Splenectomy ameliorates liver cirrhosis by restoring the gut microbiota balance.

BACKGROUND: Dysbiosis of gut microbiota is frequent in liver cirrhosis (LC) patients, and splenectomy (SP) has been reported to improve LC. Herein, we report the effects of SP on gut microbiota, especially on Veillonella parvula, a Gram-negative coccus of the gastrointestinal tract, in LC mice, and the underlying mechanism. METHODS: LC mice models were induced by tail vein injection of concanavalin A (ConA), followed by SP. 16 s rRNA sequencing was conducted to analyze the effects of ConA induction and SP on mouse gut microbiota and the gene expression affected by gut microbiota. LC mice receiving SP were gavaged with Veillonella parvula. Likewise, hepatic stellate cells (HSC) and hepatocytes (HC) were induced with conditioned medium (CM) of Veillonella parvula. RESULTS: SP alleviated LC in mice by restoring gut barrier function and maintaining gut microbiota balance, with Veillonella as the key genus. The Veillonella parvula gavage on LC mice reversed the ameliorative effect of SP. The CM of Veillonella parvula promoted the activation of HSC and the release of IL-6, IL-1ß, and TNF-α. Also, the CM of Veillonella parvula induced HC pyroptosis and the release of ALT and AST. Veillonella parvula represented an imbalance in the gut microbiota, thus enhancing gut-derived endotoxins in the liver with the main target being Tlr4/Nlrp3. Inhibition of Tlr4 blocked Veillonella parvula-induced HC damage, HSC activation, and subsequent LC progression. CONCLUSION: SP-mediated gut microbiota regulation ameliorates ConA-related LC progression by inhibiting Tlr4/Nlrp3 in the liver.

Nitrate promotes the growth and the production of short-chain fatty acids and tryptophan from commensal anaerobe Veillonella dispar in the lactate-deficient environment by facilitating the catabolism of glutamate and aspartate.

Veillonella spp. are nitrate-reducing bacteria with anaerobic respiratory activity that reduce nitrate to nitrite. They are obligate anaerobic, Gram-negative cocci that ferment lactate as the main carbon source and produce short-chain fatty acids (SCFAs). Commensal Veillonella reside in the human body site where lactate level is, however, limited for Veillonella growth. In this study, nitrate was shown to promote the anaerobic growth of Veillonella in the lactate-deficient media. We aimed to investigate the underlying mechanisms and the metabolism involved in nitrate respiration. Nitrate (15 mM) was demonstrated to promote Veillonella dispar growth and viability in the tryptone-yeast extract medium containing 0.5 mM L-lactate. Metabolite and transcriptomic analyses revealed nitrate enabled V. dispar to actively utilize glutamate and aspartate from the medium and secrete tryptophan. Glutamate or aspartate was further supplemented to a medium to investigate individual catabolism during nitrate respiration. Notably, nitrate was demonstrated to elevate SCFA production in the glutamate-supplemented medium, and further increase tryptophan production in the aspartate-supplemented medium. We proposed that the increased consumption of glutamate provided reducing power for nitrate respiration and aspartate served as a substrate for fumarate formation. Both glutamate and aspartate were incorporated into the central metabolic pathways via reverse tricarboxylic acid cycle and were linked with the increased production of acetate, propionate, and tryptophan. This study provides further understanding of the promoted growth and metabolic mechanisms by commensal V. dispar utilizing nitrate and specific amino acids to adapt to the lactate-deficient environment.IMPORTANCENitrate is a pivotal ecological factor influencing microbial community and metabolism. Dietary nitrate provides health benefits including anti-diabetic and anti-hypertensive effects via microbial-derived metabolites such as nitrite. Unraveling the impacts of nitrate on the growth and metabolism of human commensal bacteria is imperative to comprehend the intricate roles of nitrate in regulating microbial metabolism, community, and human health. Veillonella are lactate-utilizing, nitrate-reducing bacteria that are frequently found in the human body site where lactate levels are low and nitrate is at millimolar levels. Here, we comprehensively described the metabolic strategies employed by V. dispar to thrive in the lactate-deficient environment using nitrate respiration and catabolism of specific amino acids. The elevated production of SCFAs and tryptophan from amino acids during nitrate respiration of V. dispar further suggested the potential roles of nitrate and Veillonella in the promotion of human health.

