RESUMEN
Objective: To study the preventive effect of Lactobacillus helveticus (L. helveticus) on periodontitis induced by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in rats. Methods: Eighteen 8-week-old female rats were randomly divided into three groups: Sham group, Trehalose group, and L. helveticus SBT2171 (LH2171) group. We measured the distance of the cementoenamel junction-alveolar bone crest (CEJ-ABC) to evaluate alveolar bone resorption. Hematoxylin-eosin staining was used to observe the histopathological changes of rat hemimaxillary tissues. We detected the expression of ß-defensins, tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1ß, and IL-6 and the number of A. actinomycetemcomitans in rat gingival tissues by quantitative reverse transcriptase polymerase chain reaction. The levels of IL-1ß, IL-6, and TNF-α in rat gingival tissues were also measured by enzyme-linked immunosorbent assay. Results: Compared with the Trehalose group, the distance of CEJ-ABC was prominently reduced and alveolar bone resorption was notably improved in the LH2171 group. And the infiltration of inflammatory cells in the hemimaxillary tissue decreased obviously, periodontal fibers were arranged neatly, connective tissue small blood vessels proliferated, and the number of A. actinomycetemcomitans reduced significantly in the LH2171 group. In addition, the mRNA expression and release of inflammatory factors in the gingival tissues in the LH2171 group were notably lower than those in the Trehalose group. On the 21st and 36th day, the expression of ß-defensins in the gingival tissue of the LH2171 group increased significantly. Conclusion: L. helveticus improves alveolar bone resorption and increases the expression of ß-defensins thereby inhibiting the number of A. actinomycetemcomitans and thus prevents periodontitis.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Lactobacillus helveticus/fisiología , Periodontitis/prevención & control , beta-Defensinas/fisiología , Pérdida de Hueso Alveolar/etiología , Pérdida de Hueso Alveolar/patología , Pérdida de Hueso Alveolar/prevención & control , Animales , Biología Computacional , Modelos Animales de Enfermedad , Femenino , Encía/microbiología , Mediadores de Inflamación/metabolismo , Periodontitis/microbiología , Periodontitis/fisiopatología , Probióticos/farmacología , Ratas , Ratas Sprague-Dawley , Trehalosa/farmacologíaRESUMEN
OBJECTIVES: We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model. METHODS: We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans. KEY FINDINGS: EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects. CONCLUSIONS: Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.
Asunto(s)
Aggregatibacter actinomycetemcomitans , Catequina/análogos & derivados , Exotoxinas , Periodontitis , Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Aggregatibacter actinomycetemcomitans/metabolismo , Aggregatibacter actinomycetemcomitans/patogenicidad , Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Catequina/farmacología , Técnicas de Cocultivo/métodos , Relación Dosis-Respuesta a Droga , Exotoxinas/antagonistas & inhibidores , Exotoxinas/metabolismo , Humanos , Periodontitis/tratamiento farmacológico , Periodontitis/microbiología , Virulencia/efectos de los fármacosRESUMEN
Like the bacterial residents of the human gut, it is likely that many of the species in the human oral microbiota have evolved to better occupy and persist in their niche. Aggregatibacter actinomycetemcomitans (Aa) is both a common colonizer of the oral cavity and has been implicated in the pathogenesis of periodontal disease. Here, we present a whole-genome phylogenetic analysis of Aa isolates from humans and nonhuman primates that revealed an ancient origin for this species and a long history of association with the Catarrhini, the lineage that includes Old World monkeys (OWM) and humans. Further genomic analysis showed a strong association with the presence of a short-chain fatty acid (SCFA) catabolism locus (atoRDAEB) in many human isolates that was absent in almost all nonhuman OWM isolates. We show that this locus was likely acquired through horizontal gene transfer. When grown under conditions that are similar to those at the subgingival site of periodontitis (anaerobic, SCFA replete), Aa strains with atoRDAEB formed robust biofilms and showed upregulation of genes involved in virulence, colonization, and immune evasion. Both an isogenic deletion mutant and nonhuman primate isolates lacking the ato locus failed to grow in a robust biofilm under these conditions, but grew well under the carbohydrate-rich conditions similar to those found above the gumline. We propose that the acquisition of the ato locus was a key evolutionary step allowing Aa to utilize SCFAs, adapt, and modulate subgingival disease.IMPORTANCE There has been considerable interest in the impact of short-chain fatty acids (SCFAs) on inflammatory effects related to the microbiome. Here, we present evidence that SCFAs may also be important in disease by providing an energy source or disease-associated cue for colonizing pathogens. We propose that SCFAs allow Aggregatibacter actinomycetemcomitans (Aa) to adapt to the subgingival anaerobic environment, which is the site of human periodontitis. Under anaerobic, SCFA-rich conditions, human-derived Aa strains that possess butyrate metabolism genes form strong biofilms and upregulate virulence genes. Our phylogenetic analysis highlights a long history of evolution of Aa with its primate hosts and suggests that the acquisition of butyrate metabolism genes may have been a critical step in allowing Aa to colonize a new niche and cause disease in humans. Overall, this study highlights the important role that horizontal gene transfer may play in microbial adaptation and the evolution of infectious disease.
