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. 2022 Jan 26;101(6):623–631. doi: 10.1177/00220345211064571

Pathways Linking Oral Bacteria, Nitric Oxide Metabolism, and Health

E Morou-Bermúdez 1,✉, JE Torres-Colón 1, NS Bermúdez 2, RP Patel 3, KJ Joshipura 1,4
PMCID: PMC9124908  PMID: 35081826

Abstract

Nitrate-reducing oral bacteria have gained a lot of interest due to their involvement in nitric oxide (NO) synthesis and its important cardiometabolic outcomes. Consortia of nitrate-metabolizing oral bacteria associated with cardiometabolic health and cognitive function have been recently identified. Longitudinal studies and clinical trials have shown that chronic mouthwash use is associated with increased blood pressure and increased risk for prediabetes/diabetes and hypertension. Concurrently, recent studies are beginning to shed some light on the complexity of nitrate reduction pathways of oral bacteria, such as dissimilatory nitrate reduction to ammonium (DNRA), which converts nitrite into ammonium, and denitrification, which converts nitrite to NO, nitrous oxide, and dinitrogen. These pathways can affect the composition and metabolism of the oral microbiome; consequently, salivary nitrate and nitrite metabolism have been proposed as targets for probiotics and oral health. These pathways could also affect systemic NO levels because NO generated through denitrification can be oxidized back to nitrite in the saliva, thus facilitating flux along the NO3–-NO2–-NO pathway, while DNRA converts nitrite to ammonium, leading to reduced NO. It is, therefore, important to understand which pathway predominates under different oral environmental conditions, since the clinical consequences could be different for oral and systemic health. Recent studies show that oral hygiene measures such as tongue cleaning and dietary nitrate are likely to favor denitrifying bacteria such as Neisseria, which are linked with better cardiometabolic health. A vast body of literature demonstrates that redox potential, carbon-to-nitrate ratio, and nitrate-to-nitrite ratio are key environmental drivers of the competing denitrification and DNRA pathways in various natural and artificial ecosystems. Based on this information, a novel behavioral and microbial model for nitric oxide metabolism and health is proposed, which links lifestyle factors with oral and systemic health through NO metabolism.

Keywords: cardiovascular disease(s), microbial ecology, oral-systemic disease(s), microbiome, plaque/plaque biofilms, inflammation

Oral Bacteria and Nitric Oxide Synthesis

Nitric oxide (NO) is an endogenous signaling molecule that plays an important homeostatic role in biological processes. NO is synthesized from L-arginine by three isoforms of NO synthases (NOS). Endothelial NOS (eNOS or NOS-3) produces NO in response to increased intracellular Ca2+ to control vasodilation and platelet aggregation. eNOS is also activated by phosphorylation via the PI3K-Akt pathway, to facilitate insulin-dependent glucose uptake in skeletal muscle and fat. Decreased eNOS-derived NO bioavailability is a hallmark of endothelial dysfunction, a common link between cardiovascular disease, obesity, diabetes, and periodontal disease (Förstermann and Sessa 2012; Moura et al. 2017). Neuronal NOS (nNOS or NOS-1) is constitutively expressed in neural cells and is involved in memory formation and central regulation of blood pressure (Förstermann and Sessa 2012). Inducible NOS (iNOS or NOS-2) is typically activated in macrophages but also practically in every type of cell in response to inflammatory mediators/cytokines and bacterial lipopolysaccharide (LPS). iNOS activation produces large amounts of NO that have cytotoxic effects on parasitic targets through modulation of mitochondrial respiration and formation of secondary reactive nitrogen species. NO can have both proinflammatory and anti-inflammatory properties, depending on the location and concentration. Generally, low levels of eNOS or nNOS-derived NO exert anti-inflammatory effects, while higher levels of NO produced via iNOS stimulation promote inflammatory tissue injury (Iwata et al. 2020).

