism and cell death, hypothesized to be induced by the Cid/Lrg system . It is also noteworthy that another organic keto acid, e.g. α-keto glutarate, can effectively scavenge H2O2 [39, 40, 41, 42], suggesting that the level of H2O2 could be modulated by multiple metabolic products. It is an on-going study how the levels of H2O2 and pyruvate (or other organic acids) are changed in the interaction between S. mutans and S. gordonii (or S. sanguinis), and how the LrgAB pyruvate uptake system contributes to the protection from environmental H2O2 challenge.
The results of this study demonstrate that LrgAB is the first identified pyruvate uptake system in S. mutans, and provide an important new basis for how the hypothesized role of LrgAB in modulating cell death and lysis are mechanistically connected to key metabolic pathways. Overall, excreted pyruvate may play several important roles in S. mutans physiology, such as a carbon source and/or metabolic precursor for amino acid/fatty acid biosynthesis under nutrient-limited and/or stationary phase growth conditions, as well as by buffering external sources of oxidative stress, such as H2O2, which primarily appears to be modulated by LytST-LrgAB. Therefore, it is possible that the trafficking and utilization of pyruvate may significantly contribute to shaping a homeostatic mechanism and composition of oral microflora, consequently influencing the development of caries. Nevertheless, due to highly variable in vivo conditions especially in respect to nutrient availability and unknown host factors, the actual effect and role of pyruvate remain to be elucidated and are currently under investigation. This study also opens the possibility of searching for additional pyruvate uptake system as well as examining the relationship between metabolite fluxes and ecological fitness. This will be important as we explore the possible mechanisms underlying stress tolerance and programmed cell death as a bacterial survival strategy at the community level.
We thank Professor Robert A. Burne (Department of Oral Biology, University of Florida) for providing all the resources needed.
SJA designed the study and drafted the manuscript. KD and II performed the pyruvate and glucose assays. MET performed the sequence analysis and alignment of the LrgA and LrgB homologues. AW performed the microplate reporter assays. KCR and SJH participated in the conception and design of the study, and helped draft the manuscript. All authors analysed and interpreted the data, and reviewed/approved the final manuscirpt.
This work was supported by the National Institutes of Health (NIH)- National Institute of Dental and Craniofacial Research (NIDCR) grant R01 DE025237 (S.-J. A.).
Ethics approval and consent to participate
No human/animal experiment is involved in this study.
Consent for publication
The authors declare that there are no conflicts of interest.
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