Asunto(s)
Adaptación Fisiológica/genética , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/metabolismo , Butiratos/metabolismo , Transferencia de Gen Horizontal , Aggregatibacter actinomycetemcomitans/patogenicidad , Anaerobiosis , Biopelículas , Genoma Bacteriano , Factores de TiempoRESUMEN
This study aimed to focus on the simultaneous use of antimicrobial photodynamic therapy (aPDT) and sonodynamic antimicrobial chemotherapy (SACT), which is called photo-sonodynamic antimicrobial chemotherapy (PSACT) to attenuate the virulence of Aggregatibacter actinomycetemcomitans. Following the synthesis of Curcumin-decorated nanophytosomes (Cur-NPhs) as a novel photo-sonosensitizer, its particle size, polydispersity, ζ-potential surface morphology, physical stability, drug release, and entrapment efficiency were determined. In the Cur-NPhs-PSACT, the antimicrobial activities of Cur-NPhs against A. actinomycetemcomitans were investigated using cell viability, biofilm killing/degradation, metabolic activity, expression of quorum-sensing-associated qseB and qseC genes, and biofilm-associated rcpA gene under blue laser irradiation plus ultrasonic waves. Characterization tests showed the presence of a sphere-shaped vesicle and the self-closed structure of Cur-NPhs, resulting in a high drug-loading content and encapsulation efficiency. However, the antimicrobial effect of Cur-NPhs-PSACT was dose-dependent, PSACT using the high concentrations of Cur-NPhs (50 × 10-4 g/L) showed significant reductions (P < 0.05) in cell viability (13.6 log10 CFU/mL), biofilm killing/degradation (65%), metabolic activity (89.6%,), and mRNA levels of virulence determinant genes (qseB; 9.8-fold, qseC; 10.2-fold, and recA; 10.2-fold). This study concludes that the Cur-NPhs-PSACT had antimicrobial activities against A. actinomycetemcomitans by downregulating the expression of virulence genes, and may attenuate this bacterium that decreases periodontal disease severity in patients.
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Aggregatibacter actinomycetemcomitans , Antiinfecciosos , Biopelículas/efectos de los fármacos , Curcumina , Nanopartículas/química , Fotoquimioterapia , Fármacos Fotosensibilizantes , Aggregatibacter actinomycetemcomitans/patogenicidad , Aggregatibacter actinomycetemcomitans/fisiología , Antiinfecciosos/química , Antiinfecciosos/farmacología , Biopelículas/crecimiento & desarrollo , Curcumina/química , Curcumina/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Virulencia/efectos de los fármacosRESUMEN
Aggregatibacter actinomycetemcomitans is a facultative anaerobic Gram-negative bacterium associated with periodontal diseases, especially aggressive periodontitis. The virulence factors of this pathogen, including adhesins, exotoxins, and endotoxin, have been extensively studied. However, little is known about their gene expression mode in the host. Herein, we investigated whether culture conditions reflecting in vivo environments, including serum and saliva, alter expression levels of virulence genes in the strain HK1651, a JP2 clone. Under aerobic conditions, addition of calf serum (CS) into a general medium induced high expression of two outer membrane proteins (omp100 and omp64). The high expression of omp100 and omp64 was also induced by an iron-limited medium. RNA-seq analysis showed that the gene expressions of several factors involved in iron acquisition were increased in the CS-containing medium. When HK1651 was grown on agar plates, genes encoding many virulence factors, including the Omps, cytolethal distending toxin, and leukotoxin, were differentially expressed. Then, we investigated their expression in five other A. actinomycetemcomitans strains grown in general and CS-containing media. The expression pattern of virulence factors varied among strains. Compared with the other five strains, HK1561 showed high expression of omp29 regardless of the CS addition, while the gene expression of leukotoxin in HK1651 was higher only in the medium without CS. HK1651 showed reduced biofilm in both CS- and saliva-containing media. Coaggregation with Fusobacterium nucleatum was remarkably enhanced using HK1651 grown in the CS-containing medium. Our results indicate that the expression of virulence factors is altered by adaptation to different conditions during infection.