In addition to NOS-dependent catalysis, NO can be formed via NOS-independent mechanisms. Commensal oral bacteria play an important role in NOS-independent NO synthesis as key players of the enterosalivary nitrate pathway, which generates NO from the sequential reduction of nitrate (NO3–) to nitrite (NO2–) and nitric oxide (NO3-NO2-NO) (Kapil et al. 2020). In this pathway, dietary nitrate, as well as nitrate entering the oral cavity through the enterosalivary circulation, is reduced to nitrite by nitrate reductases expressed in commensal oral bacteria. Nitrite is a relatively stable vascular reserve of NO, with NO produced by this pathway exerting similar functions to those described for eNOS-derived NO. Reduction of nitrite (by 1 electron) has emerged as a viable NO production process in vivo, with several candidate metalloproteins identified as electron donors (Kapil et al. 2020). These mechanisms function most efficiently at low pH and hypoxia, conditions that can be found in the oral and gastric environment, as well as in disease states involving tissue ischemia. The enterosalivary pathway also includes mechanisms that oxidize NO back to nitrate and recirculate it to the oral cavity, as described in Figure 1. About 70% of serum nitrite is believed to be produced by eNOS (Kapil et al. 2020). The contribution of the enterosalivary pathway to serum nitrite levels can be up to 25%, based on the reduction observed when the oral microbiome is disrupted by antibacterial mouthwash (Kapil et al. 2013). However, this contribution and its clinical impact appear to differ among individuals and studies, likely due to variations in NOS activity under certain conditions (i.e., endothelial dysfunction, inflammation) but also due to significant intrapersonal and interpersonal differences in oral nitrate-reducing capacity (Liddle et al. 2019).

Figure 1.

Figure 1.

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Interconnected microbial and human pathways in nitric oxide (NO) metabolism (“enterosalivary” or NO3–-NO2–-NO pathway). 1) Endogenous NO synthesis in the blood occurs primarily via eNOS, with smaller contributions from nNOS, and iNOS in inflammatory states (Kapil et al. 2020); 2) nitric oxide in the blood is sequentially oxidized to nitrite (NO2– ) and nitrate (NO3–) by hemoglobin; 3) nitrate is concentrated into the salivary glands by sialin, a 2NO3–/H+ transporter(Qin et al. 2012); 4) salivary nitrate, mixed with ingested nitrate from the diet, gets reduced to nitrite (NO2– ) by nitrate reductase enzymes expressed in commensal oral bacteria (Nar or Nap); 5) salivary nitrite gets chemically reduced to NO in acidic and/or hypoxic environments in the mouth or in the gastrointestinal tract after swallowing; 6) salivary nitrite can be enzymatically reduced to NO, nitrous oxide (N2O), or dinitrogen (N2) in the mouth by denitrifying oral bacteria (denitrification). Nitric oxide produced via denitrification can be oxidized back to nitrite in the saliva, thus facilitating the influx of the NO3–-NO2–-NO pathway; N2O gas, if formed, is released in the breath; 7) salivary nitrite can be converted by commensal oral bacteria to ammonium via the dissimilatory nitrated reduction to ammonium pathway (DNRA). Ammonium released by the respiratory DNRA (NrfA) can end up in the liver, where it is converted to urea, while ammonium produced via fermentative DNRA (NirB) is assimilated into bacterial biomass. Details and additional references are available in the text.

The NO3-NO2-NO pathway represents a unique paradigm of a symbiotic relationship between the human body and its microbiome that also links oral and systemic health. Activity of this pathway modulates cardiometabolic health, exercise performance, oral health, and mental health (Bescos et al. 2020; Rosier et al. 2020; Vanhatalo et al. 2021). Disruption of the oral microbiome through the use of antiseptic mouthwash negatively affects NO-dependent signaling and may increase the risk of cardiometabolic disease (Senkus and Crowe-White 2019). Despite this understanding, the nitrate-reducing pathways of oral bacteria remain to be characterized in detail and there is little known regarding which pathways are expressed in different oral species under the diverse environmental conditions present in the human mouth. This is an important question as bacterial metabolism of nitrate–nitrite is more complex than simply nitrate reduction to nitrite. For example, bacterial nitrate reduction pathways may further reduce nitrite to several products such as ammonium (dissimilatory nitrate reduction to ammonium [DNRA]) or NO, nitrous oxide, and dinitrogen (denitrification), which could affect NO bioavailability (Kraft et al. 2014; Tribble et al. 2019). In natural ecosystems, the balance between DNRA and denitrification is controlled by environmental parameters, primarily availability of oxygen, nitrate versus nitrite, and carbon sources (Fig. 2) (Kraft et al. 2014; Pandey et al. 2020). It is currently unknown which factors control these competing pathways in the oral environment and whether this affects systemic NO bioavailability and associated clinical outcomes. The objective of this review is to discuss mechanisms by which interconnected human and bacterial pathways of NO production in the human body could affect oral and systemic health, as well as the potential role of modifiable lifestyle factors as modulators of this relationship. Based on this information, a novel behavioral and microbial model for nitric oxide metabolism and health is proposed, which links lifestyle factors with oral and systemic health through bacterial nitrate metabolism.

