RESUMEN
BACKGROUND: Acute cerebellitis (AC) in children and adolescents is an inflammatory disease of the cerebellum due to viral or bacterial infections but also autoimmune-mediated processes. OBJECTIVE: To investigate the frequency of autoantibodies in serum and CSF as well as the neuroradiological features in children with AC. MATERIAL AND METHODS: Children presenting with symptoms suggestive of AC defined as acute/subacute onset of cerebellar symptoms and MRI evidence of cerebellar inflammation or additional CSF pleocytosis, positive oligoclonal bands (OCBs), and/or presence of autoantibodies in case of negative cerebellar MRI. Children fulfilling the above-mentioned criteria and a complete data set including clinical presentation, CSF studies, testing for neuronal/cerebellar and MOG antibodies as well as MRI scans performed at disease onset were eligible for this retrospective multicenter study. RESULTS: 36 patients fulfilled the inclusion criteria for AC (f:m = 14:22, median age 5.5 years). Ataxia was the most common cerebellar symptom present in 30/36 (83 %) in addition to dysmetria (15/36) or dysarthria (13/36). A substantial number of children (21/36) also had signs of encephalitis such as somnolence or seizures. In 10/36 (28 %) children the following autoantibodies (abs) were found: MOG-abs (n = 5) in serum, GFAPα-abs (n = 1) in CSF, GlyR-abs (n = 1) in CSF, mGluR1-abs (n = 1) in CSF and serum. In two further children, antibodies were detected only in serum (GlyR-abs, n = 1; GFAPα-abs, n = 1). MRI signal alterations in cerebellum were found in 30/36 children (83 %). Additional supra- and/or infratentorial lesions were present in 12/36 children, including all five children with MOG-abs. Outcome after a median follow-up of 3 months (range: 1 a 75) was favorable with an mRS ≤2 in 24/36 (67 %) after therapy. Antibody (ab)-positive children were significantly more likely to have a better outcome than ab-negative children (p = .022). CONCLUSION: In nearly 30 % of children in our study with AC, a range of abs was found, underscoring that autoantibody testing in serum and CSF should be included in the work-up of a child with suspected AC. The detection of MOG-abs in AC does expand the MOGAD spectrum.
Asunto(s)
Autoanticuerpos , Encefalitis , Adolescente , Niño , Preescolar , Humanos , Ataxia , Cerebelo/diagnóstico por imagen , Encefalitis/diagnóstico por imagen , Inflamación , Estudios RetrospectivosRESUMEN
Dental composites are routinely placed as part of tooth restoration procedures. The integrity of the restoration is constantly challenged by the metabolic activities of the oral microbiome. This activity directly contributes to a less-than-desirable half-life for the dental composite formulations currently in use. Therefore, many new antimicrobial dental composites are being developed to counteract the microbial challenge. To ensure that these materials will resist microbiome-derived degradation, the model systems used for testing antimicrobial activities should be relevant to the in vivo environment. Here, we summarize the key steps in oral microbial colonization that should be considered in clinically relevant model systems. Oral microbial colonization is a clearly defined developmental process that starts with the formation of the acquired salivary pellicle on the tooth surface, a conditioned film that provides the critical attachment sites for the initial colonizers. Further development includes the integration of additional species and the formation of a diverse, polymicrobial mature biofilm. Biofilm development is discussed in the context of dental composites, and recent research is highlighted regarding the effect of antimicrobial composites on the composition of the oral microbiome. Future challenges are addressed, including the potential of antimicrobial resistance development and how this could be counteracted by detailed studies of microbiome composition and gene expression on dental composites. Ultimately, progress in this area will require interdisciplinary approaches to effectively mitigate the inevitable challenges that arise as new experimental bioactive composites are evaluated for potential clinical efficacy. Success in this area could have the added benefit of inspiring other fields in medically relevant materials research, since microbial colonization of medical implants and devices is a ubiquitous problem in the field.