Asunto(s)
Aggregatibacter actinomycetemcomitans , Proteínas de la Membrana Bacteriana Externa/metabolismo , Enfermedades Periodontales , Factores de Virulencia/metabolismo , Aggregatibacter actinomycetemcomitans/patogenicidad , Humanos , Enfermedades Periodontales/microbiología , VirulenciaRESUMEN
Aggregatibacter actinomycetemcomitans is frequently isolated from localized aggressive periodontitis and periodontitis associated with systemic diseases. A. actinomycetemcomitans produces a leukotoxin, which induces apoptosis in human leukocytes. The leukotoxin expression is dependent on the upstream sequence, likely including the promoter, of the gene encoding leukotoxin; strains with the truncated/short upstream sequence express more leukotoxin than strains with the general/long upstream. This chapter addresses the determination of the type of the leukotoxin promoter by PCR analysis, and detection of the apoptosis in the coculture of human monocyte cell line (THP-1) with A. actinomycetemcomitans by the DNA ladder formation, membrane perturbation, and lactate dehydrogenase release.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Exotoxinas/metabolismo , Leucocitos/patología , Infecciones por Pasteurellaceae/patología , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/metabolismo , Apoptosis , Línea Celular , Técnicas de Cocultivo/métodos , Exotoxinas/genética , Humanos , Leucocitos/metabolismo , Leucocitos/microbiología , Infecciones por Pasteurellaceae/microbiología , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , VirulenciaRESUMEN
Aggregatibacter actinomycetemcomitans is a Gram-negative oral bacterium with high immunostimulatory and pathogenic potential involved in the onset and progression of periodontitis, a chronic disease characterized by aberrant immune responses followed by tooth-supporting bone resorption, which eventually leads to tooth loss. While several studies have provided evidence related to the virulence factors of A. actinomycetemcomitans involved in the host cell death and immune evasion, such as its most studied primate-specific virulence factor, leukotoxin, the role of specific lipopolysaccharide (LPS) domains remain poorly understood. Here, we analyzed the role of the immunodominant domain of the LPS of A. actinomycetemcomitans termed O-polysaccharide (O-PS), which differentiates the distinct bacterial serotypes based on its antigenicity. To determine the role of the O-PS in the immunogenicity and virulence of A. actinomycetemcomitans during periodontitis, we analyzed the in vivo and in vitro effect of an O-PS-defective transposon mutant serotype b strain, characterized by the deletion of the rmlC gene encoding the α-L-rhamnose sugar biosynthetic enzyme. Induction of experimental periodontitis using the O-PS-defective rmlC mutant strain resulted in lower tooth-supporting bone resorption, infiltration of Th1, Th17, and Th22 lymphocytes, and expression of Ahr, Il1b, Il17, Il23, Tlr4, and RANKL (Tnfsf11) in the periodontal lesions as compared with the wild-type A. actinomycetemcomitans strain. In addition, the O-PS-defective rmlC mutant strain led to impaired activation of antigen-presenting cells, with less expression of the co-stimulatory molecules CD40 and CD80 in B lymphocytes and dendritic cells, and downregulated expression of Tnfa and Il1b in splenocytes. In conclusion, these data demonstrate that the O-PS from the serotype b of A. actinomycetemcomitans plays a key role in the capacity of the bacterium to prime oral innate and adaptive immune responses, by triggering the Th1 and Th17-driven tooth-supporting bone resorption during periodontitis.