Lipopolysaccharide-producing Veillonella infantium and Escherichia fergusonii cause vagus nerve-mediated cognitive impairment in mice.

Gut microbiota communicates bidirectionally with the brain through the nervous, immune, and endocrine systems of the gut. In our preliminary study, the fecal microbiota of volunteers with mild cognitive impairment (Fmci) exhibited a higher abundance of Escherichia fergusonii (NK2001), Veillonella infantium (NK2002), and Enterococcus faecium (NK2003) populations compared with those of healthy volunteers. Therefore, we examined the effects of Fmci, NK2001 (gram-negative), NK2002 (gram-negative-like), and NK2003 (gram-positive) on cognitive impairment-like behavior, neuroinflammation, and colitis in mice with or without antibiotics. Fmci transplantation increased cognitive impairment-like behavior, hippocampal tumor necrosis factor (TNF)-α expression, and the size of toll-like receptor (TLR)4+Iba1+, TLR2+Iba1+, and NF-κB+Iba1+ cell populations independent of antibiotic treatment. Oral gavage of NK2001, NK2002, or NK2003, which induced TNF-α expression in Caco-2 cells, significantly increased cognitive impairment-like behavior and hippocampal TNF-α expression and Iba1-positive cell populations and decreased brain-derived neurotrophic factor (BDNF) expression in mice. Celiac vagotomy significantly decreased NK2001- or NK2002-induced cognitive impairment-like behavior and hippocampal Iba1+ cell population and TNF-α expression and increased NK2001- or NK2002-suppressed hippocampal BDNF expression. However, NK2003-induced cognitive impairment-like behavior and hippocampal Iba1+ cell population and TNF-α expression were partially, but not significantly, attenuated by celiac vagotomy. Furthermore, celiac vagotomy did not affect NK2001-, NK2002-, or NK2003-induced lipopolysaccharide (LPS) levels in the blood and feces and TNF-α expression and NF-κB-positive cell population in the colon. In conclusion, LPS-producing NK2001 and NK2002 and LPS-nonproducing NK2003 may induce NF-κB-mediated neuroinflammation through the translocation of byproducts such as LPS and peptidoglycan into the brain through gut-blood/vagus nerve-brain and gut-blood-brain pathways, respectively, resulting in cognitive impairment.

Detecting and quantifying Veillonella by real-time quantitative PCR and droplet digital PCR.

Veillonella spp. are Gram-negative opportunistic pathogens present in the respiratory, digestive, and reproductive tracts of mammals. An abnormal increase in Veillonella relative abundance in the body is closely associated with periodontitis, inflammatory bowel disease, urinary tract infections, and many other diseases. We designed a pair of primers and a probe based on the 16S rRNA gene sequences of Veillonella and conducted real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) to quantify the abundance of Veillonella in fecal samples. These two methods were tested for specificity and sensitivity using simulated clinical samples. The sensitivity of qPCR was 100 copies/µL, allowing for the accurate detection of a wide range of Veillonella concentrations from 103 to 108 CFU/mL. The sensitivity of ddPCR was 11.3 copies/µL, only allowing for the accurate detection of Veillonella concentrations from 101 to 104 CFU/mL because of the limited number of droplets generated by ddPCR. ddPCR is therefore more suitable for the detection of low-abundance Veillonella samples. To characterize the validity of the assay system, clinical samples from children with inflammatory bowel disease were collected and analyzed, and the results were verified using isolation methods. We conclude that molecular assays targeting the 16S rRNA gene provides an important tool for the rapid diagnosis of chronic and infectious diseases caused by Veillonella and also supports the isolation and identification of Veillonella for research purposes. KEY POINTS: • With suitable primer sets, the qPCR has a wider detection range than ddPCR. • ddPCR is suitable for the detection of low-abundance samples. • Methods successfully guided the isolation of Veillonella in clinical sample.