Asunto(s)
Antiinfecciosos , Microbiota , Boca , Biopelículas , Resinas Compuestas , Película Dental , Humanos , Boca/microbiología , Streptococcus mutansRESUMEN
The pyruvate oxidase (SpxB)-dependent production of H2O2 is widely distributed among oral commensal streptococci. Several studies confirmed the ability of H2O2 to antagonize susceptible oral bacterial species, including caries-associated Streptococcus mutans as well as several periodontal pathobionts. Here we report a potential mechanism to bolster oral commensal streptococcal H2O2 production by magnesium (Mg2+) supplementation. Magnesium is a cofactor for SpxB catalytic activity, and supplementation increases the production of H2O2 in vitro. We demonstrate that Mg2+ affects spxB transcription and SpxB abundance in Streptococcus sanguinis and Streptococcus gordonii. The competitiveness of low-passage commensal streptococcal clinical isolates is positively influenced in antagonism assays against S. mutans. In growth conditions normally selective for S. mutans, Mg2+ supplementation is able to increase the abundance of S. sanguinis in dual-species biofilms. Using an in vivo biophotonic imaging platform, we further demonstrate that dietary Mg2+ supplementation significantly improves S. gordonii oral colonization in mice. In summary, our results support a role for Mg2+ supplementation as a potential prebiotic to promote establishment of oral health-associated commensal streptococci.
Asunto(s)
Boca , Animales , Biopelículas , Peróxido de Hidrógeno , Magnesio , Ratones , Streptococcus gordonii , Streptococcus mutans , Streptococcus sanguisRESUMEN
During dental caries, the dental biofilm modifies the composition of the hundreds of involved bacterial species. Changing environmental conditions influence competition. A pertinent model to exemplify the complex interplay of the microorganisms in the human dental biofilm is the competition between Streptococcus sanguinis and Streptococcus mutans. It has been reported that children and adults harbor greater numbers of S. sanguinis in the oral cavity, associated with caries-free teeth. Conversely, S. mutans is predominant in individuals with a high number of carious lesions. Competition between both microorganisms stems from the production of H2 O2 by S. sanguinis and mutacins, a type of bacteriocins, by S. mutans. There is limited evidence on how S. sanguinis survives its own H2 O2 levels, or if it has other mechanisms that might aid in the competition against S. mutans, nonetheless. We performed a genomic and metabolic pathway comparison, coupled with a comprehensive literature review, to better understand the competition between these two species. Results indicated that S. sanguinis can outcompete S. mutans by the production of an enzyme capable of metabolizing H2 O2 . S. mutans, however, lacks the enzyme and is susceptible to the peroxide from S. sanguinis. In addition, S. sanguinis can generate energy through gluconeogenesis and seems to have evolved different communication mechanisms, indicating that novel proteins may be responsible for intra-species communication.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Placa Dental/microbiología , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Streptococcus sanguis/genética , Streptococcus sanguis/metabolismo , Bacteriocinas/metabolismo , Simulación por Computador , Bases de Datos Genéticas , Caries Dental/microbiología , Genes Bacterianos/genética , Gluconeogénesis , Humanos , Peróxido de Hidrógeno/metabolismo , Redes y Vías Metabólicas , Boca/microbiologíaRESUMEN
BACKGROUND: Immune cross-reactivity between malignant and normal tissues causes the rare, so called paraneoplastic syndrome (PS). In approximately 60% of the patients, various onconeural antibodies are detectable in the cerebrospinal fluid (CSF) and are associated with typical tumour entities. METHODS: We report an unusual case of paraneoplastic limbic encephalitis (PLE) in a 17-year-old adolescent with classical Hodgkin lymphoma. RESULTS: He presented with a variety of neurologic and neuropsychiatric symptoms, profound B-symptoms and typical MRI findings including hyperintense lesions with contrast enhancement in the medial temporal lobe and limbic system. Under immunosuppressive therapy and subsequently chemotherapy the neurological situation only temporarily improved and worsened again after interruption of immunosuppression several times. Thus, multiple courses of multidrug immunosuppressive therapy were administered. To date, five years after initial presentation, the young man is able to walk with walking aids and orthoses and is still on oral prednisolone therapy. Analyses of the CSF and serum revealed anti SOX-1 antibodies at initial presentation but PCA-2 antibodies seven months after diagnosis. CONCLUSION: Neurologic and/or neuropsychiatric symptoms combined with typical MRI findings should raise the suspicion of PS and lead to further diagnostics for an underlying tumour even in children.
