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1.
Int Endod J ; 57(7): 966-980, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38387884

RESUMO

AIM: To compare the effect of different sodium hypochlorite (NaOCl) agitation techniques on an ex vivo oral multispecies biofilm during passive disinfection of simulated immature roots. METHODOLOGY: Extracted human teeth were prepared to simulate immature roots. They were infected with a dental plaque-derived multispecies biofilm and cultured for 14 days. The roots were randomly designated into four groups: (1) negative control (PBS), (2) 1.5% NaOCl (CNI), (3) CNI + Ultrasonic activation (UA), (4) CNI + EasyClean agitation (ECA), (5) CNI + XP-endo finisher agitation (XPF), and (6) positive control (6% NaOCl). Biofilm samples were collected from the root canals and used to determine the number of viable cells (colony-forming units), scanning electron microscopy, and 16S rRNA gene sequencing. The mean colony-forming units per mL (CFU/mL) were analysed using One-way anova. 16S rRNA sequencing data were analysed for alpha (observed OTUs, Shannon index, and Chao1) and beta diversity (Bray-Curtis dissimilarities). The LEfSe analysis was used to determine the effect of treatment procedures on the abundance of root canal microbiota. The significance was set at .05. RESULTS: PBS and CNI samples had significantly higher CFU/mL counts than UA, ECA, XPF, and 6% NaOCl samples (p < .05). The pre-treatment, PBS, and CNI groups had significantly greater alpha diversity than the UA, ECA, XPF, and 6% NaOCl groups (p < .05). NaOCl agitation groups and the 6% NaOCl group achieved a more pronounced reduction in bacteria from the genera Fusobacterium, Actinomyces, Porphyromonas, and Capnocytophaga. CONCLUSIONS: The effectiveness of passive disinfection protocols was enhanced by NaOCl agitation techniques, suggesting that this supplementary method can improve the outcome of revitalization procedures.


Assuntos
Biofilmes , Desinfecção , Hipoclorito de Sódio , Hipoclorito de Sódio/farmacologia , Biofilmes/efeitos dos fármacos , Humanos , Desinfecção/métodos , Irrigantes do Canal Radicular/farmacologia , Cavidade Pulpar/microbiologia , Microscopia Eletrônica de Varredura , RNA Ribossômico 16S , Técnicas In Vitro , Raiz Dentária/microbiologia , Raiz Dentária/efeitos dos fármacos
2.
Int Endod J ; 2023 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-36861850

RESUMO

AIM: To evaluate the root canal microbiome composition and bacterial functional capability in cases of primary and secondary apical periodontitis utilizing whole-metagenome shotgun sequencing. METHODOLOGY: Twenty-two samples from patients with primary root canal infections, and 18 samples obtained from previously treated teeth currently diagnosed with apical periodontitis were analysed with whole-metagenome shotgun sequencing at a depth of 20 M reads. Taxonomic and functional gene annotations were made using MetaPhlAn3 and HUMAnN3 software. The Shannon and Chao1 indices were utilized to measure alpha diversity. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarities. The Wilcoxon rank sum test was used to compare differences in taxa and functional genes. RESULTS: Microbial community variations within a community were significantly lower in secondary relative to primary infections (alpha diversity p = .001). Community composition was significantly different in primary versus secondary infection (R = .11, p = .005). The predominant taxa observed among samples (>2.5%) were Pseudopropionibacterium propionicum, Prevotella oris, Eubacterium infirmum, Tannerella forsythia, Atopobium rimae, Peptostreptococcus stomatis, Bacteroidetes bacterium oral taxon 272, Parvimonas micra, Olsenella profusa, Streptococcus anginosus, Lactobacillus rhamnosus, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, Fusobacterium nucleatum, Eubacterium brachy and Solobacterium moorei. The Wilcoxon rank test revealed no significant differences in relative abundances of functional genes in both groups. Genes with greater relative abundances (top 25) were associated with genetic, signalling and cellular processes including the iron and peptide/nickel transport system. Numerous genes encoding toxins were identified: exfoliative toxin, haemolysins, thiol-activated cytolysin, phospholipase C, cAMP factor, sialidase, and hyaluronic glucosaminidase. CONCLUSIONS: Despite taxonomic differences between primary and secondary apical periodontitis, the functional capability of the microbiomes was similar.