Veillonella faecalis sp. nov., a propionic acid-producing bacterium isolated from the faeces of an infant.

A strictly anaerobic, Gram-stain-negative, catalase-negative, cocci-shaped, and propionate-producing bacterial strain, named Ds1651T was isolated from the fecal sample collected from a South Korean infant. Through a comparison of 16S rRNA gene sequences, it was revealed that Ds1651T had the highest phylogenetic affinity with Veillonella nakazawae KCTC 25297 T (99.86%), followed by Veillonella infantium KCTC 25370 T (99.80%), and Veillonella dispar KCTC 25309 T (99.73%) in the family Veillonellaceae. Average nucleotide identity values between Ds1651T and three reference species were 95.48% for Veillonella nakazawae KCTC 25297 T, 94.46% for Veillonella infantium KCTC 25370 T, and 92.81% for Veillonella dispar KCTC 25309 T. The G + C content of Ds1651T was 38.58 mol%. Major fermentation end-products were acetic and propionic acids in Trypticase peptone glucose yeast extract broth with 1% (v/v) sodium lactate. The predominant cellular fatty acids that account for more than 10% were summed in Feature 8 (C17:1 ω8c and/or C17:2) and C13:0. Based on the findings from phylogenetic, genomic, phenotypic, and chemotaxonomic studies, we propose that the type strain Ds1651T (= KCTC 25477 T = GDMCC 1.3707 T) represents a novel bacterial species within the genus Veillonella, with the proposed name Veillonella faecalis sp. nov.

Development of a small shuttle plasmid for use in oral Veillonella and initial appraisal of potential for fluorescence-based applications.

Oral Veillonella species are among the early colonizers of the human oral cavity. We constructed a small, single-selectable-marker shuttle plasmid, examined its ability to be transformed into diverse oral Veillonella strains, and assessed its potential use for expressing a gene encoding an oxygen-independent fluorescent protein, thus generating a fluorescent Veillonella parvula strain. Because tetracycline resistance is common in Veillonella, we replaced genes encoding ampicillin- and tetracycline-resistance in a previously described shuttle plasmid (pBSJL2) with a chloramphenicol acetyltransferase gene. The resulting plasmid pCF1135 was successfully introduced into four strains representing V. parvula and V. atypica by either natural transformation or electroporation. We then modified this plasmid to express a gene encoding an oxygen-independent fluorescent protein in V. parvula SKV38. The resulting strain yielded a fluorescence signal intensity ∼16 times higher than the wild type in microplate-based fluorimetry experiments. While fluorescence microscopy demonstrated that planktonic cells, colonies, and biofilms of fluorescent V. parvula could also be imaged, photobleaching was a significant issue. In conclusion, we anticipate this genetic system and information provided here will facilitate expanded studies of oral Veillonella species' properties and behavior.

Bacteremia caused by Veillonella parvula: Two case reports and a review of the literature.

Veillonella parvula is a non-motile gram-negative coccus that forms part of the normal microbiota in several body sites and which has been rarely isolated as cause of infections in human population, particularly in bacteremias. Here we give the overview of characteristics of genus Veillonella and the summary of its role in infections, particularly in bacteremia. We additionally report two patients with bacteremia due to V. parvula. Two sets of blood cultures of each patient yielded a pure culture of an anaerobic microorganism identified as V. parvula by MALDI-TOF MS, and confirmed by 16S rRNA gene sequencing. The two patients were male and one of them had risk factors for anaerobic bacteremia. The isolates were susceptible to most antibiotics and the outcome was successful in both patients. Bacteremia due to V. parvula is still rare. MALDI-TOF MS appear to be an excellent tool for the correct identification of these species.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Safety evaluation of Veillonella atypica FB0054 with genotoxicity and subchronic toxicological studies.