Asunto(s)
Aggregatibacter actinomycetemcomitans/inmunología , Aggregatibacter actinomycetemcomitans/patogenicidad , Periodontitis/inmunología , Periodontitis/microbiología , Polisacáridos Bacterianos/inmunología , Virulencia/inmunología , Aggregatibacter actinomycetemcomitans/genética , Pérdida de Hueso Alveolar/diagnóstico por imagen , Pérdida de Hueso Alveolar/etiología , Pérdida de Hueso Alveolar/patología , Animales , Biología Computacional/métodos , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno/inmunología , Lipopolisacáridos/inmunología , Ratones , Mutación , Periodontitis/complicaciones , Periodontitis/diagnóstico , Serogrupo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Virulencia , Microtomografía por Rayos XRESUMEN
Periodontitis is a chronic inflammatory disease caused by various periodontal pathogens. Weissella cibaria CMU (oraCMU) is a probiotic that promotes oral health. However, its antiinflammatory effects against periodontal pathogens have not yet been investigated. The present study evaluated the antiinflammatory effects of live oraCMU against stimulation with the formalininactivated periodontal pathogen Aggregatibacter actinomycetemcomitans in RAW 264.7 macrophages. Cell viability was analyzed by the MTS assay in a dosedependent manner (at multiplicities of infection of 0.1, 1, 10, 100 and 1,000). Nitric oxide (NO) was monitored using the Griess test. The mRNA expression of proinflammatory cytokines such as interleukin (IL)1ß and IL6 was assessed by reverse transcriptionquantitative PCR. Western blotting was used to examine the effects of oraCMU on the phosphorylation of NFκB inhibitor α (IκBα) and IκBα kinase (IKK), the nuclear translocation of the NFκB subunit p65 and the expression of inducible NO synthase (iNOS). Live oraCMU had no cytotoxic effects on RAW 264.7 macrophages. In A. actinomycetemcomitansstimulated RAW 264.7 macrophages, oraCMU reduced NO production by suppressing iNOS expression and downregulating the mRNA expression of proinflammatory cytokines in a dosedependent manner. IKK phosphorylation and IκBα degradation were dosedependently inhibited by oraCMU and the nuclear translocation of p65 via the canonical NFκB pathway was simultaneously reduced. The results indicated that oraCMU possessed antiinflammatory activity associated with the inhibition of NFκB signal activation in response to periodontal pathogens. This suggests that oraCMU is a beneficial antiinflammatory probiotic that can aid in the maintenance of oral health.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Antiinflamatorios/farmacología , Citocinas/genética , Macrófagos/citología , Probióticos/farmacología , Weissella/fisiología , Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Aggregatibacter actinomycetemcomitans/inmunología , Animales , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Formaldehído/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/genética , Interleucina-6/genética , Macrófagos/inmunología , Ratones , FN-kappa B/metabolismo , Fosforilación , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: This study evaluated the effect of oral lactobacilli on the cytotoxicity and cytokine release from peripheral blood mononuclear cells (PBMCs) when exposed to Aggregatibacter actinomycetemcomitans subtypes in vitro. The supernatants and cell wall extracts (CWEs) of eight A. actinomycetemcomitans strains, representing different subtypes, and three Lactobacillus strains were used. The PBMCs from six blood donors were exposed to supernatants and CWEs of A. actinomycetemcomitans or Lactobacillus strains alone or combinations and untreated cells as control. The cytotoxicity was determined by trypan blue exclusion method and IL-1ß secretion by ELISA. TNF-α, IL-6, and IL-8 secretions were measured using Bioplex Multiplex Immunoassay. RESULTS: Supernatants or CWEs from all bacterial strains showed cytotoxicity and