3.
Int Endod J ; 56(2): 278-288, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36334085

RESUMO

AIM: To evaluate the root canal microbiome composition in cases of primary and secondary apical periodontitis. METHODOLOGY: Thirty-nine samples from patients with primary root canal infections obtained before root canal treatment, and 40 samples obtained during root-end resection procedures from previously filled cases with apical periodontitis were evaluated using 16S rRNA next-generation sequencing analysis (NGS). Demographic and clinical factors included age, sex, infection type, percussion sensitivity, and presence of pain. Differences in abundances of genera were evaluated using Kruskal-Wallis test. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bonferroni correction for multiple comparisons. RESULTS: Significantly fewer operational taxonomic units values were observed from samples from secondary infections (p < .0001). While no significant differences were observed in the Chao 1 index between primary and secondary infections, the Shannon alpha diversity was significantly lower in secondary relative to primary infections (p = .008). Among samples, sex, age (adult vs. older adult), percussion sensitivity, and presence of pain all showed no significant effects on community composition via an analysis of similarity (ANOSIM). However, community composition was significantly different depending on whether the sample was from a primary or secondary infection (R = .051, p = .03). Nine microbial genera comprised the predominant taxa observed among samples (>3.3%) and included Parvimonas, Fusobacterium, Campylobacter, Arachnia, Eubacterium, Prevotella, Peptostreptococcus, Fretibacterirum, and Pseudoramibacter. Significantly greater relative abundances of Prevotella, Peptostreptococcus, Veillonella, Lactucaseibacillus, and Dialister were observed in primary infections. CONCLUSIONS: Primary endodontic infections are more diverse than secondary infections. The microbial composition is not associated with the clinical manifestations of apical periodontitis.


Assuntos
Coinfecção , Cavidade Pulpar , Periodontite Periapical , Idoso , Humanos , Bactérias , Cavidade Pulpar/microbiologia , Dor , Periodontite Periapical/microbiologia , RNA Ribossômico 16S/genética
4.
Int Endod J ; 2023 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-37983635

RESUMO

AIM: To analyse the effect of ultrasonic irrigant activation (UIA) and the GentleWave (GW) multisonic irrigation (GW) with minimal instrumentation on the root canal microbial diversity in an ex vivo model that used extracted molars with a history of pulp necrosis. METHODOLOGY: Twenty-three mandibular molars were prepared ex vivo for collection of superficial (surface control), pre-treatment and post-treatment samples 24 h after extraction. Samples were divided into two groups: UIA using 6% NaOCl (n = 11) and GW group (n = 12). All samples were processed using quantitative real-time polymerase chain reaction (qPCR) and 16S rRNA next-generation sequencing to measure microbial diversity before and after the antimicrobial treatment. For qPCR, a t-test (α = .05) was used to compare the log10 reduction. The Chao1 and Shannon indices evaluated alpha diversity. Differences in community composition (beta diversity) were evaluated by analysis of similarity (ANOSIM). Kruskal-Wallis test with Bonferroni corrections was performed to evaluate the differences in abundances genera in the samples. RESULTS: Quantitative real-time polymerase chain reaction revealed an estimated 1.6 and 2.6 log10 reduction for UIA and GW groups respectively (p = .048). An average of 5 ± 4 and 3 ± 5 operational taxonomic units (OTUs) were found in surface's samples in the UIA and GW group respectively. These values were significantly lower (p < .001) compared to the number of preoperative OTUs in those groups (155 ± 79 and 187 ± 121). In assessing beta diversity, there were no significant differences found in pre-treatment samples (R = .090, p = .070 ANOSIM with Bonferroni corrections). Also, no significant differences in community composition were observed in post-treatment samples (R = -.05, p = .829). After treatment, there was a significant reduction of Eubacterium using conventional treatment with UIA and a significant reduction of Prevotella using minimal instrumentation with GW irrigation (p = .007 and p = .002 respectively). CONCLUSION: Quantitative PCR analysis revealed a significant reduction in microbial load for GW group. Overall, diversity changes were similar between UIA and GW irrigation in this ex vivo model that used extracted teeth with a history of pulp necrosis. OTUs obtained from the surface sample were negligible and did not affect the statistical outcome of the study.