Veillonella atypica is a nonmotile, nonsporulating anaerobic bacteria commonly found in various human biofilms. V. atypica FB0054 was isolated from the gastrointestinal tract of marathon runners, who have increased amounts of this species after athletic events. Interestingly, the consumption of this strain by rodents has been shown to increase their treadmill endurance, leading to the hypothesis that consumption of this species may improve athletic performance in humans as well. Further evaluation, in humans, of the usefulness of this strain should be preceded by safety studies. Therefore, the genotoxic and subchronic toxicological potential was evaluated as a contribution to this effort. Genotoxicity investigation was performed using the in vivo comet assay and in vivo mammalian micronucleus assay due to the anaerobic characteristic of the strain. A 90-day, repeated-dose oral toxicity study was performed in rats up to 2200 mg/kg bw/d to investigate general toxicity and identify any target organs. Mitsuoka buffer, a solution shown to preserve the viability of anaerobic bacteria, was used as the vehicle. All three studies revealed no toxicological effects from exposure to FB0054 was isolated from the gastrointestinal tract of marathon runners, who have increased amounts of this species after athletic events. Interestingly, the consumption of this strain by rodents has been shown to increase their treadmill endurance, leading to the hypothesis that consumption of this species may improve athletic performance in humans as well. Further evaluation, in humans, of the usefulness of this strain should be preceded by safety studies. Therefore, the genotoxic and subchronic toxicological potential was evaluated as a contribution to this effort. Genotoxicity investigation was performed using the in vivo comet assay and in vivo mammalian micronucleus assay due to the anaerobic characteristic of the strain. A 90-day, repeated-dose oral toxicity study was performed in rats up to 2200 mg/kg bw/d to investigate general toxicity and identify any target organs. Mitsuoka buffer, a solution shown to preserve the viability of anaerobic bacteria, was used as the vehicle. All three studies revealed no toxicological effects from exposure to FB0054 at the highest doses tested.

Veillonella atypica causing retropharyngeal abscess: A rare case presentation.

Veillonella species are obligate anaerobes which are part of the human oral, gut and vaginal microbiota. The genus Veillonella consists of 16 characterized species. Very few infections due to Veillonella atypica have been reported till date. Here we present a case of retropharyngeal abscess due to this organism in a 55-year-old lady.

A Co-Association of Streptococcus mutans and Veillonella parvula/dispar in Root Caries Patients and In Vitro Biofilms.

Root caries in geriatric patients is a growing problem as more people are maintaining their natural teeth into advanced age. We determined the levels of various bacterial species previously implicated in root caries disease or health using quantitative real-time PCR in a pilot study of 7 patients with 1 to 4 root caries lesions per person. Levels of 12 different species on diseased roots compared to healthy (contralateral control) roots were measured. Four species were found at significantly higher levels on diseased roots (Streptococcus mutans, Veillonella parvula/dispar, Actinomyces naeslundii/viscosus, and Capnocytophaga granulosa) compared across all plaque samples. The level of colonization by these species varied dramatically (up to 1,000-fold) between patients, indicating different patients have different bacteria contributing to root caries disease. Neither of the two species previously reported to correlate with healthy roots (C. granulosa and Delftia acidovorans) showed statistically significant protective roles in our population, although D. acidovorans showed a trend toward higher levels on healthy teeth (P = 0.08). There was a significant positive correlation between higher levels of S. mutans and V. parvula/dispar on the same diseased teeth. In vitro mixed biofilm studies demonstrated that co-culturing S. mutans and V. parvula leads to a 50 to 150% increase in sucrose-dependent biofilm mass compared to S. mutans alone, depending on the growth conditions, while V. parvula alone did not form in vitro biofilms. The presence of V. parvula also decreased the acidification of S. mutans biofilms when grown in artificial saliva and enhanced the health of mixed biofilms.