Figure 2.

Figure 2.

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Dissimilatory bacterial nitrate reduction and environmental conditions that control the competing denitrification and dissimilatory nitrate reduction to ammonium (DNRA) pathways. Nar, cytoplasmic nitrate reductase; Nap, periplasmic nitrate reductase (located in the periplasmic space, between the inner and outer membrane of Gram-negative bacteria); Nir, nitrite reductase; Nor, nitric oxide reductase; Nos, nitrous oxide reductase; Nrf, ammonia-producing nitrite reductase, respiratory DNRA (generates energy via electron transport; dissimilatory); NirB, ammonia-producing nitrite reductase, fermentative DNRA (energy is produced via substrate-level phosphorylation; dissimilatory or assimilatory).

Nitrate Reduction Pathways in Oral Bacteria

Dissimilatory nitrate reduction is an essential component of the environmental nitrogen cycle, and it involves respiratory pathways via which microorganisms use nitrate (NO3–) and nitrite (NO2–) as terminal electron acceptors to generate energy in oxygen-limiting environments. Two microbial respiratory processes compete for nitrate as an electron acceptor. Denitrification is a 4-step reductive process that converts nitrate into nitrous oxide (N2O) and dinitrogen gases (N2) via the intermediate formation of NO2– and NO; DNRA, on the other hand, is a 2-step process, which converts nitrate into ammonium (NH4+) via intermediate formation of NO2– (Sparacino-Watkins et al. 2014) (Fig. 2). Both pathways share a common first step, the reduction of nitrate to nitrite via nitrate reductase (Nap or Nar). Denitrification is carried out by the Nar/Nir protein system, where Nar is the cytoplasmic nitrate reductase and Nir the nitrite reductase. The denitrification pathway also includes a nitric oxide reductase (Nor) and a nitrous oxide reductase (Nos), which catalyze the further reduction of NO to nitrous oxide and dinitrogen, respectively (Sparacino-Watkins et al. 2014). Respiratory DNRA is carried out by the Nap/Nrf protein system, where Nap is the periplasmic nitrate reductase and Nrf the respiratory nitrite reductase. In some bacteria, DNRA can also be fermentative; in contrast to the respiratory DNRA, which is always dissimilatory, ammonium generated via the fermentative nitrite reductase (NirB) can be assimilated to increase bacterial biomass (Pandey et al. 2020).

Several commensal oral bacteria are able to reduce nitrate, primarily the genera Neisseria, Haemophilus, Granulicatella, Veillonella, Prevotella, Corynebacterium, Actinomyces, and Rothia (Doel et al. 2005; Hyde et al. 2014; Liddle et al. 2019). Nitrate reductase activity is highest in the posterior of the dorsum of the tongue, especially under anaerobic conditions, but is also present in dental plaque and in saliva and under aerobic conditions (Doel et al. 2005). There is significant inter- and intrapersonal variability in the abundance of nitrate-reducing species; the most variable nitrate-reducing species on the human tongue are Rothia dentocariosa and Haemophilus parainfluenzae, while Prevotella melaninogenica and Veillonella dispar are the most constant and most abundant nitrate-reducing species (Liddle et al. 2019).

Several oral species have been shown to reduce nitrite, including Streptococcus mutans, Fusobacterium nucleatum, and V. dispar (Choudhury et al. 2007; Hyde et al. 2014), but the specific nitrite reduction pathways present in oral bacteria have not been characterized. Nitrate reductase genes (narG or napA) have been identified in several oral taxa, but DNRA-related genes appear to be much more prevalent compared to denitrification genes (Table). Nitrate-reducing Gram-positive oral bacteria appear to have genes related to fermentative nitrite reduction via DNRA but not respiratory denitrification, except for NorB and NosZ in some cases. These pathways could serve as a protective mechanism against cytotoxic levels of nitrite and also for nitrogen assimilation in these bacteria. Respiratory denitrification genes have been found in Neisseria, Haemophilus, and Aggregatibacter species; notably, Aggregatibacter actinomycetemcomitans and H. parainfluenzae appear to have both denitrification and DNRA genes. DNRA genes appear to be more frequent compared to denitrification genes in oral bacteria. Neisseria is the only taxon in the Table where only denitrification genes and not DNRA genes have been so far identified. Genes involved in both pathways have been identified in human dental plaque and tongue scrapings (Schreiber et al. 2010; Tribble et al. 2019); denitrification genes were more abundant in participants with predominance of Neisseria species, while participants with predominant Prevotella species had higher abundance of genes involved in ammonium-producing nitrate reduction (Tribble et al. 2019). Dental plaque was shown to perform denitrification of nitrate to NO, N2O, and N2 in situ under aerobic conditions, and production of N2O from nitrate in the mouth has been clinically demonstrated (Schreiber et al. 2010). Ammonium generation from nitrate has been recently demonstrated in human saliva and tongue scrapings (Morou-Bermúdez et al. 2019; Tribble et al. 2019; Rosier et al. 2020). There is a remarkable lack of knowledge regarding these important metabolic pathways in oral bacteria, the factors that regulate them, and their impact in oral and systemic health.