5.
J Bacteriol ; 204(6): e0011822, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35652671

RESUMO

Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.


Assuntos
Aderência Bacteriana , Streptococcus gordonii , Adesinas Bacterianas/metabolismo , Lipopolissacarídeos , Mucinas/metabolismo , Streptococcus gordonii/metabolismo , Ácidos Teicoicos/metabolismo
6.
Int Endod J ; 55(7): 772-783, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35383959

RESUMO

AIM: To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque-derived polymicrobial community. METHODOLOGY: Dental plaque-derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio-C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy-dispersive spectroscopy. Data normality was assessed using the Shapiro-Wilk test. One-way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two-way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray-Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05). RESULTS: The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity. CONCLUSION: Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.


Assuntos
Placa Dentária , Materiais Restauradores do Canal Radicular , Biofilmes , Compostos de Cálcio/química , Cavidade Pulpar , Resinas Epóxi/química , Humanos , Teste de Materiais , RNA Ribossômico 16S , Materiais Restauradores do Canal Radicular/química , Materiais Restauradores do Canal Radicular/farmacologia , Silicatos/química
7.
J Bacteriol ; 203(2)2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33106345

RESUMO

Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent.IMPORTANCES. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Cisteína Endopeptidases/metabolismo , Streptococcus gordonii/fisiologia , Aminoaciltransferases/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/química , Placa Dentária/microbiologia , Deleção de Genes , Hemaglutinação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Boca/microbiologia , Saliva/microbiologia , Ovinos/sangue , Streptococcus gordonii/genética , Streptococcus gordonii/crescimento & desenvolvimento
8.
Microb Ecol ; 78(2): 336-347, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30474730

RESUMO

Staphylococcus aureus, an opportunistic pathogen member of the nasal and skin microbiota, can also be found in human oral samples and has been linked to infectious diseases of the oral cavity. As the nasal and oral cavities are anatomically connected, it is currently unclear whether S. aureus can colonize the oral cavity and become part of the oral microbiota, or if its presence in the oral cavity is simply transient. To start addressing this question, we assessed S. aureus ability to directly bind selected members of the oral microbiota as well as its ability to integrate into a human-derived complex oral microbial community in vitro. Our data show that S. aureus forms aggregates with Fusobacterium nucleatum and Porphyromonas gingivalis and that it can incorporate into the human-derived in vitro oral community. Further analysis of the F. nucleatum-S. aureus interaction revealed that the outer-membrane adhesin RadD is partially involved in aggregate formation and that the RadD-mediated interaction leads to an increase in expression of the staphylococcal global regulator gene sarA. Our findings lend support to the notion that S. aureus can become part of the complex microbiota of the human mouth, which could serve as a reservoir for S. aureus. Furthermore, direct interaction with key members of the oral microbiota could affect S. aureus pathogenicity contributing to the development of several S. aureus associated oral infections.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fusobacterium nucleatum/metabolismo , Microbiota , Boca/microbiologia , Staphylococcus aureus/metabolismo , Transativadores/genética , Transativadores/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Biofilmes , Fusobacterium nucleatum/genética , Humanos , Ligação Proteica , Staphylococcus aureus/genética
9.
Antimicrob Agents Chemother ; 59(7): 3789-99, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25870061

RESUMO

CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/agonistas , Carbazóis/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Descoberta de Drogas , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Genes Reporter/genética , Haemophilus ducreyi/genética , Haemophilus ducreyi/metabolismo , Células Hep G2 , Humanos , Óperon Lac/genética , Testes de Sensibilidade Microbiana , Proteínas Quinases/genética , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta-Glucosidase/metabolismo
10.
bioRxiv ; 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38559107