Asunto(s)
Enfermedad de Hodgkin/complicaciones , Encefalitis Límbica/etiología , Síndromes Paraneoplásicos/etiología , Síndromes Paraneoplásicos/inmunología , Adolescente , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Humanos , Encefalitis Límbica/inmunología , Imagen por Resonancia Magnética , Masculino , Factores de Transcripción SOXB1/inmunologíaRESUMEN
The 5' untranslated region (5' UTR) of an mRNA molecule embeds important determinants that modify its stability and translation efficiency. In Streptococcus pyogenes, a strict human pathogen, a gene encoding a secreted protease (speB) has a large 5' UTR with unknown functions. Here we describe that a partial deletion of the speB 5' UTR caused a general accumulation of mRNA in the stationary phase, and that the mRNA accumulation was due to retarded mRNA degradation. The phenotype was observed in several M serotypes harboring the partial deletion of the speB 5' UTR. The phenotype was triggered by the production of the truncated speB 5' UTR, but not by the disruption of the intact speB 5' UTR. RNase Y, a major endoribonuclease, was previously shown to play a central role in bulk mRNA turnover in stationary phase. However, in contrast to our expectations, we observed a weaker interaction between the truncated speB 5' UTR and RNase Y compared with the wild-type, which suggests that other unidentified RNA degrading components are required for the pleiotropic effects observed from the speB UTR truncation. Our study demonstrates how S. pyogenes uses distinct mRNA degradation schemes in exponential and stationary growth phases.
Asunto(s)
Regiones no Traducidas 5'/genética , Proteínas Bacterianas/genética , Exotoxinas/genética , Estabilidad del ARN , Streptococcus pyogenes/genética , Proteínas Bacterianas/química , Cisteína Endopeptidasas/metabolismo , Exotoxinas/química , Exotoxinas/metabolismo , Regulación Bacteriana de la Expresión Génica , Fenotipo , Procesamiento Postranscripcional del ARN , Eliminación de Secuencia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidadRESUMEN
The commensal oral microbial flora has evolved with the human host to support colonization of the various intraoral sites without triggering a significant immune response. In exchange, the commensal microbes provide critical protection against invading pathogens. The intrinsic ability of the oral flora to create a symbiotic microbial community with the host can be disturbed, selecting for the overgrowth of a dysbiotic community that can result in dental diseases, such as caries and periodontitis. Although the mechanisms of molecular pathogenesis in oral diseases are well characterized, much less is known about the molecular mechanisms used by the commensal flora to maintain oral health. Here we focus on the commensal species Streptococcus sanguinis, which is found in abundance in the early oral biofilm and is strongly correlated with oral health. Streptococcus sanguinis exhibits a variety of features that make it ideally suited as a model organism to explore the molecular basis for commensalism. As such, this review will describe our current mechanistic understanding of S. sanguinis commensalism and speculate upon its molecular traits that may be exploitable to maintain or restore oral health under conditions that would otherwise lead to disease.
Asunto(s)
Boca/microbiología , Streptococcus sanguis/genética , Streptococcus sanguis/fisiología , Simbiosis , Biopelículas/crecimiento & desarrollo , Caries Dental , Humanos , Periodontitis , Streptococcus mutans/fisiología , Streptococcus sanguis/crecimiento & desarrolloRESUMEN
Salivary agglutination is an important host defense mechanism to aggregate oral commensal bacteria as well as invading pathogens. Saliva flow and subsequent swallowing more easily clear aggregated bacteria compared with single cells. Phagocytic clearance of bacteria through polymorphonuclear neutrophil granulocytes also seems to increase to a certain extent with the size of bacterial aggregates. To determine a connection between salivary agglutination and the host innate immune response by phagocytosis, an in vitro agglutination assay was developed reproducing the average size of salivary bacterial aggregates. Using the oral commensal Streptococcus gordonii as a model organism, the effect of salivary agglutination on phagocytic clearance through polymorphonuclear neutrophil granulocytes was investigated. Here we describe how salivary aggregates of S. gordonii are readily cleared through phagocytosis, whereas single bacterial cells showed a significant delay in being phagocytosed and killed. Furthermore, before phagocytosis the polymorphonuclear neutrophil granulocytes were able to induce a specific de-aggregation, which was dependent on serine protease activity. The data presented suggest that salivary agglutination of bacterial cells leads to an ideal size for recognition by polymorphonuclear neutrophil granulocytes. As a first line of defense, these phagocytic cells are able to recognize the aggregates and de-aggregate them via serine proteases to a more manageable size for efficient phagocytosis and subsequent killing in the phagolysosome. This observed mechanism not only prevents the rapid spreading of oral bacterial cells while entering the bloodstream but would also avoid degranulation of involved polymorphonuclear neutrophil granulocytes, so preventing collateral damage to nearby tissue.