The Commensal Microbe Veillonella as a Marker for Response to an FGF19 Analog in NASH.

BACKGROUND AND AIMS: The composition of the human gut microbiota is linked to health and disease, and knowledge of the impact of therapeutics on the microbiota is essential to decipher their biological roles and to gain new mechanistic insights. Here we report the effect of aldafermin, an analog of the gut hormone FGF19, versus placebo on the gut microbiota in a prospective, phase 2 study in patients with NASH. APPROACH AND RESULTS: A total of 176 patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH) (nonalcoholic fatty liver disease activity score ≥ 4), fibrosis (F1-F3 by NASH Clinical Research Network criteria), and elevated liver fat content (≥ 8% by magnetic resonance imaging-proton density fat fraction) received 0.3 mg (n = 23), 1 mg (n = 49), 3 mg (n = 49), and 6 mg (n = 28) aldafermin or placebo (n = 27) for 12 weeks. Stool samples were collected on day 1 and week 12 and profiled using 16S ribosomal RNA gene sequencing; 122 patients had paired stool microbiome profiles at both day 1 and week 12. Overall, the state of the gut microbial community was distinctly stable in patients treated with aldafermin, with all major phyla and genera unaltered during therapy. Patients treated with aldafermin showed a significant, dose-dependent enrichment in the rare genus Veillonella, a commensal microbe known to have lactate-degrading and performance-enhancing properties, which correlated with changes in serum bile acid profile. CONCLUSIONS: Veillonella may be a bile acid-sensitive bacteria whose enrichment is enabled by aldafermin-mediated suppression of bile acid synthesis and, in particular, decreases in toxic bile acids. This study provides an integrated analysis of gut microbiome, serum bile acid metabolome, imaging, and histological measurements in clinical trials testing aldafermin for NASH. Our results provide a better understanding of the intricacies of microbiome-host interactions (clinicaltrials.gov trial No. NCT02443116).

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to Streptococcus pneumoniae.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.

The first case of Veillonella atypica bacteremia in a patient with renal pelvic tumor.

We report the first case of bacteremia caused by Veillonella atypica in a morbid elderly female patient who developed obstructive pyelonephritis. She was treated with ceftriaxone and ureteral stenting; this is the first report of V. atypica infection in humans. Species identification was performed by multiplex PCR and sequencing of rpoB. The strain was susceptible to metronidazole and clindamycin but resistant to benzylpenicillin, ampicillin, ampicillin/sulbactam, and moxifloxacin.

Polymicrobial bacteremia due to Lactobacillus jensenii and Veillonella montpellierensis in a pregnant patient; case report and review of literature.

Prompt and accurate diagnosis of polymicrobial bacteremia, which causes the difficulty in anti-infective treatments, poor treatment outcome and high mortality, is essential for initiating effective antimicrobial therapy. Here we present a case of bacteremia caused by two types of uncommon bacteria, Lactobacillus jenseniand and Veillonella montpellierensis in a 29-year-old pregnant woman at 33 weeks of gestation with anemia due to iron deficiency. She had no comorbidity or other chronic illnesses and was successfully treated with appropriate antibiotic use.

Antibiotic susceptibility and biofilm-forming ability of Veillonella strains.

