RESUMO
Each HACEK group pathogen, which can cause infective endocarditis, expresses type IVa pili. The type IVa major pilin PilA plays a role in bacterial colonization, virulence, twitching motility, and the uptake of extracellular DNA. The type IV prepilin homolog PilA of the periodontal pathogen A. actinomycetemcomitans (AaPilA) is linked to DNA uptake and natural competence. Our aim was to investigate the virulence properties and immunogenic potential of AaPilA. Since Neisseria meningitidis PilE, which shares sequence similarity with AaPilA, participates in sequestering host cytokines, we examined the ability of AaPilA to interact with various cytokines. Moreover, we investigated the structural characteristics of AaPilA with molecular modeling. AaPilA was conserved among A. actinomycetemcomitans strains. One of the 18 different natural variants, PilAD7S, is present in naturally competent strains. This variant interacted with DNA and bound interleukin (IL)-8 and tumor necrosis factor (TNF)-α. Specific anti-AaPilA antibodies were present in A. actinomycetemcomitans-positive periodontitis patient sera, and the production of reactive oxygen species from human neutrophils was less effectively induced by the ΔpilA mutant than by the wild-type strains. However, AaPilA did not stimulate human macrophages to produce proinflammatory cytokines, nor was it cytotoxic. The results strengthen our earlier hypothesis that the DNA uptake machinery of A. actinomycetemcomitans is involved in the sequestration of inflammatory cytokines. Furthermore, AaPilA stimulates host immune cells, such as B cells and neutrophils, making it a potential virulence factor.
Assuntos
Aggregatibacter actinomycetemcomitans , Citocinas , Humanos , Aggregatibacter actinomycetemcomitans/química , Citocinas/metabolismo , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , DNA/metabolismo , Leucócitos/metabolismoRESUMO
Late embryogenesis abundant (LEA) proteins are important players in the management of responses to stressful conditions, such as drought, high salinity, and changes in temperature. Many LEA proteins do not have defined three-dimensional structures, so they are intrinsically disordered proteins (IDPs) and are often highly hydrophilic. Although LEA-like sequences have been identified in bacterial genomes, the functions of bacterial LEA proteins have been studied only recently. Sequence analysis of outer membrane interleukin receptor I (BilRI) from the oral pathogen Aggregatibacter actinomycetemcomitans indicated that it shared sequence similarity with group 3/3b/4 LEA proteins. Comprehensive nuclearcgq magnetic resonance (NMR) studies confirmed its IDP nature, and expression studies in A. actinomycetemcomitans harboring a red fluorescence reporter protein-encoding gene revealed that bilRI promoter expression was increased at decreased temperatures. The amino acid backbone of BilRI did not stimulate either the production of reactive oxygen species from human leukocytes or the production of interleukin-6 from human macrophages. Moreover, BilRI-specific IgG antibodies could not be detected in the sera of A. actinomycetemcomitans culture-positive periodontitis patients. Since the bilRI gene is located near genes involved in natural competence (i.e., genes associated with the uptake of extracellular (eDNA) and its incorporation into the genome), we also investigated the role of BilRI in these events. Compared to wild-type cells, the ΔbilRI mutants showed a lower transformation efficiency, which indicates either a direct or indirect role in natural competence. In conclusion, A. actinomycetemcomitans might express BilRI, especially outside the host, to survive under stressful conditions and improve its transmission potential.
Assuntos
Proteínas de Bactérias , Proteínas Intrinsicamente Desordenadas , Aggregatibacter actinomycetemcomitans , Desenvolvimento Embrionário , Humanos , Proteínas de Plantas , TemperaturaRESUMO
Resonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three Hα-detected 4D experiments with Cα as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D Hα-start, HN-detect experiments which have two NH dimensions to enhance peak dispersion in a sequential walk through C', NH and HN, and provide more accurate NH/HN chemical shifts than those that can be obtained from a crowded 1H, 15N-HSQC spectrum. Application of these 4D experiments is demonstrated using BilRI (165 aa), an outer-membrane intrinsically disordered protein from the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans. BilRI amino acid sequence encompasses three very similar repeats with a 13-residue identical stretch in two of them.