IL-1ß secretion and the subtypes of A. actinomycetemcomitans showed generally a significantly higher effect on PBMCs than that of the Lactobacillus strains. Two highly toxic A. actinomycetemcomitans strains (JP2 and JP2-like) induced a higher response than all other strains. When combined, Lactobacillus significantly reduced the toxicity and the IL-1ß secretion induced by A. acinomycetemcomitans. The effect varied between the subtypes and the reduction was highest for the JP2 and JP2-like strains. The Lactobacillus paracasei strain SD1 had a higher reducing effect than the other Lactobacillus strains. This strain had a consistent reducing effect on all subtypes of A. actinomycetemcomitans cytotoxicity, and release of IL-1ß, IL-6, IL-8, and TNF-α from PBMCs of the blood donors. A strong and significant variation in cytokine release between the six blood donors was noticed. CONCLUSIONS: Lactobacillus spp. and L. paracasei SD1 in particular, showed a limited but statistically significant reducing interaction with A. actinomycetemcomitans toxicity and release of cytokines in vitro.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Citocinas/metabolismo , Lactobacillus , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Pared Celular/química , Humanos , Lacticaseibacillus paracasei , Boca/microbiología , Infecciones por Pasteurellaceae/microbiología , Probióticos/administración & dosificaciónRESUMEN
OBJECTIVE: This 6-year study evaluatedAggregatibacter actinomycetemcomitans outcomes and their relationship to clinical status. DESIGN: From the eligible individuals (23-70 years of age), 31 regular compliers (between-visit interval < 6 months) were randomly selected and matched for age/sex with 31 irregular compliers (between-visit interval > 6 months). Periodontal clinical examination and subgingival samples were obtained 5 times: T1 (baseline), T2 (after active periodontal therapy), T3 (2 years), T4 (4 years), and T5 (6 years). Total bacteria load, A. actinomycetemcomitans, and red complex species Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola levels were determined by qPCR while PCR was used to determine the occurrence of the a-b-c-d-e-f-g serotypes and the JP2 clone of A. actinomycetemcomitans. Data between groups was compared over time. RESULTS: At baseline PCR revealed A. actinomycetemcomitans prevalence of 9.7 % and JP2 prevalence of 6.7 %. A. actinomycetemcomitans qPCR levels were higher among individuals < 35 years of age and increased at T2 in irregular compliers. At in irregular compliers at the three follow-up visits. Serotypes a, d, and f showed greater values in at least one follow-up visit in regular compliers. A. actinomycetemcomitans showed negative correlation with probing depth (PD) while serotype b showed negative correlations with PD, PI, clinical attachment level and red complex. CONCLUSIONS: Longitudinally, compliance during PMT contributed to lower A. actinomycetemcomitans levels with some degree of correlation with clinical status. However, this study failed to report any positive effect on the occurrence of the most virulent representatives, i.e. serotype b and the JP2 clone.
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Aggregatibacter actinomycetemcomitans , Infecciones por Pasteurellaceae , Treponema denticola , Adulto , Anciano , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Aggregatibacter actinomycetemcomitans/patogenicidad , Humanos , Persona de Mediana Edad , Infecciones por Pasteurellaceae/complicaciones , Periodoncia , Porphyromonas gingivalis , Serogrupo , Tannerella forsythia , Adulto JovenRESUMEN
Medical literature on the microbiology of kinkajou bites is scarce. We present a patient presenting with hand cellulitis and abscess after a kinkajou bite that grew Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. We review the literature regarding microbiology and management of kinkajou bites.
