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Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: implications for nitric oxide homeostasis.

ABSTRACT: The microbiota of the human lower intestinal tract helps maintain healthy host physiology, for example through nutrient acquisition and bile acid recycling, but specific positive contributions of the oral microbiota to host health are not well established. Nitric oxide (NO) homeostasis is crucial to mammalian physiology. The recently described entero-salivary nitrate-nitrite-nitric oxide pathway has been shown to provide bioactive NO from dietary nitrate sources. Interestingly, this pathway is dependent upon oral nitrate-reducing bacteria, since humans lack this enzyme activity. This pathway appears to represent a newly recognized symbiosis between oral nitrate-reducing bacteria and their human hosts in which the bacteria provide nitrite and nitric oxide from nitrate reduction. Here we measure the nitrate-reducing capacity of tongue-scraping samples from six healthy human volunteers, and analyze metagenomes of the bacterial communities to identify bacteria contributing to nitrate reduction. We identified 14 candidate species, seven of which were not previously believed to contribute to nitrate reduction. We cultivated isolates of four candidate species in single- and mixed-species biofilms, revealing that they have substantial nitrate- and nitrite-reduction capabilities. Colonization by specific oral bacteria may thus contribute to host NO homeostasis by providing nitrite and nitric oxide. Conversely, the lack of specific nitrate-reducing communities may disrupt the nitrate-nitrite-nitric oxide pathway and lead to a state of NO insufficiency. These findings may also provide mechanistic evidence for the oral systemic link. Our results provide a possible new therapeutic target and paradigm for NO restoration in humans by specific oral bacteria.

SUBMITTER: Hyde ER

PROVIDER: S-EPMC3966736 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: implications for nitric oxide homeostasis.

Hyde Embriette R ER Andrade Fernando F Vaksman Zalman Z Parthasarathy Kavitha K Jiang Hong H Parthasarathy Deepa K DK Torregrossa Ashley C AC Tribble Gena G Kaplan Heidi B HB Petrosino Joseph F JF Bryan Nathan S NS

PloS one 20140326 3

The microbiota of the human lower intestinal tract helps maintain healthy host physiology, for example through nutrient acquisition and bile acid recycling, but specific positive contributions of the oral microbiota to host health are not well established. Nitric oxide (NO) homeostasis is crucial to mammalian physiology. The recently described entero-salivary nitrate-nitrite-nitric oxide pathway has been shown to provide bioactive NO from dietary nitrate sources. Interestingly, this pathway is d ...[more]

PMID: 24670812

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Project description:Background The enterosalivary nitrate-nitrite-nitric oxide pathway is an alternative pathway of nitric oxide generation, potentially linking the oral microbiome to insulin resistance and blood pressure (BP). We hypothesized that increased abundance of nitrate-reducing oral bacteria would be associated with lower levels of cardiometabolic risk cross-sectionally. Methods and Results ORIGINS (Oral Infections, Glucose Intolerance, and Insulin Resistance Study) enrolled 300 diabetes mellitus-free adults aged 20 to 55 years (mean=34±10 years) (78% women). Microbial DNA was extracted from subgingival dental plaque (n=281) and V3-V4 regions of the 16S rRNA gene were sequenced to measure the relative abundances of 20 a priori-selected taxa with nitrate-reducing capacity. Standardized scores of each taxon's relative abundance were summed, producing a nitrate-reducing taxa summary score (NO3TSS) for each participant. Natural log-transformed homeostatic model assessment of insulin resistance, plasma glucose, systolic BP, and diastolic BP were regressed on NO3TSS in multivariable linear regressions; prediabetes mellitus and hypertension prevalence were regressed on NO3TSS using modified Poisson regression models. Nitrate-reducing bacterial species represented 20±16% of all measured taxa. After multivariable adjustment, a 1-SD increase in NO3TSS, was associated with a -0.09 (95% CI, -0.15 to -0.03) and -1.03 mg/dL (95% CI, -1.903 to -0.16) lower natural log-transformed homeostatic model assessment of insulin resistance and plasma glucose, respectively. NO3TSS was associated with systolic BP only among patients without hypertension; 1-SD increase in NO3TSS was associated with -1.53 (95% CI, -2.82 to -0.24) mm Hg lower mean systolic BP. No associations were observed with prediabetes mellitus and hypertension. Conclusions A higher relative abundance of oral nitrate-reducing bacteria was associated with lower insulin resistance and plasma glucose in the full cohort and with mean systolic BP in participants with normotension.