RESUMO

N-acyl homoserine lactones (AHLs) are small diffusible signaling molecules that mediate a cell density-dependent bacterial communication system known as quorum sensing (QS). AHL-mediated QS regulates gene expression to control many critical bacterial behaviors including biofilm formation, pathogenicity, and antimicrobial resistance. Dental plaque is a complex multispecies oral biofilm formed by successive colonization of the tooth surface by groups of commensal, symbiotic, and pathogenic bacteria, which can contribute to tooth decay and periodontal diseases. While the existence and roles of AHL-mediated QS in oral microbiota have been debated, recent evidence indicates that AHLs play significant roles in oral biofilm development and community dysbiosis. The underlying mechanisms, however, remain poorly characterized. To better understand the importance of AHL signaling in dental plaque formation, we manipulated AHL signaling by adding AHL lactonases or exogenous AHL signaling molecules. We find that AHLs can be detected in dental plaque grown under 5% CO2 conditions, but not when grown under anaerobic conditions, and yet anaerobic cultures are still responsive to AHLs. QS signal disruption using lactonases leads to changes in microbial population structures in both planktonic and biofilm states, changes that are dependent on the substrate preference of the used lactonase but mainly result in the increase in the abundance of commensal and pioneer colonizer species. Remarkably, the opposite manipulation, that is the addition of exogenous AHLs increases the abundance of late colonizer bacterial species. Hence, this work highlights the importance of AHL-mediated QS in dental plaque communities, its potential different roles in anaerobic and aerobic parts of dental plaque, and underscores the potential of QS interference in the control of periodontal diseases.

11.
J Endod ; 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38815857

RESUMO

INTRODUCTION: To evaluate the antimicrobial activity of Triton irrigation versus 4% sodium hypochlorite (NaOCl) utilizing a direct contact test and an extracted tooth model. METHODS: In the first experiment, a direct contact test was conducted to compare bacterial DNA removal and microbial diversity changes following irrigation with 4% NaOCl or Triton. Hydroxyapatite and dentin discs were inoculated with subgingival human-derived dental plaque for 2 weeks utilizing the Center for Disease Control biofilm reactor and subsequently challenged with the root canal irrigants for 5 minutes. In the second experiment, teeth contaminated with a multispecies biofilm (n = 24) were assigned into two treatment groups, NaOCl or Triton irrigation. Samples were obtained for quantitative real-time polymerase chain reaction and next-generation sequencing analysis before and after instrumentation. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity and ANOSIM was used to measure beta diversity. Differences in abundances of genera were evaluated using Kruskal-Wallis test with Bonferroni corrections. RESULTS: The direct contact test revealed no significant differences in the bacterial load based on 16S rRNA gene molecules/µL, reads, or differences in the Shannon index among groups. In the extracted tooth model, a bacterial load reduction of log10 3.08 ± 0.69 and 2.76 ± 0.91 were found for NaOCl and Triton, respectively (P = .348). Next-generation sequencing showed fewer reads, lower Chao1, and beta diversity values when pretreatment and post-treatment samples were assessed in both experimental groups (P < .0001). The Kruskal-Wallis analysis found that 17 genera of bacteria were over-represented in minimal values in the Triton post-treatment group, 14 of these genera represented less than 1% of the microbial community. CONCLUSIONS: Both irrigants had limited antimicrobial activity in the direct contact test. When used in conjunction with mechanical instrumentation both irrigants were able to reduce the bacterial DNA load and diversity in comparison with pretreatment communities. The NaOCl irrigation, followed by ethylenediaminetetraacetic acid flush, was more effective in decreasing DNA counts from low-abundance organisms.

12.
J Biol Chem ; 287(38): 32147-60, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22829598

RESUMO

The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment.


Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Lisina/química , Proteínas de Membrana/genética , Fosfatos/química , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose/química , Íons , Modelos Químicos , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Fosforilação , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional
13.
Cell Rep ; 42(5): 112299, 2023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37080202

RESUMO

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during microgravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the microgravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.


Assuntos
Microbioma Gastrointestinal , Voo Espacial , Humanos , RNA Ribossômico 16S/genética , Cromatografia Líquida , Viagem , Espectrometria de Massas em Tandem
14.
Mol Microbiol ; 81(5): 1190-204, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21696463

RESUMO

In eukaryotes, lysine acetylation is a well-established post-translational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologues of the enzymes that catalyse protein acetylation are widely conserved and distributed among bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase subunits: ß, ß' and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Because the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression.