Asunto(s)
Absceso/microbiología , Mordeduras y Picaduras/complicaciones , Celulitis (Flemón)/microbiología , Mano/microbiología , Mano/patología , Procyonidae/microbiología , Adolescente , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Aggregatibacter actinomycetemcomitans/patogenicidad , Animales , Femenino , Fusobacterium nucleatum/aislamiento & purificación , Fusobacterium nucleatum/patogenicidad , Humanos , Mascotas/microbiologíaRESUMEN
OBJECTIVE: The serotype b of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) induces higher cytokine production in dendritic cells (DCs) compared with the other serotypes. However, this increased immunostimulatory potential was modified when DCs were co-infected with the other A. actinomycetemcomitans serotypes. This study aimed to analyze whether the production of interferon gamma (IFN-γ), C-reactive protein (CRP), matrix metalloproteinase (MMP)-2, and MMP-9, as well as the activity of osteoclasts, also varies when DCs are co-infected with the A. actinomycetemcomitans serotypes. MATERIALS AND METHODS: Human DCs were stimulated with the A. actinomycetemcomitans serotypes using the following stimulatory conditions: serotype a/b/c/a+b/a+c/b+c/a+b+c. The IFN-γ, CRP, and MMP-2 levels were quantified by ELISA. The active form of MMP-9 was quantified using fluorescent functional assays. The MMP-2 gelatinolytic activity was identified by zymogram. The osteoclast activity was determined by quantifying the TRAP expression and resorption-pit formation using cytochemistry and osteoassays. RESULTS: Higher levels of IFN-γ, CRP, MMP-2, MMP-9, and osteoclast activity were detected when DCs were stimulated with the serotype b of A. actinomycetemcomitans compared with the others. This increased immunostimulatory potential attributed to serotype b diminished when DCs were co-infected with the serotype a. CONCLUSIONS: This study provides new insights into the virulence of A. actinomycetemcomitans and reveals important differences in the immunostimulatory and pro-destructive potential among its serotypes.
Asunto(s)
Aggregatibacter actinomycetemcomitans/clasificación , Aggregatibacter actinomycetemcomitans/patogenicidad , Células Dendríticas/microbiología , Proteína C-Reactiva/metabolismo , Humanos , Interferón gamma/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Osteoclastos , SerogrupoRESUMEN
Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Boca/microbiología , Periodontitis/microbiología , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/metabolismo , Aggregatibacter actinomycetemcomitans/fisiología , Animales , Adhesión Bacteriana/efectos de los fármacos , Biopelículas , Durapatita/farmacología , Exotoxinas/genética , Exotoxinas/metabolismo , Ácido Láctico/metabolismo , Macaca mulatta , Masculino , MicrobiotaRESUMEN
Recent evidence suggests that the genes an organism needs to survive in an environment drastically differ when alone or in a community. However, it is not known if there are universal functions that enable microbes to persist in a community and if there are functions specific to interactions between microbes native to the same (sympatric) or different (allopatric) environments. Here, we ask how the essential functions of the oral pathogen Aggregatibacter actinomycetemcomitans change during pairwise coinfection in a murine abscess with each of 15 microbes commonly found in the oral cavity and 10 microbes that are not. A. actinomycetemcomitans was more abundant when coinfected with allopatric than with sympatric microbes, and this increased fitness correlated with expanded metabolic capacity of the coinfecting microbes. Using transposon sequencing, we discovered that 33% of the A. actinomycetemcomitans genome is required for coinfection fitness. Fifty-nine "core" genes were required across all coinfections and included genes necessary for aerobic respiration. The core genes were also all required in monoinfection, indicating the essentiality of these genes cannot be alleviated by a coinfecting microbe. Furthermore, coinfection with some microbes, predominately sympatric species, induced the requirement for over 100 new community-dependent essential genes. In contrast, in other coinfections, predominately with nonoral species, A. actinomycetemcomitans required 50 fewer genes than in monoinfection, demonstrating that some allopatric microbes can drastically alleviate gene essentialities. These results expand our understanding of how diverse microbes alter growth and gene essentiality within polymicrobial infections.
Asunto(s)
Aggregatibacter actinomycetemcomitans/genética , Genes Esenciales/genética , Microbiota/genética , Simpatría/genética , Aggregatibacter/genética , Aggregatibacter actinomycetemcomitans/patogenicidad , Animales , Coinfección , Aptitud Genética/genética , Ratones , ARN Ribosómico 16S/genéticaRESUMEN
Aggregatibacter actinomycetemcomitans is an oral pathogen that produces the RTX toxin, leukotoxin (LtxA; Leukothera®). A. actinomycetemcomitans is strongly associated with the development of localized aggressive periodontitis. LtxA acts as a virulence factor for A. actinomycetemcomitans to subvert the host immune response by binding to the ß2 integrin lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) on white blood cells (WBCs), causing cell death. In this paper, we reviewed the state of knowledge on LtxA interaction with WBCs and the subsequent mechanisms of induced cell death. Finally, we touched on the potential therapeutic applications of LtxA (trade name Leukothera®) toxin therapy for the treatment of hematological malignancies and immune-mediated diseases.
