|Home | About | Journals | Submit | Contact Us | Français|
The NAD+ and NADH-sensing transcriptional regulator Rex is widely conserved across Gram-positive bacteria. Rex monitors cellular redox poise and controls the expression of genes/operons involved in diverse pathways including alternative fermentation, oxidative stress responses, and biofilm formation. The oral cavity undergoes frequent and drastic fluctuations in nutrient availability, pH, temperature, oxygen tension, saliva, and shear forces. The oral streptococci are major colonizers of oral mucosa and tooth surfaces and include commensals as well as opportunistic pathogens, including the primary etiological agent of dental caries, Streptococcus mutans. Current understanding of the Rex regulon in oral bacteria is mostly based on studies in S. mutans and endodontic pathogen Enterococcus faecalis. Indeed, other oral bacteria encode homologs of the Rex protein and much is to be gleaned from more in-depth studies. Our current understanding has Rex positioned at the interface of oxygen and energy metabolism. In biofilms, heterogeneous oxygen tension influences the ratio of intracellular NADH and NAD+, which is finely tuned through glycolysis and fermentation. In S. mutans, Rex regulates the expression of glycolytic enzyme NAD+-dependent glyceraldehyde 3-phosphate dehydrogenase, and NADH-dependent fermentation enzymes/complexes lactate dehydrogenase, pyruvate dehydrogenase, alcohol-acetylaldehyde dehydrogenase, and fumarate reductase. In addition, Rex controls the expression of NADH oxidase, a major enzyme used to eliminate oxidative stress and regenerate NAD+. Here, we strive to summarize recent studies carried out on the Rex regulators in S. mutans and E. faecalis. This research has important implications for understanding how Rex monitors redox balance and optimizes fermentation pathways for survival and subsequent pathogenicity.
The oral cavity houses over 700 different bacterial species (Jenkinson and Lamont 2005), many of which live in communities of heterologous biofilms on the tooth surface called dental plaque. The complexity of oral biofilms prevents homogenous diffusion of environmental compounds including antimicrobials, oxygen and reactive oxygen species (ROS), and metabolic byproducts including organic acids. Thus, biofilms can be considered heterogeneous in terms of genetic regulation at the species level depending on localized conditions (Son et al. 2012). Previous studies have demonstrated that reduction potential values of supragingival plaque drops from +294 to −141 mV over the course of seven days (Kenney and Ash 1969) and oxygen tension ranges from 5 to 27 mm Hg depending on the depth of periodontal pockets (Mettraux et al. 1984). Oxidative stress occurs when ROS, including superoxide, hydrogen peroxide (H2O2), and the hydroxyl radical accumulate at a faster rate than the rate at which they are detoxified (Imlay 2013). Researchers have shown that endogenous H2O2 is generated in oral biofilms through metabolic oxygen reduction by some streptococci including Streptococcus sanguinis, Streptococcus sobrinus, and Streptococcus gordonii (Ryan and Kleinberg 1995). In oral streptococci, fermentation pathways and ultimately, acidogenesis are linked to oxygen and ROS through changes in the redox poise (Baldeck and Marquis 2008). Bacteria can sense oxygen tension through monitoring the accumulation of metabolites or the redox state of specific compounds as a result of changes in cellular homeostasis (Wang et al. 2008).
Gram-positive oral streptococci nearly ubiquitously express homologs of the NADH- and NAD+-sensing transcriptional regulator Rex (Ravcheev et al. 2012). Rex was first identified in Streptomyces coelicolor for being a redox-responsive transcriptional regulator capable of controlling the transcription of NADH dehydrogenase operon nuoA-nuoN, the heme biosynthesis genes hemACD, and the cytochrome bd terminal oxidase operon cydABCD (Brekasis and Paget 2003). Rex proteins have since been characterized in several model microorganisms including Bacillus subtilis, Staphylococcus aureus, S. mutans, and E. faecalis. Brekasis and Paget first showed Rex-regulated genes were found to have a consensus cis binding site 5′-TGTGNNCNNNTTCACA-3′ called the Rex operator (ROP) sites (Brekasis and Paget 2003). More recently, a bioinformatics study has identified optimized ROP sites for eleven phylogenetic taxa comprising more than 110 genomes (Ravcheev et al. 2012). Moreover, Rex-mediated transcriptional regulation has been implicated in species-specific genetic targets of diverse organisms (Ravcheev et al. 2012). In S. aureus, Rex controls the expression of genes linked to nitrate/nitrite respiration, fermentation, and amino acid metabolism (Pagels et al. 2010). In cariogenic S. mutans, Rex controls the expression of genes linked to fermentation, oxidative stress response, and biofilm formation (Baker et al. 2014; Bitoun et al. 2012; Bitoun et al. 2011). Rex-deficiency in S. mutans causes increased sensitivity to exogenous H2O2 and increased end-point pH values of stationary phase culture medium. Indeed other commensals commonly found in the oral cavity encode homologs of Rex (Table 1). In E. faecalis, Rex-deficiency causes impaired growth of aerated cultures, which can be rescued by the addition of catalase to the medium suggesting the rex-deficient mutant has an impaired ability to cope with oxidative stress (Mehmeti et al. 2011; Vesic and Kristich 2013). Recently, a Rex-regulated gene product of Streptococcus pneumoniae, AdhE, was shown to increase pneumolysin production under alcohol stress conditions, thereby increasing pneumococcal virulence (Luong et al. 2015).