Assuntos
Acetiltransferases/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Proteínas de Membrana/biossíntese , Regiões Promotoras Genéticas , Sirtuínas/genética , Estresse Fisiológico/genética , Transcrição Gênica , Acetilcoenzima A/metabolismo , Acetilação , Acetiltransferases/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Lisina/química , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Sirtuínas/metabolismo
15.
Mol Microbiol ; 77(1): 15-21, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20487279

RESUMO

Protein acetylation has historically been considered a predominantly eukaryotic phenomenon. Recent evidence, however, supports the hypothesis that acetylation broadly impacts bacterial physiology. To explore more rapidly the impact of protein acetylation in bacteria, microbiologists can benefit from the strong foundation established by investigators of protein acetylation in eukaryotes. To help advance this learning process, we will summarize the current understanding of protein acetylation in eukaryotes, discuss the emerging link between acetylation and metabolism and highlight the best-studied examples of protein acetylation in bacteria.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Eucariotos/metabolismo
16.
mSphere ; 4(6)2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31801844

RESUMO

Lipoteichoic acid (LTA) is an abundant polymer of the Gram-positive bacterial cell envelope and is essential for many species. Whereas the exact function of LTA has not been elucidated, loss of LTA in some species affects hydrophobicity, biofilm formation, and cell division. Using a viable LTA-deficient strain of the human oral commensal Streptococcus gordonii, we demonstrated that LTA plays an important role in surface protein presentation. Cell wall fractions derived from the wild-type and LTA-deficient strains of S. gordonii were analyzed using label-free mass spectroscopy. Comparisons showed that the abundances of many proteins differed, including (i) SspA, SspB, and S. gordonii 0707 (SGO_0707) (biofilm formation); (ii) FtsE (cell division); (iii) Pbp1a and Pbp2a (cell wall biosynthesis and remodeling); and (iv) DegP (envelope stress response). These changes in cell surface protein presentation appear to explain our observations of altered cell envelope homeostasis, biofilm formation, and adhesion to eukaryotic cells, without affecting binding and coaggregation with other bacterial species, and provide insight into the phenotypes revealed by the loss of LTA in other species of Gram-positive bacteria. We also characterized the chemical structure of the LTA expressed by S. gordonii Similarly to Streptococcus suis, S. gordonii produced a complex type I LTA, decorated with multiple d-alanylations and glycosylations. Hence, the S. gordonii LTA appears to orchestrate expression and presentation of cell surface-associated proteins and functions.IMPORTANCE Discovered over a half-century ago, lipoteichoic acid (LTA) is an abundant polymer found on the surface of Gram-positive bacteria. Although LTA is essential for the survival of many Gram-positive species, knowledge of how LTA contributes to bacterial physiology has remained elusive. Recently, LTA-deficient strains have been generated in some Gram-positive species, including the human oral commensal Streptococcus gordonii The significance of our research is that we utilized an LTA-deficient strain of S. gordonii to address why LTA is physiologically important to Gram-positive bacteria. We demonstrate that in S. gordonii, LTA plays an important role in the presentation of many cell surface-associated proteins, contributing to cell envelope homeostasis, cell-to-cell interactions in biofilms, and adhesion to eukaryotic cells. These data may broadly reflect a physiological role of LTA in Gram-positive bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana/metabolismo , Streptococcus gordonii/metabolismo , Ácidos Teicoicos/metabolismo , Parede Celular/química , Lipopolissacarídeos/deficiência , Espectrometria de Massas
17.
Sci Signal ; 12(580)2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064885