Assuntos
Aggregatibacter actinomycetemcomitans/química , Proteínas de Bactérias/química , Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear BiomolecularRESUMO
Epithelial cells express keratins, which are essential for the structural integrity and mechanical strength of the cells. In the junctional epithelium (JE) of the tooth, keratins such as K16, K18, and K19, are expressed, which is typical for non-differentiated and rapidly dividing cells. The expression of K17, K4, and K13 keratins can be induced by injury, bacterial irritation, smoking, and inflammation. In addition, these keratins can be found in the sulcular epithelium and in the JE. Our aim was to estimate the changes in K4, K13, K17, and K19 expression in gingival epithelial cells exposed to Aggregatibacter actinomycetemcomitans. An organotypic gingival mucosa and biofilm co-culture was used as a model system. The effect of the biofilm after 24 h was assessed using immunohistochemistry. The structure of the epithelium was also studied with transmission electron microscopy (TEM). The expression of K17 and K19, as well as total keratin expression, decreased in the suprabasal layers of epithelium, which were in close contact with the A. actinomycetemcomitans biofilm. The effect on keratin expression was biofilm specific. The expression of K4 and K13 was low in all of the tested conditions. When stimulated with the A. actinomycetemcomitans biofilm, the epithelial contact site displayed a thick necrotic layer on the top of the epithelium. The A. actinomycetemcomitans biofilm released vesicles, which were found in close contact with the epithelium. After A. actinomycetemcomitans irritation, gingival epithelial cells may lose their resistance and become more vulnerable to bacterial infection.
RESUMO
Aggregatibacter actinomycetemcomitans genome can be divided into an accessory gene pool (found in some but not all strains) and a core gene pool (found in all strains). The functions of the accessory genes (genomic islands and non-island accessory genes) are largely unknown. We hypothesize that accessory genes confer critical functions for A. actinomycetemcomitans in vivo. This study examined the expression patterns of accessory and core genes of A. actinomycetemcomitans in distinct growth conditions. We found similar expression patterns of island and non-island accessory genes, which were generally lower than the core genes in all growth conditions. The median expression levels of genomic islands were 29%-37% of the core genes in enriched medium but elevated to as high as 63% of the core genes in nutrient-limited media. Several putative virulence genes, including the cytolethal distending toxin operon, were found to be activated in nutrient-limited conditions. In conclusion, genomic islands and non-island accessory genes exhibited distinct patterns of expression from the core genes and may play a role in the survival of A. actinomycetemcomitans in nutrient-limited environments.
RESUMO
Aggregatibacter actinomycetemcomitans is a periodontal pathogen colonizing the oral cavity of a large proportion of the human population. It is equipped with several potent virulence factors that can cause cell death and induce or evade inflammation. Because of the large genetic diversity within the species, both harmless and highly virulent genotypes of the bacterium have emerged. The oral condition and age, as well as the geographic origin of the individual, influence the risk to be colonized by a virulent genotype of the bacterium. In the present review, the virulence and pathogenicity properties of A. actinomycetemcomitans will be addressed.
RESUMO
Various gram-negative species sequester host cytokines using outer membrane proteins or surface modulation by sulfated polysaccharides. An outer membrane lipoprotein (BilRI) of the periodontal pathogen Aggregatibacter actinomycetemcomitans binds several cytokines, including interleukin (IL)-8. Because IL-8 is positively charged at physiological pH, we aimed to determine whether IL-8 interacts with negatively charged lipopolysaccharide (LPS). Binding was investigated using electrophoretic mobility shift assays and microwell-based time-resolved fluorometric immunoassay. LPS from each tested strain of A. actinomycetemcomitans (N = 13), Pseudomonas aeruginosa (N = 1) and Escherichia coli (N = 1) bound IL-8. The Kd value of the A. actinomycetemcomitans LPS-IL-8 interaction varied between 1.2-17 µM irrespective of the serotype and the amount of phosphorus in LPS and was significantly lower than that of the BilRI-IL-8 interaction. Moreover, IL-8 interacted with whole A. actinomycetemcomitans cells and outer membrane vesicles. Hence, LPS might be involved in binding of IL-8 to the outer membrane of A. actinomycetemcomitans. This raises an interesting question regarding whether other gram-negative periodontal pathogens use LPS for IL-8 sequestering in vivo.
RESUMO
Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43 nM in static settings and 2.4 µM in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested ß-lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.