INTRODUCTION: Veillonella, known as early colonizers in oral biofilms, take part in some infections in human. Biofilm refers to complex, sessile communities of microbes, which function as strong barriers for bacteria to survive. Biofilm matrixes surrounding bacteria enable them to withstand harsh conditions, protect against immune cells, etc., and also make them resistant to antimicrobial treatments. Thus, the knowledge of antibiotic susceptibility and biofilm formation of Veillonella will shed light on their resistance mechanism. MATERIALS AND METHOD: Their morphology was observed by scanning electron microscopy (SEM). According to the performance standards for antibiotic susceptibility testing of the Clinical & Laboratory Standards Institute, the Agar dilution method was used to study the susceptibility of Veillonella strains to eight antibiotics (ampicillin, piperacillin-tazobactam, cefoxitin, tetracycline, moxifloxacin, clindamycin, metronidazole, and vancomycin). In addition, we applied the crystal violet staining method to reveal the processes of biofilm formation of these Veillonella strains. RESULTS: V. rogosae, V. nakazawae, and V. parvula were isolated from oral cavities of healthy adults and V. ratti was isolated from dairy goat droppings. Observations by scanning electron microscopy showed that Veillonella were spherical and arranged in single or short chains. The diameter of a single cell was about 0.3-0.5 µm. The Minimum Inhibitory Concentrations (MICs) of the antibiotics were determined and the results showed that these four strains were all sensitive to cefoxitin, tetracycline, moxifloxacin, clindamycin and metronidazole. Among the four strains, V. ratti was resistant to piperacillin-tazobactam, and V. rogosae and V. nakazawae were resistant to ampicillin. The vancomycin susceptibility of the four Veillonella strains varied greatly. The MICs of vancomycin against V. rogosae and V. ratti were greater than 256 µg/mL but the MICs of vancomycin against V. nakazawae and V. parvula were less than 2 µg/mL. V. parvula had significantly higher biofilm-forming ability than the other three strains (p < 0.05) and V. nakazawae had the weakest biofilm-forming ability. CONCLUSION: In this study, V. rogosae, V. nakazawae, V. parvula and V. ratti were isolated and identified. The four strains of Veillonella showed differences in MIC values for different antibiotics and biofilm-forming ability.

An Overview of Adenoid Microbiome Using 16S rRNA Gene Sequencing-Based Metagenomic Analysis.

Background and Objectives: the upper respiratory tract harbors the highest bacterial density in the whole respiratory system. Adenoids, which are located in the nasopharynx, are a major site of bacterial colonies in the upper airways. Our goal was to use culture-independent molecular techniques to identify the breadth of bacterial diversity in the adenoid vegetations of children suffering from chronic rhinosinusitis and obstructive sleep apnea. Materials and methods: in total, 21 adenoid samples were investigated using amplification and sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene. Results: among the most common bacterial species found were Veillonella atypica, Fusobactrium nucelatum, Shaalia odontolytica, and Moraxella catarrhalis. Veillonella atypica and Fusbacteriumnucelatum dominated the microbiome in all 21 samples, attributing to more than 60% of all detected genetic material. Conclusions: since both Veillonella atypica and Fusobacterium nucleatum are, predominantly, oral cavity and dental microorganisms, our findings may suggest oral microbiome migration deeper into the oropharynx and nasopharynx where these bacteria colonize adenoid vegetations.

Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria.

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown "route I" nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.

Early host-microbe interaction in a peri-implant oral mucosa-biofilm model.

The host-microbe relationship is pivotal for oral health as well as for peri-implant diseases. Peri-implant mucosa and commensal biofilm play important roles in the maintenance of host-microbe homeostasis, but little is known about how they interact. We have therefore investigated the early host-microbe interaction between commensal multispecies biofilm (Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar, Porphyromonas gingivalis) and organotypic peri-implant mucosa using our three-dimensional model. After 24 hr, biofilms induced weak inflammatory reaction in the peri-implant mucosa by upregulation of five genes related to immune response and increased secretion of IL-6 and CCL20. Biofilm volume was reduced which might be explained by secretion of ß-Defensins-1, -2, and CCL20. The specific tissue reaction without intrinsic overreaction might contribute to intact mucosa. Thus, a relationship similar to homeostasis and oral health was established within the first 24 hr. In contrast, the mucosa was damaged and the bacterial distribution was altered after 48 hr. These were accompanied by an enhanced immune response with upregulation of additional inflammatory-related genes and increased cytokine secretion. Thus, the homeostasis-like relationship was disrupted. Such profound knowledge of the host-microbe interaction at the peri-implant site may provide the basis to improve strategies for prevention and therapy of peri-implant diseases.