Table.

GenBank Search for Genes with Homology to Proteins Involved in Nitrate Reduction in Oral Bacteria.

Nitrate Reduction Denitrification DNRA
Genus Oral Species NarG NapA NirK NirS NorB NosZ NrfA NirB
Gram positive
 Actinomyces Multispecies/oral taxon + – – – ~ + – +
naeslundii + – – – + – – +
viscosus + – – – – – – –
Corynebacterium + – – – + + – +
  Corynebacterium matruchotii + – – – – – – +
 Rothia + – – – ~ + – +
  Rothia mucilaginosa + – – – + – – +
  Rothia dentocariosa + – – – + + – +
 Streptococcus ~ – – – – – ~ +
  Streptococcus mutans – – – – – – – +
  Streptococcus mitis – – – – – – – +
  Streptococcus parasanguinis – – – – – – – +
Gram negative
 Aggregatibacter ~ + ~ – + – ~ –
  Aggregatibacter actinomycetemcomitans – + + – – – + –
 Fusobacterium – – – – – – – +
  Fusobacterium nucleatum – – – – – – – +
 Haemophilus ~ + + – + – + –
  Haemophilus parainfluenzae + + + – + – + –
 Neisseria + + + – + + – –
  Neisseria subflava – – + – + + – –
flavescens – – + – + + – +
 Porphyromonas – – – – ~ ~ + ~
  Porphyromonas gingivalis – – – – – + + +
 Prevotella ~ – – – ~ ~ + ~
  Prevotella melaninogenica – – – – + – + –
denticola – – – – – + + –
 Veillonella + – – – + – ~ +
  Veillonella parvula + – – – + – – +
  Veillonella dispar + – – – + – – +
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Search performed on GenBank (https://www.ncbi.nlm.nih.gov/genbank), accessed on September 15, 2021. Taxa were selected based on previous reports in the literature that they are involved in nitrate/nitrite reduction (see text for explanations and references). This list does not preclude that additional taxa may have the specific genes or that additional genes involved in nitrate/nitrite reduction may be present in these or other oral taxa. –, gene was not found in that particular genus or species; +, gene was found in that particular species, and in the case of the genus rows, it means that the gene is found in multiple species of that genus; ~, within a certain genus, that particular gene is not found in multiple species but was found in other species apart from those listed in the table.

Role of Nitrate-Reducing Oral Bacteria in Oral and Systemic Health

Bacterial nitrate reduction is coupled to the oxidation of organic electron donors, including glucose and lactate (van den Berg et al. 2017), which leads to significant decreases in acid content of dental plaques in vitro (Li et al. 2007; Rosier et al. 2020) and in vivo (Rosier et al. 2021). The metabolic link between lactate oxidation and nitrate reduction to nitrite was recently shown in Veillonella sp., indicating that lactate is initially oxidized to pyruvate and subsequently to weaker acids, such as acetate, propionate, and others, depending on the redox balance (Wicaksono et al. 2020). Nitrite and its metabolites can also be inhibitory to oral bacteria, including S. mutans and periodontal pathogens (Backlund et al. 2015). Based on these observations, nitrate has been suggested as a potential probiotic for oral health (Rosier et al. 2020). In clinical studies, salivary nitrite levels are inversely associated with numbers of cariogenic bacteria, such as S. mutans and lactobacilli (Doel et al. 2004), and with caries levels in children, including early childhood caries (ECC) (Senthil Eagappan et al. 2016; Syed et al. 2016). On the other hand, increased nitrite levels in saliva and in gingival crevicular fluid are positively associated with periodontal inflammation (Han et al. 2013; Toczewska et al. 2020). This could be the result of iNOS activation in oral keratinocytes, as well as in macrophages, neutrophils, and dendritic cells by oral pathogens (Hussain et al. 2015). In addition, the association between salivary nitrite and periodontal inflammation could be due to production of ammonium, which has been long associated with the development of periodontal disease (Niederman et al. 1990). Clinical improvement following periodontal treatment appears to be associated with a reduction in salivary nitrate and nitrite levels (Sánchez et al. 2014).