RESUMO

Bacterial adhesins mediate adhesion to substrates and biofilm formation. Adhesins of the LPXTG family are posttranslationally processed by the cell membrane-localized peptidase sortase A, which cleaves the LPXTG motif. This generates a short C-terminal peptide (C-pep) that remains in the cell membrane, whereas the mature adhesin is incorporated into the cell wall. Genes encoding adhesins of the oral bacterium Streptococcus gordonii were differentially expressed depending on whether the bacteria were isolated from saliva or dental plaque and appeared to be coordinately regulated. Deletion of sspA and sspB (sspAB), both of which encode LPXTG-containing adhesins, unexpectedly enhanced adhesion and biofilm formation. C-peps produced from a model LPXTG-containing adhesin localized to the cell membrane and bound to and inhibited the intramembrane sensor histidine kinase SGO_1180, thus preventing activation of the cognate response regulator SGO_1181. The absence of SspAB C-peps induced the expression of the scaCBA operon encoding the lipoprotein adhesin ScaA, which was sufficient to preserve and even enhance biofilm formation. This C-pep-driven regulatory circuit also exists in pathogenic streptococci and is likely conserved among Gram-positive bacteria. This quality control mechanism ensures that the bacteria can form biofilms under diverse environmental conditions and may play a role in optimizing adhesion and biofilm formation.


Assuntos
Adesinas Bacterianas/metabolismo , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Streptococcus gordonii/metabolismo , Adesinas Bacterianas/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Biofilmes , Cisteína Endopeptidases/genética , Placa Dentária/microbiologia , Regulação Bacteriana da Expressão Gênica , Mutação , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Saliva/microbiologia , Homologia de Sequência de Aminoácidos , Streptococcus gordonii/genética , Streptococcus gordonii/fisiologia
18.
J Bacteriol ; 190(7): 2314-22, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18223085

RESUMO

The CpxAR two-component signal transduction system in Escherichia coli and other pathogens senses diverse envelope stresses and promotes the transcription of a variety of genes that remedy these stresses. An important member of the CpxAR regulon is cpxP. The CpxA-dependent transcription of cpxP has been linked to stresses such as misfolded proteins and alkaline pH. It also has been proposed that acetyl phosphate, the intermediate of the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway, can activate the transcription of cpxP in a CpxA-independent manner by donating its phosphoryl group to CpxR. We tested this hypothesis by measuring the transcription of cpxP using mutants with mutations in the CpxAR pathway, mutants with mutations in the Pta-AckA pathway, and mutants with a combination of both types of mutations. From this epistasis analysis, we learned that CpxR integrates diverse stimuli. The stimuli that originate in the envelope depend on CpxA, while those associated with growth and central metabolism depend on the Pta-AckA pathway. While CpxR could receive a phosphoryl group from acetyl phosphate, this global signal was not the primary trigger for CpxR activation associated with the Pta-AckA pathway. On the strength of these results, we contend that the interactions between central metabolism and signal transduction can be quite complex and that successful investigations of such interactions must include a complete epistatic analysis.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Transdução de Sinais/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Modelos Biológicos , Mutação , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Regulon/genética , Transdução de Sinais/fisiologia , Transcrição Gênica
19.
Microbiologyopen ; 6(3)2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28173636

RESUMO

To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Fusobacterium nucleatum/fisiologia , Interações Microbianas , Streptococcus gordonii/fisiologia , Adesinas Bacterianas/genética , Fusobacterium nucleatum/metabolismo
20.
FEMS Microbiol Lett ; 363(5): fnw011, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26790713

RESUMO

The central metabolite acetyl phosphate (acP) has long been proposed to influence transcription regulation by directly transferring its phosphoryl group to a number of response regulators in many bacterial species. Here, we provide in vitro evidence for this proposition and demonstrate, using an in vitro transcription system, that acP-dependent phosphorylation of aspartate 51 of CpxR induces transcription of one of its regulon members in E. coli, cpxP. We also used this in vitro transcription system to extend our previously reported in vivo data that hypothesized that acetylation of RNA polymerase (RNAP) influences acP-dependent cpxP transcription, using glutamine as a genetic mimic for acetylated arginine 291 of the carboxy-terminal domain of RNAP α subunit. The data we present here lend strong support to the hypothesis that acP has a direct effect on transcription regulation in E. coli via phosphorylation of CpxR, and that RNAP acetylation can modulate this response.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/biossíntese , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas de Membrana/biossíntese , Organofosfatos/química , Acetilação , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional , Regulon
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