Rex proteins are homodimers with N-terminal winged helix DNA-binding motifs and C-terminal Rossmann fold NADH/NAD+-binding motifs. The Rex protein of S. aureus was shown to have a Kd of 95 nM for NADH and 150 μM for NAD+ (Pagels et al. 2010). The higher binding affinity of Rex to NADH suggests that Rex is poised to modulate gene expression when subtle changes are made to the redox poise. Under basal growth conditions, the high cellular NAD+ concentration outcompetes NADH for Rex-binding. Rex-NAD+ complex binds the cis ROP sites of regulated promoters and negatively regulates the transcription of target genes including adhE, ldh, and frdC, for alcohol-acetylaldehyde dehydrogenase (AdhE), lactate dehydrogenase (Ldh), and fumarate reductase (FrdC), respectively. On the other hand, when the NADH concentration rises against the threshold, NADH outcompetes NAD+ for Rex-binding, changes the allosteric conformation of Rex protein, and alleviates genetic repression of target genes (McLaughlin et al. 2010; Wang et al. 2011). Rex homologs from select oral cocci show species-specific differences in primary amino acid sequence (Figure 1). As seen, helix-turn-helix and Rossmann fold motifs are conserved in Rex homologs among the oral cocci. However, differences in primary amino acid sequences suggest that Rex has evolved to regulate the expression of different genes/operons among the oral cocci. When Rex primary amino acid sequences of select Gram-positive cocci were parsed using Rossmann-fold prediction algorithm Cofactory (Geertz-Hansen et al. 2014), there were notable differences in NAD(H) binding potential among Rex homologs (Table 1). Understanding differences in NAD(H) binding to Rex homologues of oral streptococci may help researchers fine-tune species-specific Rex regulons.
The oral streptococci predominate in dental plaque and use fermentation pathways to generate ATP (Cole 1977). The oral streptococci metabolize a wide range of sugars and sugar alcohols into pyruvate through glycolysis (Abranches et al. 2004; Grossiord et al. 2003; Lemos et al. 2005). NAD+ and NADH are the gatekeepers of glycolysis and fermentation, respectively, in the oral streptococci. Recently, glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was shown to be regulated by Rex in S. mutans (Ravcheev et al. 2012). In addition, Rex regulates the expression of genes encoding proteins that use the reducing power of NADH during fermentation, including lactate dehydrogenase, pyruvate dehydrogenase, alcohol-acetylaldehyde dehydrogenase, and anaerobic fumarate reductase.
Like other lactic acid bacteria, the default fermentation pathway of S. mutans is the catabolism of excess glucose through lactate dehydrogenase to form lactic acid (Korithoski et al. 2008). Lactate dehydrogenase uses NADH as an electron donor to convert pyruvate into lactic acid, thus priming the next cycle of glycolysis by oxidizing NADH back into NAD+. The lactate generated is the culprit in the dissolution of the tooth enamel leading to dental caries. However, under conditions of anaerobiosis, glucose limitation, or low dilution rates in continuous culture, fermentation products shift from mainly lactate towards a mixture of lactate, formate, acetate, and ethanol (Yamada and Carlsson 1975). When grown in galactose, S. mutans yields a blend of formate, acetate, and ethanol end-products (Abbe et al. 1982). S. mutans also ferments sugar alcohols, mannitol and sorbitol, into a large quantity of ethanol (Brown and Patterson 1973).