While salivary nitrite levels are invariably positively associated with periodontal inflammation, serum nitrite levels appear to be inversely associated with periodontal disease and with C-reactive protein (CRP) levels, suggesting a protective role of systemic NO against periodontal inflammation (Andrukhov et al. 2013). Our preliminary analyses, using data from more than 1,000 overweight adults from the San Juan Overweight Adult Longitudinal Study (SOALS), suggest a significant inverse association between serum nitrite levels and gingival bleeding, probing depth, and clinical attachment loss, after adjusting for multiple confounders (Morou-Bermúdez et al. 2021). Earlier smaller studies, however, have found positive associations between systemic NO levels and aggressive periodontitis (Sundar et al. 2013). Consistent with the general principals of NO function (Iwata et al. 2020), low levels of NO produced via eNOS or nNOS could play a protective homeostatic role by improving endothelial function and preventing inflammation in the periodontal tissues, while iNOS stimulation by periodontal pathogens could increase nitric oxide production thus exacerbating periodontal inflammation.

Nitrate-reducing oral bacteria are associated with salivary nitrate and nitrite levels, but they are not consistently associated with systemic NO levels and cardiometabolic outcomes (Burleigh et al. 2018). Dietary nitrate supplementation increases the numbers of denitrifying species such as Neisseria flavescens and Rothia sp., while DNRA organisms like Veillonella and Prevotella decrease. These changes are associated with increased plasma nitrite levels and lower blood pressure in older participants, as well as a significant improvement of vascular function in patients with hypercholesterolemia (Velmurugan et al. 2016; Vanhatalo et al. 2018; Vanhatalo et al. 2021). Increased numbers of Neisseria and Haemophilus sp. in the saliva following dietary nitrate supplementation have been recently associated with improved cognitive function in older adults (Vanhatalo et al. 2021). The sum of nitrate-reducing bacteria in supragingival plaque was found to be inversely associated with blood glucose levels and insulin resistance in diabetes-free adults in the ORIGINS study, after adjusting for multiple confounders (Goh et al. 2019). Nitrate-reducing bacteria were also negatively associated with diastolic blood pressure in normotensive subjects but not in the subjects with hypertension. In terms of specific species, higher abundance of H. parainfluenzae was associated with reduced systolic and diastolic blood pressure and blood glucose levels, while N. flavescens was inversely associated with insulin resistance and mean systolic blood pressure (Goh et al. 2019). Overall, there is increasing evidence that nitrate supplementation favors denitrifying species like Neisseria and that this increase is associated with improved cardiometabolic and neurocognitive clinical outcomes.

Antibacterial mouthwash use induces compositional changes in the oral microbiome, which negatively affect salivary and systemic NO levels and blood pressure in small human trials and animal models (Senkus and Crowe-White 2019). Use of chlorhexidine for up to 7 d was shown to lower oral nitrate reduction by 90% and reduce plasma nitrite levels by up to 25%, with a concomitant small (2–3.5 mm Hg) significant increase in systolic and diastolic blood pressure in healthy volunteers (Kapil et al. 2013; Bescos et al. 2020). Use of the same mouthwash by hypertensive individuals for 3 d resulted in increased systolic but not diastolic blood pressure (Bondonno et al. 2015). In small clinical trials, antibacterial mouthwash abolished the beneficial effects of nitrate supplementation on blood pressure (Woessner et al. 2016), postexercise blood pressure (Cutler et al. 2019), and insulin sensitivity (Beals et al. 2017). However, some clinical trials failed to demonstrate significant changes in blood pressure in healthy subjects after short-term mouthwash use, despite significant reductions in salivary nitrite levels (Sundqvist et al. 2016; Ashworth et al. 2019). This could be due to small sample sizes and lack of power to detect small differences in the blood pressure outcome, but it could also be due to the short-term impact of the mouthwash on the oral microbiome. The impact of chlorhexidine on the oral microbiome appears to be transient with a fast recovery; furthermore, the recovery is associated with an increase in metabolic activity, including nitrate reduction (Tribble et al. 2019). Recent publications from SOALS, a longitudinal study of over 1,000 overweight/obese adults in Puerto Rico, showed that routine long-term twice-daily or more frequent use of over-the-counter (OTC) mouthwash was associated with increased risk for prediabetes/diabetes and hypertension over a 3-y period, after adjusting for important confounders (Joshipura et al. 2017, 2020). These associations could be mediated by the impact of mouthwash on the NO3-NO2-NO pathway, but the NO metabolites or nitrate-reducing bacteria were not available to evaluate these pathways. Small clinical trials suggest that antibacterial agents commonly found in OTC mouthwash, such essential oils or povidone iodine, do not significantly affect oral nitrate reduction (Mitsui and Harasawa 2017). However, chronic use could have a more long-term impact on the oral microbiome and on systemic NO levels. A recent clinical study found that the impact of antibacterial mouthwash on blood pressure was stronger among individuals who brushed their tongue frequently (Tribble et al. 2019). The tongue microbiome of these individuals had a greater capacity to reduce nitrate to NO via denitrification. On the other hand, subjects who did not regularly brush their tongues had a tongue microbiome that predominantly reduced nitrate to ammonium; mouthwash use did have a significant impact on blood pressure in these individuals. Overall, the available evidence suggests that the impact of the oral microbiome on the NO3-NO2-NO pathway and its associated clinical outcomes would largely depend on which nitrate-reducing species and nitrate reduction pathways are predominant under specific conditions.