Assuntos
Aggregatibacter actinomycetemcomitans/química , Proteínas de Bactérias/genética , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Interleucina-8/metabolismo , Secretina/metabolismo , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/patogenicidade , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/imunologia , Biofilmes/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Humanos , Interleucina-8/imunologia , Periodontite/imunologia , Periodontite/microbiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Secretina/imunologia , Virulência , beta-Lactamas/farmacologiaRESUMO
Phosphorylcholine (ChoP) is covalently incorporated into bacterial surface structures, contributing to host mimicry and promoting adhesion to surfaces. Our aims were to determine the frequency of ChoP display among Aggregatibacter actinomycetemcomitans strains, to clarify which surface structures bear ChoP, and whether ChoP-positivity relates to serum killing. The tested oral (N = 67) and blood isolates (N = 27) represented 6 serotypes. Mab TEPC-15 was used for immunoblotting of cell lysates and fractions and for immunofluorescence microscopy of cell surface-bound ChoP. The lysates were denatured with urea for hidden ChoP or treated with proteinase K to test whether it binds to a protein. Three ChoP-positive and two ChoP-negative strains were subjected to serum killing in the presence/absence of CRP and using Ig-depleted serum as complement source. Cell lysates and the first soluble cellular fraction revealed a < 10 kDa band in immunoblots. Among 94 strains, 27 were ChoP positive. No difference was found in the prevalence of ChoP-positive oral (21/67) and blood (6/27) strains. Immunofluorescence microscopy corresponded to the immunoblot results. Proteinase K abolished ChoP reactivity, whereas urea did not change the negative result. The TEPC-15-reactive protein was undetectable in Δflp1 mutant strain. The survival rate of serotype-b strains in serum was 100% irrespective of ChoP, but that of serotype-a was higher in ChoP-positive (85%) than ChoP-negative (71%) strains. The results suggest that a third of rough-colony strains harbor ChoP and that ChoP is attached to fimbrial subunit protein Flp1. It further seems that ChoP-positivity does not enhance but may reduce A. actinomycetemcomitans susceptibility to serum killing.
Assuntos
Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/imunologia , Proteínas de Bactérias/química , Fosforilcolina/análise , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Proteínas de Bactérias/genética , Sangue/microbiologia , Atividade Bactericida do Sangue , Deleção de Genes , Humanos , Immunoblotting , Viabilidade Microbiana , Microscopia de Fluorescência , Boca/microbiologia , Infecções por Pasteurellaceae/microbiologia , SorogrupoRESUMO
Peptidoglycan-associated lipoprotein (PAL) is a conserved pro-inflammatory outer membrane lipoprotein in Gram-negative bacteria. Compared to systemic pathogens, little is known about the virulence properties of PAL in Aggregatibacter actinomycetemcomitans (AaPAL). The aims of this study were to investigate the cytolethality of AaPAL and its ability to induce pro-inflammatory cytokine production in macrophages. Mouse macrophages were stimulated with AaPAL, and the production of IL-1ß, IL-6, TNF-α, and MCP-1 was measured after 6, 24, and 48 h. To investigate which receptor AaPAL employs for its interaction with macrophages, anti-toll-like receptor (TLR)2 and anti-TLR4 antibodies were used to block respective TLRs on macrophages. Metabolic activity and apoptosis of the macrophages were investigated after stimulation with AaPAL. AaPAL induced the production of MCP-1, TNF-α, IL-6, and IL-1ß from mouse macrophages in order of decreasing abundance. The pre-treatment of macrophages with an anti-TLR2 antibody significantly diminished cytokine production. Under AaPAL stimulation, the metabolic activity of macrophages decreased in a dose- and time-dependent manner. Furthermore, AaPAL induced apoptosis in 56% of macrophages after 48 h of incubation. Our data suggest that AaPAL can kill macrophages by apoptosis. The results also emphasize the role of AaPAL as a potent pro-inflammatory agent in A. actinomycetemcomitans-associated infections.
RESUMO
Several human pathogens bind and respond to host cytokines, which can be considered a virulence mechanism that communicates defensive actions of the host to the pathogen. This review summarizes the current knowledge of bacterial cytokine-binding proteins, with a particular focus on their functional and structural characteristics. Many bacterial cytokine-binding proteins function in the development of infection and inflammation and mediate adhesion to host cells, suggesting multiple roles in pathogen-host interactions. The regions of the bacterial proteins that interact with host cytokines can display structural similarities to other proteins involved in cytokine signaling. However, there appears to be no central shared structural themes for bacterial cytokine-binding proteins, and they appear to possess structures that are different from the cytokine receptors of the host. Atomic-level information regarding receptor-cytokine interactions is needed to be able to disrupt these interactions and to elucidate the specific consequences of cytokine binding in a pathogen and host.