The major point of divergence in S. mutans fermentation comes at the point of pyruvate (Guest et al. 1989) (Figure 2). Pyruvate can be metabolized into organic acids lactate, acetate, and formate and non-acid ethanol. The differences in byproduct pKa significantly contribute pathogenicity. For example, if pyruvate is metabolized exclusively by pyruvate dehydrogenase (PDH) and bifunctional alcohol/acetylaldehyde dehydrogenase (AdhE), major quantities of ethanol would be formed, reducing S. mutans acidogenicity. Under strictly anaerobic conditions, pyruvate-formate lyase (PFL) converts a portion of pyruvate to formate and acetyl-CoA. Acetyl-CoA catabolism can yield ethanol or acetate through the enzymatic activities of alcohol-acetylaldehyde dehydrogenase (AdhE) or phosphotransacetylase (PTA) and acetate kinase (ACK), respectively. Pyruvate-formate lyase activating enzyme (PFL-AE) contains an extremely oxygen-labile iron-sulphur cluster and is inactivated under aerobic conditions (Takahashi-Abbe et al. 2003). On the other hand, under aerobic conditions, pyruvate is catabolized to lactate, acetate, or ethanol via the activities of lactate dehydrogenase (LDH), pyruvate oxidase (POX), or the pyruvate dehydrogenase (PDH) complex (Korithoski et al. 2008). Previous studies have shown that pyruvate dehydrogenase is regulated by redox potential (Snoep et al. 1992). Interestingly, pdhA expression was more highly induced in an anaerobic environment in S. mutans (Korithoski et al. 2008).
S. mutans contains a single rex gene (SMU.1053) that encodes for a protein of high similarity to the Rex family of NADH- and NAD+ transcription factors. In S. mutans, microarray experiments comparing gene expression of a rex-deficient mutant to the wild-type showed Rex-deficiency up-regulated 25 genes at P <0.001, when the cultures were grown statically at 37°C in an aerobic incubator with 5% CO2 (Bitoun et al. 2011). As shown in other organisms, many of the up-regulated genes/operons in response to Rex-deficiency in S. mutans were part of fermentation pathways. S. mutans encodes multiple fermentative genes/operons including lactate dehydrogenase (ldh, SMU.1115), acetoin dehydrogenase (adhABCD, SMU.127-SMU.130), pyruvate dehydrogenase (pdhABCD, SMU.1421-SMU.1424), alcohol-acetylaldehyde dehydrogenase (adhE, SMU.148), pyruvate-formate lyase (pfl, SMU. 402 and pfl-2, SMU.493) and fumarate dehydrogenase (frdC, SMU.490). Based on microarray data, pdhA, adhE, adhA, and frdC were up-regulated by 2.7-fold, 10.0-fold, 2.9-fold, and 4.7-fold, respectively, in a rex-deficient mutant as compared to wild-type. In a subsequent study, researchers used electrophoretic mobility binding assays to show Rex binds specifically to the promoters of rex, ldh, frdC, and adhE (Bitoun et al. 2012). Researchers demonstrated downstream rex operon member guaA was also up-regulated in response to Rex-deficiency, suggesting auto-regulation of the two gene rex operon (Bitoun et al. 2012). A model is shown in Figure 3 with the ROPs of the Rex-regulated genes adhE, rex, and ldh demonstrating the ROPs are found within the -10 and -35 regions of the respective operators. Interestingly, ldh was not shown to be differentially regulated in the microarray study despite having two ROP sites upstream of the transcription start site.
Details concerning Rex-mediated transcriptional activation have remained elusive. Using microarray analysis, researchers have shown at least 28 genes down-regulated in S. mutans in response to Rex-deficiency. Based on microarray data, mleS, mleP, gshR, and tpn, were down-regulated 17.6-fold, 15.4-fold, 18.7-fold, and 40.6-fold respectively, in a rex-deficient mutant as compared to wild-type (Bitoun et al. 2011). Interestingly, gshR encodes a putative glutathione reductase which uses NADPH for the reduction of oxidized glutathione yet Rex has not been shown to be responsive to NADPH. The microarray data was confirmed with Realtime PCR experiments that demonstrated almost 20-fold down-regulation of gshR in a rex-deficient mutant of S. mutans as compared to wild-type (Bitoun et al. 2012). In addition, researchers showed that purified recombinant Rex could indeed bind to a region in the promoter of gshR (Bitoun et al. 2012). No previous Rex-mediated transcriptional activation had since been reported, and further studies are needed to elucidate the cis element for Rex-mediated genetic activation.