Disruption of oral nitrate reduction significantly attenuates but does not completely eliminate the rise in serum nitrite levels induced by nitrate ingestion, suggesting that a small amount of nitrate reduction can occur elsewhere, likely in the gut. The amount of nitrate that reaches the intestine depends on how much of the ingested nitrate is reduced by the oral microbiome, which is characterized by significant intra- and interpersonal variability (Liddle et al. 2019). Studies on pure cultures of gastrointestinal bacteria, such as Escherichia coli, Lactobacillus, and Bifidobacterium, or using clinical stool samples indicate that the predominant nitrate reduction pathway in gut bacteria is the DNRA; therefore, any nitrate that makes it to the gut will be predominantly reduced to ammonium, which is then converted to urea in the liver (Vermeiren et al. 2009; Tiso et al. 2015; Rocha and Laranjinha 2020). However, DNRA has the potential to generate some NO, because in this pathway, nitrate is first converted to nitrite, which, together with swallowed nitrite from the mouth, can be chemically reduced in NO in acidic and hypoxic environments. Nitric oxide plays an important role in protecting the integrity of the gastric mucosa through its antibacterial properties and by stimulating mucosal blood flow and mucus secretion (González-Soltero et al. 2020). There is very limited knowledge regarding the contribution of the gut microbiome to the systemic NO levels and corresponding physiological outcomes.

Environmental Control of Competing Denitrification and DNRA Pathways in Bacteria

Denitrification and DNRA compete for substrate nitrite. Understanding the specific conditions that favor one versus the other could be of biological and clinical importance, therefore. Environmental factors that regulate the competing denitrification and DNRA pathways in mixed microbial communities have been extensively studied in a variety of natural ecosystems, and the physiological principals that govern these mechanisms appear to be universal. Both metabolic pathways are under the control of transcriptional regulators of the fumarate/nitrate reduction (FNR) superfamily, which regulates gene expression in response to environmental signals such as oxygen, nitrate, and nitrite availability (Pandey et al. 2020).

DNRA is energetically more favorable for bacteria because the reduction of NO3– to ammonium transfers more electrons (8e–) and yields more free energy per molecule of NO3– reduced, compared to complete denitrification to N2 (5e–) (Fig. 2) (Pandey et al. 2020). Consequently, DNRA would be favored in oral sites with limited oxygen availability, while denitrification has been shown to occur in the dental plaque under more aerobic conditions (Schreiber et al. 2010). Poor oral hygiene could promote DNRA over denitrification by creating thicker biofilms with lower redox potential; effective plaque control, on the other hand, stimulates fast bacterial growth and population recovery, which has been shown to favor denitrification, while longer generation times favor the reduction of nitrite into ammonium in controlled in vitro studies (Kraft et al. 2014; van den Berg et al. 2015). As mentioned previously, regular tongue cleaning was associated with a tongue microbiome that had a higher capacity to reduce nitrate to NO in a recent clinical study, while the tongue microbiome of individuals who did not clean their tongue frequently predominantly metabolized nitrate to ammonium (Tribble et al. 2019).