Assuntos
Bactérias/metabolismo , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocinas/metabolismo , Animais , Bactérias/genética , Infecções Bacterianas/genética , Infecções Bacterianas/metabolismo , Proteínas de Bactérias/genética , Citocinas/genética , Interações Hospedeiro-Patógeno , Humanos , Ligação ProteicaRESUMO
Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1ß. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1ß and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI- mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1ß in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1ß internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes/crescimento & desenvolvimento , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Receptores de Interleucina-1/metabolismo , Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Gengiva/microbiologia , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-10/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/farmacologia , Interleucina-8/genética , Interleucina-8/farmacologia , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/imunologia , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1ß. Some bacterial species can alter their physiological properties as a result of sensing IL-1ß. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1ß sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1ß through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1ß than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1ß, but this binding was not specific, as a control protein for IL-1ß also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1ß-binding surface-exposed lipoprotein that may be part of the bacterial IL-1ß-sensing system.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Interleucina-1beta/metabolismo , Proteínas Recombinantes/metabolismo , Aggregatibacter actinomycetemcomitans/genética , Aggregatibacter actinomycetemcomitans/fisiologia , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Biofilmes , Membrana Celular/metabolismo , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Immunoblotting , Dados de Sequência Molecular , Infecções por Pasteurellaceae/microbiologia , Ligação Proteica , Sinais Direcionadores de Proteínas/genéticaRESUMO
Periodontitis is a common inflammatory disease affecting the tooth-supporting structures. It is initiated by bacteria growing as a biofilm at the gingival margin, and communication of the biofilms differs in health and disease. The bacterial composition of periodontitis-associated biofilms has been well documented and is under continual investigation. However, the roles of several host response and inflammation driven environmental stimuli on biofilm formation is not well understood. This review article addresses the effects of environmental factors such as pH, temperature, cytokines, hormones, and oxidative stress on periodontal biofilm formation and bacterial virulence.
Assuntos
Biofilmes , Bactérias Gram-Negativas/fisiologia , Bactérias Gram-Positivas/fisiologia , Periodontite/microbiologia , Animais , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/patogenicidade , Hemina/metabolismo , Hormônios/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/metabolismo , Ferro/metabolismo , Estresse Oxidativo , Periodontite/imunologia , Periodontite/metabolismo , VirulênciaRESUMO
The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1ß, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1ß; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1ß when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1ß and caused IL-1ß leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1ß localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1ß interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1ßin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1ß. The interaction of IL-1ß with conserved HU provides a potential mechanism for shaping bacterial gene expression.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Biofilmes , DNA Bacteriano/metabolismo , Endocitose , Interleucina-1beta/metabolismo , Viabilidade Microbiana , Infecções por Actinobacillus/microbiologia , Infecções por Actinobacillus/patologia , Aggregatibacter actinomycetemcomitans/efeitos dos fármacos , Aggregatibacter actinomycetemcomitans/ultraestrutura , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas ELAV/química , Proteínas ELAV/metabolismo , Endocitose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Gengiva/microbiologia , Gengiva/patologia , Humanos , Queratinócitos/microbiologia , Queratinócitos/patologia , Viabilidade Microbiana/efeitos dos fármacos , Dados de Sequência Molecular , Mucosa/efeitos dos fármacos , Mucosa/microbiologia , Mucosa/patologia , Mucosa/ultraestrutura , Penicilinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Estreptomicina/farmacologiaRESUMO
We studied the effects of xylitol on biofilms containing xylitol-resistant (Xr) and xylitol-sensitive (Xs) Streptococcus mutans, Actinomyces naeslundii and S. sanguinis. The biofilms were grown for 8 and 24 h on hydroxyapatite discs. The viable microorganisms were determined by plate culturing techniques and fluorescence in situ hybridization (FISH) was performed using a S. mutans-specific probe. Extracellular cell-bound polysaccharides (EPS) were determined by spectrofluorometry from single-species S. mutans biofilms. In the presence of 5 % xylitol, the counts of the Xs S. mutans decreased tenfold in the young (8 h) biofilm (p < 0.05) but no effect was seen in the mature (24 h) biofilm. No decrease was observed for the Xr strains, and FISH confirmed these results. No differences were detected in the EPS production of the Xs S. mutans grown with or without xylitol, nor between Xr and Xs S. mutans strains. Thus, it seems that xylitol did not affect the EPS synthesis of the S. mutans strains. Since the Xr S. mutans strains, not inhibited by xylitol, showed no xylitol-induced decrease in the biofilms, we conclude that growth inhibition could be responsible for the decrease of the counts of the Xs S. mutans strains in the clinically relevant young biofilms.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos , Xilitol/farmacologia , Análise de Variância , Biofilmes/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Humanos , Hibridização in Situ Fluorescente , Saliva/microbiologia , Streptococcus mutans/fisiologiaRESUMO
Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1ß, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1ß receptor has been identified, current knowledge of the bacterial IL-1ß sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1ß than inactive ones. The effect of IL-1ß on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1ß to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1ß, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1ß and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1ß slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1ß. Our results suggest that IL-1ß might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit ß interacted with IL-1ß, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1ß during inflammation.