Researchers also demonstrated that a rex-deficient mutant of S. mutans was at least 1-log more sensitive to H2O2-mediated killing than the wild-type. In addition, the rex-deficient mutant displayed an extended lag phase when incubated with paraquat, an oxidative stress inducing agent (Bitoun et al. 2011). Researchers have found that the NADH:NAD+ ratio is the highest under anaerobic conditions (de Graef et al. 1999; Snoep et al. 1994). In some oral streptococci, overlapping regulatory mechanisms of NADH oxidase, encoded by the nox gene, have linked oxidative stress response with the redox balance (Baker et al. 2014; Derr et al. 2012; Sheng et al. 2010). NADH oxidase is an important enzyme that can restore the basal redox balance by oxidizing existing NADH to NAD+ with the concomitant reduction of molecular oxygen to water. This enzyme uses FAD cofactor and cycling disulphides to perform the electron transfer reactions to ultimately replenish the NAD+ pools necessary for glycolysis (Derr et al. 2012). Researchers tested the ability of Rex to bind the nox promoter using electrophoretic mobility shift analysis, and found that Rex can bind the nox promoter (Pnox) (Baker et al. 2014). Co-incubation of Rex with NAD+ increased the ability of Rex to bind the nox promoter and co-incubation of Rex with NADH resulted in alleviating Rex-Pnox interactions. Interestingly, using microarray analysis researchers found that a nox-deficient mutant of S. mutans had nearly 3-fold elevation in rex transcript levels, as compared to wild-type, suggesting NADH oxidase activity changes the NADH:NAD+ ratio with downstream alterations in the ability of Rex to regulate itself.
Biofilm formation is a hallmark of S. mutans virulence. The glucosyltransferases GtfB, GtfC, and GtfD catalyse extracellular glucose polymer (known as glucan) formation with sucrose as the substrate (Bowen and Koo 2011). The glucans, including both soluble and insoluble ones varying with glyosidic linkages (α-1,3, α-1,4, α-1,6), are used by S. mutans to adhere to tooth enamel and the surfaces of other microorganisms in the plaque community. Researchers noticed that biofilms of the rex-deficient strain accumulated 3.5-fold less biomass than wild-type when grown in glucose and 3-fold less biomass than wild-type when grown in sucrose. When grown in glucose and sucrose, biofilms of the rex-deficient strain displayed a more porous and rugged architecture than wild-type UA159 with a 2-fold higher glucan to cell ratio. Realtime PCR was carried out on the glucosyltransferase genes, gtfB, gtfC, and gtfD in both the wild-type and rex-deficient strain. Studies showed up-regulation of gtfC by more than 13-fold in biofilms of the rex-deficient strain and subsequent analysis of the gtfC promoter revealed a potential ROP site, linking biofilm formation to redox poise (Bitoun et al. 2011).
The overlap in the control of redox and oxidative stress response genes is not surprising. Researchers have shown that both hydrogen peroxide and paraquat are more difficult for a rex-deficient mutant to overcome as compared to wild-type. In addition, Rex can bind to its own promoter and is subject to auto-regulation.
E. faecalis is a facultative anaerobe, low-GC Gram-positive opportunistic pathogen. E. faecalis is a natural commensal of the gastrointestinal tract (Nallapareddy et al. 2005). However, due to faecal dissemination and its adaptability to different environments, E. faecalis can be routinely associated with enterococcal nosocomial infections including urinary tract infections, surgical wound infections, and bacterial endocarditis (Jett et al. 1994; Reffuveille et al. 2011). In the oral cavity, E. faecalis has been shown to be associated with root caries and with periodontal disease (Preza et al. 2008; Sun et al. 2009). Virulence factors of E. faecalis include bile salt hydrolase, cytolysin toxin, capsular polysaccharides, gelatinase, lipoproteins and other surface-associated LPxTG aggregation substances (Nallapareddy et al. 2006; Theilacker et al. 2009). Recent studies have shown that cell wall lipoteichoic acid of E. faecalis can suppress Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced IL-8 expression in human periodontal cells (Im et al. 2015).