Asunto(s)
Aglutinación , Boca/microbiología , Neutrófilos/inmunología , Saliva/fisiología , Streptococcus gordonii/inmunología , Humanos , Inmunidad Innata , Boca/inmunología , Fagocitosis , Serina Proteasas/metabolismo , Streptococcus gordonii/fisiologíaRESUMEN
Streptococcus gordonii is an important member of the oral biofilm community. As an oral commensal streptococcus, S. gordonii is considered beneficial in promoting biofilm homeostasis. CcpA is known as the central regulator of carbon catabolite repression in Gram-positive bacteria and is also involved in the control of virulence gene expression. To further establish the role of CcpA as central regulator in S. gordonii, the effect of CcpA on biofilm formation and natural competence of S. gordonii was investigated. These phenotypic traits have been suggested to be important to oral streptococci in coping with environmental stress. Here we demonstrate that a CcpA mutant was severely impaired in its biofilm-forming ability, showed a defect in extracellular polysaccharide production and reduced competence. The data suggest that CcpA is involved in the regulation of biofilm formation and competence development in S. gordonii.
Asunto(s)
Proteínas Bacterianas/fisiología , Biopelículas/crecimiento & desarrollo , Competencia de la Transformación por ADN/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Represoras/fisiología , Streptococcus gordonii/metabolismo , Adaptación Fisiológica/genética , Adhesinas Bacterianas/biosíntesis , Adhesinas Bacterianas/genética , Proteínas Bacterianas/genética , Eliminación de Gen , Resistencia a la Kanamicina/genética , Polisacáridos Bacterianos/biosíntesis , Proteínas Represoras/genética , Estrés Fisiológico , Factores de Virulencia/biosíntesisRESUMEN
OBJECTIVE: As shown in the quantitative suspension test adding lactoperoxidase to a thiocyanate (SCN(-)) hydrogen peroxide (H(2)O(2)) combination over the physiological saliva level has significant positive antimicrobial effects to a level of totally killing Streptococcus mutans, Streptococcus sanguinis, and Candida albicans. The aim of this study was to evaluate this positive effect under human saliva loading. METHODS: The bactericidal and fungicidal effect of lactoperoxidase was evaluated in a quantitative suspension test by using two test mixtures of a 2.0% thiocyanate and 1.2% hydrogen peroxide solution, one without (Group A) and one with (Group B) lactoperoxidase under saliva loading. Following the quantitative suspension tests (EN-13727/EN-13624), the growth of surviving bacteria and fungi in a nutrient broth was measured. The exposure times were restricted to 1, 3, 5, and 15 min. All statistical analyses were carried out with SPSS 11.5. RESULTS: In the quantitative suspension test, the combination of thiocyanate and hydrogen peroxide showed relatively low antimicrobial effectiveness on S. mutans, S. sanguinis, and C. albicans in the presence of human saliva at measured time points in comparison to the mixture with lactoperoxidase, which showed a high bactericidal activity within 15 min (S. mutans and S. sanguinis) and fungicidal activity within 3 min (C. albicans). CONCLUSION: The antimicrobial effectiveness of the tested thiocyanate hydrogen peroxide combination was increased significantly by adding lactoperoxidase in the quantitative suspension test under human saliva loading.
Asunto(s)
Antiinfecciosos Locales/farmacología , Candida albicans/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Lactoperoxidasa/farmacología , Saliva/microbiología , Streptococcus mutans/efectos de los fármacos , Streptococcus sanguis/efectos de los fármacos , Tiocianatos/farmacología , Candida albicans/crecimiento & desarrollo , Humanos , Estadísticas no Paramétricas , Streptococcus mutans/crecimiento & desarrollo , Streptococcus sanguis/crecimiento & desarrollo , Suspensiones/farmacologíaRESUMEN
Streptococcus sanguinis is an oral commensal bacterium and endogenous pathogen in the blood, which is generally naturally competent to take up extracellular DNA. Regarded as a stress response, competence development enables S. sanguinis to acquire new genetic material. The sequenced reference strain SK36 encodes and expresses the genes required for competence (com) and uptake of DNA. Isolated from blood cultures of a confirmed case of infective endocarditis, strain 133-79 encodes all necessary com genes but is not transformable under conditions permissive for competence development in SK36. Using synthetic competence-stimulating peptides (sCSP) based on sequences of SK36 and 133-79 comC, both strains developed competence at similar frequencies in cross-transformation experiments. Furthermore, downstream response pathways are similar in strains SK36 and 133-79 because platelet aggregation and biofilm formation appeared unaffected by CSP. Collectively, the data indicate that strains SK36 and 133-79 respond to CSP similarly, strongly suggesting that endogenous production or release of CSP from 133-79 is impaired.