A key mechanism controlling the competition of denitrification versus DNRA in natural ecosystems is the relative proportions of the electron donor to the electron acceptor, or carbon-to-nitrate (C/NO3–) ratio. In that regard, DNRA bacteria have a competitive advantage of over-denitrifying bacteria when there is an excess of electron donors, because they consume more electrons per molecule of nitrate (Fig. 2); consequently, a high C/NO3– ratio generally favors the conversion of NO3– into ammonium, while low C/NO3– ratios favor denitrification (Kraft et al. 2014; Heo et al. 2020; Pandey et al. 2020). Nitrate-reducing bacteria use a variety of fermentable (i.e., glucose, lactate) and nonfermentable (i.e., acetate) electron donors, and the availability of these donors can influence the competition between DNRA and denitrification in similar ways. In a continuous culture system, van den Berg et al. (2017) observed that when lactate was at high excess compared to nitrate (Lac/N 2.97 molar ratio), DNRA was the dominant pathway for nitrate reduction, but when lactate was reduced to a 0.63 Lac/N ratio, all the nitrate and lactate were used for denitrification. DNRA could have a greater impact in preventing oral acidification compared to denitrification when there is high lactate availability, because it consumes more protons per molecule of nitrate reduced (Fig. 2).

When C/NO3– proportions are equal, the relative amounts of NO3– versus NO2– (NO3–/NO2– ratio) modulate the competition between denitrification and DNRA (Kraft et al. 2014; Pandey et al. 2020). The substrate affinity (μmax) of DNRA bacteria for nitrate is 3 times higher compared to that of denitrifying bacteria; therefore, DNRA bacteria tend to predominate over denitrifying species under nitrate-limiting conditions (Pandey et al. 2020). DNRA predominates in low NO3–/NO2– ratios due to reduced expression of Nir proteins, while high NO3– concentrations repress DNRA activity by downregulating NrfA (Heo et al. 2020; Pandey et al. 2020). The rate of denitrification in the dental plaque was shown to increase with increasing salivary NO3–/NO2– ratio and with dietary nitrate supplementation (Schreiber et al. 2010). The NO3–/NO2– ratio in the oral cavity could also be influenced by diet. About 60% to 80% of dietary nitrate is from vegetables, especially green leafy vegetables and beetroot; nitrite, on the other hand, is mostly found in processed meats (Kapil et al. 2020). “Nitrate(s)” and “nitrite(s)” are frequently considered in nutritional sciences together, and there has been a lot of controversy regarding their consumption because of their potential to be converted to nitrosamines, which are linked to cancer, versus their potential beneficial role in increasing NO levels (Kapil et al. 2020). Based on the observations mentioned previously, a diet low in green leafy vegetables but high in processed meats could lower the NO3–/NO2– ratio, favoring the conversion of the limited nitrate to ammonium via DNRA, while a diet rich in green leafy vegetables and low in processed meats (higher NO3–/NO2– ratio) could enhance NO production through denitrification. Dietary supplementation with nitrate indeed increases the proportions of denitrifying species, such as Neisseria, and reduces the proportions of Veillonella, a DNRA organism in human clinical trials; these changes are associated with a concomitant increase in systemic nitrite levels and improved cardiometabolic outcomes (Velmurugan et al. 2016; Vanhatalo et al. 2018).

Additional environmental factors involved in the control of denitrification versus DNRA are temperature, pH, and also concentrations of sulfide (S2–) and iron (Fe2–) (Pandey et al. 2020). Generally, temperatures over 30°C and alkaline pH favor DNRA over denitrification, while acidic pH also promotes the chemical conversion of nitrite to NO in the oral cavity (Schreiber et al. 2010; Pandey et al. 2020). At the protein activity level, NirK has optimum activity at pH <7 while the optimum activity of NrfA is >7.5. These factors can be influenced by diet, in particular sugar consumption, but also potentially by smoking and other behaviors. It is unclear, though, how releant these factors could be in the oral cavity or how they can be influenced by lifestyle factors.