E. faecalis contains two homologs of Rex, encoded at loci EF2638 and EF2933 (Mehmeti et al. 2011). The interplay of multiple Rex proteins in a host remains unknown. Researchers characterized the transcriptome of a rex-deficient mutant (Δrex, ΔEF2638) in E. faecalis and found that Rex-deficiency up-regulated genes involved in anaerobic fermentation pathways including pyruvate-formate lyase (EF1613), pyruvate-formate lyase activating enzyme (EF1612), alcohol/aldehyde dehydrogenase (EF0900), anaerobic ribonucleotide reductase (EF2754-5), and fumarate reductase (EF1226-7) In addition, genes encoding abortive infection protein (EF2637) and cell envelope-associated acid phosphatse were also up-regulated in response to Rex-deficiency (Vesic and Kristich 2013). Recently, researchers created a strain of E. faecalis V583 deficient of both ldh1 and ldh2 (Δldh1.2), both genes encoding lactate dehydrogenases. Under anaerobiosis, they showed changes in the end-product metabolites of Δldh1.2 as compared to wild-type V583. Researchers showed drastic increases in formate, ethanol, pyruvate, and acetoin production as a result of Δldh1.2 lactate dehydrogenase deficiency (Mehmeti et al. 2011). In other studies, researchers showed that the NADH:NAD+ ratio decreased more than 2-fold in Δrex as compared to wild-type (Vesic and Kristich 2013). On the other hand, deletion of EF2933 (Δrex2) maintained a NADH:NAD+ ratio similar to wild-type (Vesic and Kristich 2013). In addition, aerobiosis impairs the growth of Δrex through the production of excess hydrogen peroxide and the addition of catalase to the culture medium rescued the aerobic slow-growth phenotype of the Δrex mutant (Vesic and Kristich 2013).
Increased production of ethanol and pyruvate has been shown to correlate to an increased NADH/NAD+ ratio in the lactate dehydrogenase-deficient mutant (Snoep et al. 1994). Researchers found at least 22 putative ROP sites upstream of 22 genes/operons that were found to be differentially expressed in their proteome or transcriptome studies of the lactate dehydrogenase deficient mutant (Mehmeti et al. 2011). Interestingly, virulence plasmid pAD1 mating pheromone cAD1 precursor protein (EF3256) and virulence factor LPxTG cell wall surface anchor family protein (EF3314) contain ROP sites in their respective promoters, although direct experimental Rex-mediated regulation of these genes has not been studied (An and Clewell 2002; Creti et al. 2009). Like in S. mutans, lactate dehydrogenase and alcohol-acetylaldehyde dehydrogenase showed the strongest up-regulation in Δrex and are both preceded by two ROP sites (Bitoun et al. 2012; Mehmeti et al. 2011).
Accumulating reports in the literature have provided a more complete understanding of Rex-mediated transcriptional regulation in the oral pathogens S. mutans and E. faecalis. Like other Gram-positive bacteria, in the oral pathogens the Rex transcription factor negatively regulates alternative energy metabolism pathways by monitoring the NADH:NAD+ ratio. In S. mutans, Rex-deficiency causes a pH increase of stationary phase culture medium, suggesting activation of alternative fermentation pathways generating non-acid byproducts. More studies testing the end-product metabolite ratios in response to Rex-deficiency would provide pertinent information to understanding alternative fermentation in oral pathogens. Unlike previous studies, Rex has also been postulated to activate the transcription of genes in oral pathogens S. mutans and E. faecalis (Bitoun et al. 2012; Mehmeti et al. 2011). Further studies are needed to discern the direct mechanism of gene activations. In S. mutans, Rex-activated genes gshR (SMU.140), mleR (SMU.135), and tpn (SMU.1363c) have palindromic ROP sites in their promoters of 5′-AAACACCGCCGCCGATGA-3′, 5′-AAAGTAACAAGATAGTGG-3′, and 5′-AAACTACTTCGATATTGA-3′, respectively (Bitoun, pers. comm.). In addition, other transcription factors encoded at loci SMU.1409, SMU.1419, and SMU.1361c were differentially regulated in response to Rex-deficiency (Bitoun et al. 2011). It is also possible that Rex activates gene expression by modulating the expression of other transcription factors. In S. mutans, many genes including ptsG-rgfB, pulA, gluQHMP, mntABC, and nox have been shown to be regulated by more than one transcription factor (Baker et al. 2014; Ravcheev et al. 2013). Using a combination of mRNA and proteomic analysis, researchers also suggest that Rex may function as a transcriptional activator in E. faecalis (Mehmeti et al. 2011; Vesic and Kristich 2013). Continued research into Rex-mediated transcriptional modulation of oral pathogens will yield new insights into the pathogenicity and the mechanisms that regulate.
Work in the Wen laboratory was supported by NIH/NIDCR grant DE19452 to ZTW. Additional funds were provided by Tulane University SOM.