Assuntos
Proteínas de Bactérias/metabolismo , Biopolímeros/metabolismo , Interleucina-1beta/metabolismo , Pasteurellaceae/enzimologia , Biofilmes , Citometria de Fluxo , Humanos , Microscopia Eletrônica de Transmissão , Ligação ProteicaRESUMO
Secretins form large oligomeric assemblies in the membrane that control both macromolecular secretion and uptake. Several Pasteurellaceae are naturally competent for transformation, but the mechanism for DNA assimilation is largely unknown. In Haemophilus influenzae, the secretin ComE has been demonstrated to be essential for DNA uptake. In closely related Aggregatibacter actinomycetemcomitans, an opportunistic pathogen in periodontitis, the ComE homolog HofQ is believed to be the outer membrane DNA translocase. Here, we report the structure of the extra-membranous domains of HofQ at 2.3 Å resolution by X-ray crystallography. We also show that the extra-membranous domains of HofQ are capable of DNA binding. The structure reveals two secretin-like folds, the first of which is formed by means of a domain swap. The second domain displays extensive structural similarity to K homology (KH) domains, including the presence of a GxxG motif, which is essential for the nucleotide-binding function of KH domains, suggesting a possible mechanism for DNA binding by HofQ. The data indicate a direct involvement in DNA acquisition and provide insight into the molecular basis for natural competence.
Assuntos
Proteínas de Bactérias/química , DNA/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/química , Bactérias Gram-Negativas/química , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Alinhamento de SequênciaRESUMO
BACKGROUND: Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressively progressing periodontitis. Extracellular release of bacterial outer membrane proteins has been suggested to mainly occur via outer membrane vesicles. This study investigated the presence and conservation of peptidoglycan-associated lipoprotein (AaPAL) among A. actinomycetemcomitans strains, the immunostimulatory effect of AaPAL, and whether live cells release this structural outer membrane lipoprotein in free-soluble form independent of vesicles. RESULTS: The pal locus and its gene product were confirmed in clinical A. actinomycetemcomitans strains by PCR-restriction fragment length polymorphism and immunoblotting. Culturing under different growth conditions revealed no apparent requirement for the AaPAL expression. Inactivation of pal in a wild-type strain (D7S) and in its spontaneous laboratory variant (D7SS) resulted in pleiotropic cellular effects. In a cell culture insert model (filter pore size 0.02 mum), AaPAL was detected from filtrates when strains D7S and D7SS were incubated in serum or broth in the inserts. Electron microscopy showed that A. actinomycetemcomitans vesicles (0.05-0.2 mum) were larger than the filter pores and that there were no vesicles in the filtrates. The filtrates were immunoblot negative for a cytoplasmic marker, cyclic AMP (cAMP) receptor protein. An ex vivo model indicated cytokine production from human whole blood stimulated by AaPAL. CONCLUSION: Free-soluble AaPAL can be extracellularly released in a process independent of vesicles.
Assuntos
Aggregatibacter actinomycetemcomitans/fisiologia , Aggregatibacter actinomycetemcomitans/patogenicidade , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Aggregatibacter actinomycetemcomitans/ultraestrutura , Técnicas de Cultura de Células , Membrana Celular/ultraestrutura , Humanos , Lipoproteínas/imunologiaRESUMO
In a search for novel bioactive cell surface structures of periodontal pathogens, it was found that sera from two patients with Actinobacillus actinomycetemcomitans-associated infections reacted strongly at 17 kDa on immunoblots of A. actinomycetemcomitans outer-membrane protein (OMP) preparations. The 17 kDa antigen was also recognized by anti-CsgA (Escherichia coli curli major subunit) antibody. The 17 kDa A. actinomycetemcomitans protein was identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by two-dimensional immunoblotting and subsequent sequence analysis by mass spectrometry and bioinformatics tools. AaPAL was an OMP and a lipoprotein, and it had an OmpA-like domain. In a group of middle-aged subjects (n = 26), serum reactivity to AaPAL was associated with the presence of periodontitis but not with the oral detection of A. actinomycetemcomitans. Both human sera and rabbit antisera against three different types of antigens, the gel-purified AaPAL, A. actinomycetemcomitans whole-cell antigens, and CsgA, recognized putative PALs of oral haemophili in addition to AaPAL. The results demonstrated that the novel AaPAL is a conserved bacterial lipoprotein. It is expressed in vivo and is strongly immunoreactive. The antigenic cross-reactivity found between AaPAL and oral haemophili may enhance local and systemic immuno-inflammatory reactions in periodontitis.