Asunto(s)
Aggregatibacter actinomycetemcomitans/metabolismo , Exotoxinas/farmacología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Factores de Virulencia/farmacología , Aggregatibacter actinomycetemcomitans/patogenicidad , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Exotoxinas/aislamiento & purificación , Exotoxinas/uso terapéutico , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Leucocitos/efectos de los fármacos , Leucocitos/patología , Boca/microbiología , Unión Proteica , Factores de Virulencia/aislamiento & purificación , Factores de Virulencia/uso terapéuticoRESUMEN
The impact of oral commensal and pathogenic bacteria on peri-implant mucosa is not well understood, despite the high prevalence of peri-implant infections. Hence, we investigated responses of the peri-implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri-implant mucosa-biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri-implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin-6 (IL-6), interleukin-8 (CXCL8), and monocyte chemoattractant protein-1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor-alpha (TNF-α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri-implant mucosa-biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri-implant disease.
Asunto(s)
Aggregatibacter actinomycetemcomitans/fisiología , Biopelículas/crecimiento & desarrollo , Modelos Inmunológicos , Mucosa Bucal/inmunología , Mucosa Bucal/microbiología , Periimplantitis/inmunología , Streptococcus oralis/fisiología , Aggregatibacter actinomycetemcomitans/patogenicidad , Células Cultivadas , Quimiocina CCL2/metabolismo , Implantes Dentales/efectos adversos , Implantes Dentales/microbiología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mucosa Bucal/metabolismo , Mucosa Bucal/patología , Periimplantitis/microbiología , Periimplantitis/patología , Infecciones Relacionadas con Prótesis/inmunología , Titanio/química , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
ß-glucan is an abundant cell wall component of fungi and yeast. Dectin-1, a ß-glucan receptor, plays an important regulatory role in the natural immunity. In the present study, we investigated the effect of ß-glucan on mouse macrophages that had been invaded by the periodontopathic bacterium, Aggregatibacter actinomycetemcomitans. Exposure to curdlan, a type of ß-glucan, suppressed cell death and led to the accumulation of a sub-G1-phase population upon A. actinomycetemcomitans invasion under conditions of constitutive expression of dectin-1. Members of the nucleotide-binding domain leucine-rich repeat-containing (NLR) protein family, such as NLR protein 3 (NLRP3), NLR family apoptosis inhibitory protein (NAIP), and NLR family CARD domain-containing protein 4 (NLRC4), as well as an associated protein, caspase-11, were clearly detected in A. actinomycetemcomitans-invaded control RAW cells (c-RAW cells; negative control). Interestingly, NAIP expression was upregulated and caspase-11 expression was downregulated by dectin-1 activity in A. actinomycetemcomitans-invaded dectin-1 overexpressing RAW 264.7 cells (d-RAW cells), suggesting that dectin-1 in macrophages regulates cell death upon A. actinomycetemcomitans invasion. These results support a potential correlation between dectin-1 and regulation of cell death in macrophages.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Caspasas Iniciadoras/metabolismo , Muerte Celular/genética , Lectinas Tipo C/genética , Macrófagos/microbiología , beta-Glucanos/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Regulación de la Expresión Génica , Macrófagos/efectos de los fármacos , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Células RAW 264.7 , Transducción de Señal , beta-Glucanos/farmacologíaRESUMEN
