Oral Prevotella Species and Their Connection to Events of Clinical Relevance in Gastrointestinal and Respiratory Tracts

Prevotella is recognized as one of the core anaerobic genera in the oral microbiome. In addition, members of this genus belong to microbial communities of the gastrointestinal and respiratory tracts. Several novel Prevotella species, most of them of oral origin, have been described, but limited knowledge is still available of their clinical relevance. Prevotella melaninogenica is among the anaerobic commensals on oral mucosae from early months of life onward, and other early colonizing Prevotella species in the oral cavity include Prevotella nigrescens and Prevotella pallens. Oral Prevotella species get constant access to the gastrointestinal tract via saliva swallowing and to lower airways via microaspiration. At these extra-oral sites, they play a role as commensals but also as potentially harmful agents on mucosal surfaces. The aim of this narrative review is to give an updated overview on the involvement of oral Prevotella species in gastrointestinal and respiratory health and disease.

Structural and Functional Studies of 3-Deoxy-D-arabino- Heptulosonate 7-Phosphate Synthase from Prevotella nigrescens

<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>

Structural and Functional Studies of 3-Deoxy-D-arabino- Heptulosonate 7-Phosphate Synthase from Prevotella nigrescens

<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>

Variations in the oral microbiome are associated with depression in young adults

A growing body of evidence supports an important role for alterations in the brain-gut-microbiome axis in the aetiology of depression and other psychiatric disorders. The potential role of the oral microbiome in mental health has received little attention, even though it is one of the most diverse microbiomes in the body and oral dysbiosis has been linked to systemic diseases with an underlying inflammatory aetiology. This study examines the structure and composition of the salivary microbiome for the first time in young adults who met the DSM-IV criteria for depression (n = 40) and matched controls (n = 43) using 16S rRNA gene-based next generation sequencing. Subtle but significant differences in alpha and beta diversity of the salivary microbiome were observed, with clear separation of depressed and healthy control cohorts into distinct clusters. A total of 21 bacterial taxa were found to be differentially abundant in the depressed cohort, including increased Neisseria spp. and Prevotella nigrescens, while 19 taxa had a decreased abundance. In this preliminary study we have shown that the composition of the oral microbiome is associated with depression in young adults. Further studies are now warranted, particuarly investigations into whether such shifts play any role in the underling aetiology of depression.

Reciprocal allostery arising from a bi-enzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens

ABSTRACTModular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively. The interactions between these two distinct catalytic domains support functional inter-reliance within this bifunctional enzyme. The binding of prephenate, the product of CM-catalyzed reaction, to the CM domain is associated with a striking rearrangement of overall protein conformation that alters the interdomain interactions and allosterically inhibits the DAH7PS activity. In this study, we observed allosteric activation of CM activity in the presence of all DAH7PS substrates. Using small angle X-ray scattering (SAXS) experiments we show that changes in overall protein conformations and dynamics are associated with the presence of different DAH7PS substrates and the allosteric inhibitor prephenate. Furthermore, we have identified an extended interhelix loop located in CM domain, loopC320-F333, as a crucial segment for the interdomain structural and catalytic communications. Our results suggest that the dual function enzyme PniDAH7PS contains a reciprocal allosteric system between the two enzymatic moieties, as a result of this bidirectional interdomain communication. This arrangement allows for a complex feedback and feedforward system for control of pathway flux by connecting the initiation and branch point of aromatic amino acid biosynthesis.

Quantification of Bacteria in Mouth-Rinsing Solution for the Diagnosis of Periodontal Disease

This study aimed to evaluate the feasibility of diagnosing periodontitis via the identification of 18 bacterial species in mouth-rinse samples. Patients (n = 110) who underwent dental examinations in the Department of Periodontology at the Veterans Health Service Medical Center between 2018 and 2019 were included. They were divided into healthy and periodontitis groups. The overall number of bacteria, and those of 18 specific bacteria, were determined via real-time polymerase chain reaction in 92 mouth-rinse samples. Differences between groups were evaluated through logistic regression after adjusting for sex, age, and smoking history. There was a significant difference in the prevalence (healthy vs. periodontitis group) of Aggregatibacter actinomycetemcomitans (2.9% vs. 13.5%), Treponema denticola (42.9% vs. 69.2%), and Prevotella nigrescens (80% vs. 2.7%). Levels of Treponema denticola, Prevotella nigrescens, and Streptococcus mitis were significantly associated with severe periodontitis. We demonstrated the feasibility of detecting periopathogenic bacteria in mouth-rinse samples obtained from patients with periodontitis. As we did not comprehensively assess all periopathogenic bacteria, further studies are required to assess the potential of oral-rinsing solutions to indicate oral infection risk and the need to improve oral hygiene, and to serve as a complementary method for periodontal disease diagnosis.

Evaluation of the Microbiological Profile of Alveolar Residual Screws and Cleft-Adjacent Teeth in Individuals With Complete Unilateral Fissures

Objective: To evaluate the microbiota profile of residual alveolar slits and teeth adjacent to the cleft in fissured individuals. Designs: This study used a cross-sectional design. Participants: Twenty individuals, aged 14 to 24 years, who had a residual fissure in the maxillary alveolar ridge region were selected. Main outcome measures: Three sites per individual were selected for microbiological collection (the site of the residual cleft and the 2 nearest teeth). The samples were analyzed using the Checkerboard DNA–DNA hybridization technique for 73 species of bacteria. Results: All the species analyzed were found in the 2 niches (slits and teeth). The bacterial species present in the largest number in the residual cracks were Prevotella melaninogenica, Prevotella nigrescens, and Streptococcus mitis. With regard to the bacterial profiles in the mesial and distal faces, the most prevalent species were P nigrescens, Veillonella parvula, and Fusobacterium nucleatum sp vicentii. The analysis of all the collected samples demonstrated very similar profiles for the mesial and distal faces, with these 2 sites even presenting the same species in greater frequencies. Higher counts of 20 bacterial species (Wilcoxon test) were observed in the dental niche, in relation to the fissure, particularly, P nigrescens, V parvula, F nucleatum sp vicentii, and Neisseria mucosa. Conclusion: Some species were significantly more prevalent in the residual alveolar fissures and in adjacent teeth. The comparison between the profiles of the 2 niches demonstrated large differences in the most frequent species in the teeth, and no qualitative differences with regard to specific pathogens.

Биофармацевтические исследования по выбору основы для стоматологического геля с экстрактом барбариса

Исследованы биофармацевтические свойства модельных гелевых композиций c экстрактом корня барбариса и использованием в качестве гелеобразователей метилцеллюлозы, натриевой соли карбоксиметилцеллюлозы, натриевой соли альгиновой кислоты и поливинилового спирта методами прямой диффузии в агаровый гель и диализа через полупроницаемую мембрану. Определены кинетические закономерности процесса и скорости набухания исследуемых гелевых основ. Показана целесообразность использования натриевой соли альгиновой кислоты для получения стоматологического геля с экстрактом корня барбариса, обладающего специфической бактериостатической активностью в отношении таких пародонтопатогенных микроорганизмов, как Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens и Actinobacillus actinomycetemcomitans, для профилактики и лечения воспалительных заболеваний пародонта.