Competition between Denitrification and DNRA in Oral Bacteria: A Behavioral and Microbial Model for Nitric Oxide Metabolism and Health

The processes of denitrification and DNRA compete for oral nitrate, and both processes can negatively affect the NO3-NO2-NO pathway by shunting nitrite to N2 or NH4+, respectively. However, denitrification generates NO as an intermediate product, which can be rapidly oxidized back to NO2 in the saliva, thus facilitating the flux through the NO3-NO2-NO pathway (Ignarro et al. 1993). In the relatively aerobic salivary environment, the expression of NO reductase (Nor) could be inhibited by NNR (nitrate reductase and NO reductase regulator), resulting in accumulation of NO (Gaimster et al. 2018). Ammonium generated from respiratory DNRA in the mouth or gut can be transferred to the liver and enter the urea cycle, while ammonium generated via the fermentative DNRA can be assimilated to bacterial biomass (Tiso et al. 2015; Pandey et al. 2020). Therefore, we can postulate that the denitrification process may be more favorable for NO generation through the NO3-NO2-NO pathway compared to the DNRA. Denitrifying oral bacteria, such as Neisseria, are consistently associated with increased systemic NO levels and improved cardiometabolic and cognitive health, as well as oral health, while DNRA bacteria, such as Prevotella and Veillonella, are associated with lower NO levels, poorer cardiometabolic health, and increased periodontal inflammation (Velmurugan et al. 2016; Yamashita and Takeshita 2017; Vanhatalo et al. 2018; Tribble et al. 2019; Vanhatalo et al. 2021).

Available knowledge from natural ecosystems, supported by the findings of recent clinical studies, indicates that diet, oral hygiene, and other modifiable lifestyle factors could influence key environmental drivers of the competing pathways in oral bacteria, including redox potential, carbon-to-nitrate ratio, and nitrate-to-nitrite ratio. Based on this information, we are proposing a Behavioral and Microbial Model for Nitric Oxide Metabolism and Health (Fig. 3); according to this model, a low-sugar diet with a high content in nitrate-rich foods, such as green leafy vegetables, and good oral hygiene/plaque control will increase NO levels through denitrification promoting oral and systemic health. In contrast, high-sugar diets low in green leafy vegetables and poor oral hygiene/plaque control may shift oral nitrate reduction toward the production of ammonium via DNRA, potentially resulting in reduced systemic NO levels, increased risk for cardiometabolic disease, and higher periodontal inflammation.

Figure 3.

Figure 3.

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Behavioral and microbial model for nitric oxide (NO) metabolism and health. A low-sugar diet with a high content in nitrate-rich foods, such as green leafy vegetables, and good oral hygiene/plaque control will increase NO levels through denitrification promoting oral and systemic health. In contrast, high-sugar diets low in green leafy vegetables and poor oral hygiene/plaque control may shift oral nitrate reduction toward the production of ammonium via dissimilatory nitrate reduction to ammonium, potentially resulting in reduced systemic NO levels, increased risk for cardiometabolic disease, and higher periodontal inflammation. For further explanations, see text.

This model provides a novel mechanism linking lifestyle factors with oral and systemic health through NO metabolism. Increased sugar consumption has been proposed as a common risk factor for dental caries, periodontal disease, obesity, and systemic inflammation, while the benefits of green leafy vegetables are also well established (Nyvad and Takahashi 2020). Our proposed behavioral and microbial model for nitric oxide metabolism and health provides an additional possible mechanism mediating these associations through nitric oxide metabolism, which incorporates additional modifiable lifestyle factors. Although the physiological principles behind this model are ubiquitous in nature, the actual mechanistic pathways involved can be more intricate in the complex oral environment. Additional dietary components and behaviors, such as smoking and mouthwash use, and endogenous NO production via the 3 NOS isoforms in the oral tissues could further modulate the proposed mechanisms. The salivary glands could play an important role in this model as regulators of nitrate circulation between the oral cavity and also as a principal source of NO via NOS-dependent synthesis (Hezel and Weitzberg 2015). This review provides a scientific framework for future studies that are urgently needed to elucidate the potentially crucial role of the oral microbiome in NO metabolism as related to cardiometabolic and oral health, as well as other clinical outcomes.

Author Contributions

E. Morou-Bermúdez, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; J.E. Torres-Colón, N.S. Bermúdez, contributed to data acquisition and analysis, drafted the manuscript; R.P. Patel, K.J. Joshipura, contributed to conception, data interpretation, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Acknowledgments

We thank Ana Maria Bermúdez for the artwork.

Footnotes

Declaration of Conflicting Interests: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: R.P. Patel is a coinventor on the use of nitrite salts for the treatment of cardiovascular conditions and chronic ischemia and a coinventor on a provisional patent for methods to diagnose and predict chronic lung and bowel disease in preterm infants. Other authors do not have any conflicts of interest to report.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Funding for this manuscript was provided by grant R01DE028195 from the National Institute for Dental and Craniofacial Research (Principal Investigators: K.J. Joshipura and E. Morou-Bermúdez). The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services, nor does mention by trade names, commercial practices, or organizations imply endorsement by the US government.

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