Project description:Nitrate reductases (NRs) are molybdoenzymes that reduce nitrate (NO3 -) to nitrite (NO2 -) in both mammals and plants. In mammals, the salival microbes take part in the generation of the NO2 - from NO3 -, which further produces nitric oxide (NO) either in acid-induced NO2 - reduction or in the presence of nitrite reductases (NiRs). Here, we report a new approach of VCl3 (V3+ ion source) induced step-wise reduction of NO3 - in a CoII-nitrato complex, [(12-TMC)CoII(NO3 -)]+ (2,{CoII-NO3 -}), to a CoIII-nitrosyl complex, [(12-TMC)CoIII(NO)]2+ (4,{CoNO}8), bearing an N-tetramethylated cyclam (TMC) ligand. The VCl3 inspired reduction of NO3 - to NO is believed to occur in two consecutive oxygen atom transfer (OAT) reactions, i.e., OAT-1 = NO3 - → NO2 - (r1) and OAT-2 = NO2 - → NO (r2). In these OAT reactions, VCl3 functions as an O-atom abstracting species, and the reaction of 2 with VCl3 produces a CoIII-nitrosyl ({CoNO}8) with VV-Oxo ({VV[double bond, length as m-dash]O}3+) species, via a proposed CoII-nitrito (3, {CoII-NO2 -}) intermediate species. Further, in a separate experiment, we explored the reaction of isolated complex 3 with VCl3, which showed the generation of 4 with VV-Oxo, validating our proposed reaction sequences of OAT reactions. We ensured and characterized 3 using VCl3 as a limiting reagent, as the second-order rate constant of OAT-2 (k 2 /) is found to be ∼1420 times faster than that of the OAT-1 (k 2) reaction. Binding constant (K b) calculations also support our proposition of NO3 - to NO transformation in two successive OAT reactions, as K b(CoII-NO2 -) is higher than K b(CoII-NO3 -), hence the reaction moves in the forward direction (OAT-1). However, K b(CoII-NO2 -) is comparable to K b{CoNO}8 , and therefore sequenced the second OAT reaction (OAT-2). Mechanistic investigations of these reactions using 15N-labeled-15NO3 - and 15NO2 - revealed that the N-atom in the {CoNO}8 is derived from NO3 - ligand. This work highlights the first-ever report of VCl3 induced step-wise NO3 - reduction (NRs activity) followed by the OAT induced NO2 - reduction and then the generation of Co-nitrosyl species {CoNO}8.

Project description:Recent evidence indicates that the reduction of salivary nitrate by oral bacteria can contribute to prevent oral diseases, as well as increase systemic nitric oxide levels that can improve conditions such as hypertension and diabetes. The objective of the current manuscript was to isolate nitrate-reducing bacteria from the oral cavity of healthy donors and test their in vitro probiotic potential to increase the nitrate-reduction capacity (NRC) of oral communities. Sixty-two isolates were obtained from five different donors of which 53 were confirmed to be nitrate-reducers. Ten isolates were selected based on high NRC as well as high growth rates and low acidogenicity, all being Rothia species. The genomes of these ten isolates confirmed the presence of nitrate- and nitrite reductase genes, as well as lactate utilization genes, and the absence of antimicrobial resistance, mobile genetic elements and virulence genes. The pH at which most nitrate was reduced differed between strains. However, acidic pH 6 always stimulated the reduction of nitrite compared to neutral pH 7 or slightly alkaline pH 7.5 (p < 0.01). We tested the effect of six out of 10 isolates on in vitro oral biofilm development in the presence or absence of 6.5 mM nitrate. The integration of the isolates into in vitro communities was confirmed by Illumina sequencing. The NRC of the bacterial communities increased when adding the isolates compared to controls without isolates (p < 0.05). When adding nitrate (prebiotic treatment) or isolates in combination with nitrate (symbiotic treatment), a smaller decrease in pH derived from sugar metabolism was observed (p < 0.05), which for some symbiotic combinations appeared to be due to lactate consumption. Interestingly, there was a strong correlation between the NRC of oral communities and ammonia production even in the absence of nitrate (R = 0.814, p < 0.01), which indicates that bacteria involved in these processes are related. As observed in our study, individuals differ in their NRC. Thus, some may have direct benefits from nitrate as a prebiotic as their microbiota naturally reduces significant amounts, while others may benefit more from a symbiotic combination (nitrate + nitrate-reducing probiotic). Future clinical studies should test the effects of these treatments on oral and systemic health.

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Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: implications for nitric oxide homeostasis.

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Metagenomic analysis of nitrate-reducing bacteria in the oral cavity: implications for nitric oxide homeostasis.

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