Asunto(s)
Proteínas Bacterianas/genética , Biopelículas , Endocarditis Bacteriana/microbiología , Regulación Bacteriana de la Expresión Génica/genética , Infecciones Estreptocócicas/microbiología , Streptococcus sanguis/fisiología , Carga Bacteriana , Proteínas Bacterianas/farmacología , Técnicas Bacteriológicas , Biopelículas/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/genética , Humanos , Fenotipo , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Streptococcus sanguis/genética , Streptococcus sanguis/crecimiento & desarrollo , Factores de Transcripción/genética , Transcripción Genética/genética , Activación Transcripcional/genética , Transformación Bacteriana/efectos de los fármacos , Transformación Bacteriana/genéticaRESUMEN
Methionine sulphoxide reductase maintains adhesin function during oxidative stress. Using Streptococcus gordonii as a model, we now show the mechanistic basis of adhesin maintenance provided by MsrA. In biofilms, S. gordonii selectively expresses the msrA gene. When the wild-type strain was grown with exogenous hydrogen peroxide (H(2)O(2)), msrA-specific mRNA expression significantly increased, while acid production was unaffected. In the presence of H(2)O(2), a msrA-deletion mutant (ΔMsrA) showed a 6 h delay in lag phase growth, a 30% lower yield of H(2)O(2), significantly greater inhibition by H(2)O(2) on agar plates (reversed by complementation), 30% less adhesion to saliva-coated hydroxyapatite, 87% less biofilm formation and an altered electrophoretic pattern of SspAB protein adhesins. Using mass spectrometry, methionine residues in the Met-rich central region of SspB were shown to be oxidized by H(2)O(2) and reduced by MsrA. In intact wild-type cells, MsrA colocalized with a cell wall-staining dye, and MsrA was detected in both cell wall and cytosolic fractions. To maintain normal adhesion and biofilm function of S. gordonii in response to exogenous oxidants therefore msrA is upregulated, methionine oxidation of adhesins and perhaps other proteins is reversed, and adhesion and biofilm formation is maintained.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Metionina Sulfóxido Reductasas/metabolismo , Streptococcus gordonii/enzimología , Streptococcus gordonii/fisiología , Biopelículas/crecimiento & desarrollo , Pared Celular/enzimología , Citoplasma/enzimología , Eliminación de Gen , Prueba de Complementación Genética , Peróxido de Hidrógeno/toxicidad , Metionina Sulfóxido Reductasas/genética , Streptococcus gordonii/crecimiento & desarrolloRESUMEN
Pulpal and periradicular diseases are primarily caused by bacterial invasion of the root canal system as a result of caries progression. The presence of residual bacteria at the time of root canal completion (obturation) is associated with significantly higher rate of treatment failure. Re-infection of obturated root canals can be potentially prevented by enhancing the antibacterial activities of root canal obturation materials. We evaluated, in an in vitro model, the antimicrobial efficacy of silver ions added to a common endodontic sealer. For that purpose we performed growth inhibition studies and bacterial viability tests. We measured the zone of inhibition, optical density and performed confocal laser scanning microscopy. Our results show that the silver ions enhance the antimicrobial activity of the root canal sealer against Streptococcus mutans. This study approach may hold promise for studying other biologically based therapies and therefore increasing the success rate of routine orthograde root canal treatment.
RESUMEN
INTRODUCTION: Dental caries has been closely linked to fermentable carbohydrates as key environmental factors. Sucrose has been identified as the most cariogenic carbohydrate. Streptococcus mutans, considered to be the primary pathogen causing dental caries, is able to utilize sucrose as a nutrient source, partially for the production of intracellular storage components and for the production of extracellular glucans via the glucosyltransferases GtfB, GtfC, and GtfD. The following study explores the competitiveness and fitness of S. mutans when grown with different concentrations of sucrose. METHODS: Growth competition with oral streptococci and antimicrobial susceptibility in static biofilm models grown without sucrose or with 0.1% or 0.5% sucrose were investigated using confocal laser scanning microscopy. The numbers of surviving S. mutans of both wild-type and an isogenic Gtf-negative mutant after antimicrobial treatment were determined as colony-forming units. RESULTS: S. mutans was able to establish microcolonies with increasing sucrose concentration in the presence of other streptococcal competitors during biofilm development. The antimicrobial susceptibility decreased when sucrose was available as substrate and was dependent on the presence of the Gtfs. CONCLUSION: The increased resistance against antimicrobial treatment was associated with the availability of sucrose, but was not influenced much by the concentration used during this study. The resistance was strongly associated with the Gtf activity, excluding any intracellular metabolic effect of sucrose in the resistance mechanism.