Aggregatibacter actinomycetemcomitans (Aa) is a low-abundance Gram-negative oral pathobiont that is highly associated with a silent but aggressive orphan disease that results in periodontitis and tooth loss in adolescents of African heritage. For the most part Aa conducts its business by utilizing strategies allowing it to conceal itself below the radar of the host mucosal immune defense system. A great deal of misinformation has been conveyed with respect to Aa biology in health and disease. The purpose of this review is to present misconceptions about Aa and the strategies that it uses to colonize, survive, and evade the host. In the process Aa manages to undermine host mucosal defenses and contribute to disease initiation. This review will present clinical observational, molecular, and interventional studies that illustrate genetic, phenotypic, and biogeographical tactics that have been recently clarified and demonstrate how Aa survives and suppresses host mucosal defenses to take part in disease pathogenesis. At one point in time Aa was considered to be the causative agent of Localized Aggressive Periodontitis. Currently, it is most accurate to look at Aa as a community activist and necessary partner of a pathogenic consortium that suppresses the initial host response so as to encourage overgrowth of its partners. The data for Aa's activist role stems from molecular genetic studies complemented by experimental animal investigations that demonstrate how Aa establishes a habitat (housing), nutritional sustenance in that habitat (food), and biogeographical mobilization and/or relocation from its initial habitat (transportation). In this manner Aa can transfer to a protected but vulnerable domain (pocket or sulcus) where its community activism is most useful. Aa's "strategy" includes obtaining housing, food, and transportation at no cost to its partners challenging the economic theory that "there ain't no such thing as a free lunch." This "strategy" illustrates how co-evolution can promote Aa's survival, on one hand, and overgrowth of community members, on the other, which can result in local host dysbiosis and susceptibility to infection.
Asunto(s)
Aggregatibacter actinomycetemcomitans/patogenicidad , Periodontitis Agresiva/microbiología , Infecciones por Pasteurellaceae/microbiología , Adolescente , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/inmunología , Periodontitis Agresiva/etiología , Periodontitis Agresiva/inmunología , Animales , Biopelículas/crecimiento & desarrollo , Genes Bacterianos , Interacciones Microbiota-Huesped/inmunología , Humanos , Inmunidad Mucosa , Modelos Inmunológicos , Infecciones por Pasteurellaceae/etiología , Infecciones por Pasteurellaceae/inmunologíaRESUMEN
Periodontitis, an inflammatory disease caused by subgingival Gram-negative (G-) bacteria, is linked with loss of the connective tissue and destruction of the alveolar bone. In the regulation of inflammatory response, chemokine receptor 2 (CXCR2), a specific receptor for interleukin-8 and neutrophil chemoattractant, plays an important role. The first aim of this study was to investigate the CXCR2 gene variability in chronic periodontitis (CP) patients and healthy nonperiodontitis controls in the Czech population. The second aim was to find a relation between CXCR2 gene variants and the presence of periodontal bacteria. A total of 500 unrelated subjects participated in this case-control study. 329 CP patients and 171 healthy nonperiodontitis controls were analyzed using polymerase chain reaction techniques for three single-nucleotide polymorphisms (SNPs): +785C/T (rs2230054), +1208T/C (rs1126579), and +1440A/G (rs1126580). A DNA microarray detection kit was used for the investigation of the subgingival bacterial colonization, in a subgroup of CP subjects (N = 162). No significant differences in allele, genotype, haplotype, or haplogenotype frequencies of CXCR2 gene variants between patients with CP and healthy controls (P > 0.05) were determined. Nevertheless, Aggregatibacter actinomycetemcomitans was detected more frequently in men positive for the C allele of the CXCR2 +785C/T polymorphism (61.8% vs. 41.1%, P < 0.05; OR = 2.31, 95% CI = 1.03-5.20) and for the T allele of the CXCR2 +1208C/T variant (61.8% vs. 38.9%, P < 0.05; OR = 2.54, 95% CI = 1.13-5.71). In contrast, no statistically significant associations of CXCR2 variants with seven selected periodontal bacteria were found in women. Although none of the investigated SNPs in the CXCR2 gene was associated with CP, the CXCR2 gene variants can be associated with subgingival colonization of G- bacteria in men with CP in the Czech population.
Asunto(s)
Periodontitis Crónica/genética , Receptores de Interleucina-8B/genética , Adulto , Aggregatibacter actinomycetemcomitans/patogenicidad , Alelos , Estudios de Casos y Controles , Periodontitis Crónica/microbiología , República Checa/epidemiología , Femenino , Genotipo , Haplotipos/genética , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.