Asunto(s)
Cariogénicos/farmacología , Streptococcus mutans/crecimiento & desarrollo , Sacarosa/farmacología , Antiinfecciosos Locales/administración & dosificación , Antiinfecciosos Locales/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Cariogénicos/administración & dosificación , Clorhexidina/administración & dosificación , Clorhexidina/análogos & derivados , Clorhexidina/farmacología , Recuento de Colonia Microbiana , Combinación de Medicamentos , Glucosiltransferasas/genética , Humanos , Metaloproteínas/administración & dosificación , Metaloproteínas/farmacología , Pruebas de Sensibilidad Microbiana , Microscopía Confocal , Boca/microbiología , Mutación/genética , Salicilatos/administración & dosificación , Salicilatos/farmacología , Streptococcus gordonii/efectos de los fármacos , Streptococcus gordonii/crecimiento & desarrollo , Streptococcus mitis/efectos de los fármacos , Streptococcus mitis/crecimiento & desarrollo , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/enzimología , Streptococcus oralis/efectos de los fármacos , Streptococcus oralis/crecimiento & desarrollo , Streptococcus sobrinus/efectos de los fármacos , Streptococcus sobrinus/crecimiento & desarrollo , Sacarosa/administración & dosificación , Terpenos/administración & dosificación , Terpenos/farmacologíaRESUMEN
Streptococcus mutans is considered one of the main causative agents of human dental caries. Cell-cell communication through two-component signal transduction systems (TCSTS) plays an important role in the pathogenesis of S. mutans. One of the S. mutans TCSTS, ComDE, controls both competence development and biofilm formation. In this study, we showed that addition of exogenous competence-stimulating peptide (CSP) beyond the levels necessary for competence inhibited the growth of S. mutans in a ComDE-dependent manner. We also demonstrated that further increases of CSP stopped S. mutans cell division leading to cell death. Use of CSP as a possible therapeutic agent is discussed.
Asunto(s)
Feromonas/farmacología , Streptococcus mutans/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Biopelículas , Muerte Celular , Péptidos , Streptococcus mutans/metabolismo , Streptococcus mutans/fisiología , Transformación BacterianaRESUMEN
Streptococcus mutans is implicated as the primary pathogen involved in the development of dental caries. The production of specific bacteriocins (called mutacins) by S. mutans is one of the major virulence factors which facilitate the dominance of the bacterium within dental plaque. While much has been revealed about the biochemical structures of mutacins, little is known about the expression and regulation of mutacin genes, largely due to the lack of proper methods to monitor mutacin gene expression, especially under biofilm conditions. In this study, a set of reporter systems with the green fluorescent protein (gfp), the monomeric red fluorescent protein (mrfp1), and the glucuronidase (gusA) are introduced to S. mutans to study the transcriptional activities of the mutacin I gene (mutA). Although the mutA-reporter fusions are in single copy on the chromosome, these reporter systems display strong signals that allow us to effectively monitor mutA gene expression in S. mutans. Using these reporter systems, we show that mutA is expressed in both planktonic and biofilm cells, even though mutacin activities are normally detected only in biofilm cells. Furthermore, we confirm that mutR, the gene upstream of the mutacin operon, is required for mutacin I gene expression. The success of this study validates the feasibility of using these reporter systems to study gene expression and regulation in S. mutans.
Asunto(s)
Bacteriocinas/genética , Genes Reporteros , Streptococcus mutans/genética , Transcripción Genética , Fusión Artificial Génica , Plásmidos de Bacteriocinas , Bacteriocinas/metabolismo , Biopelículas , Estudios de Factibilidad , Fluorescencia , Perfilación de la Expresión Génica , Glucuronidasa/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Mutagénesis , Plancton/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Streptococcus mutans/patogenicidad , Virulencia , Proteína Fluorescente RojaRESUMEN
Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.
RESUMEN
The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.