RESUMO
Despite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation. Porphyromonas gingivalis is an important pathogen implicated in the development of periodontitis. We isolated several strains of Lactobacillus delbrueckii from dairy products and examined their ability to inhibit P. gingivalis growth in vitro We observed strain-specific inhibition of P. gingivalis growth in vitro Whole-genome sequencing of inhibitory and noninhibitory strains of L. delbrueckii revealed significant genetic differences supporting the strain specificity of the interaction. Extracts of the L. delbrueckii STYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) from L. delbrueckii STYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibit P. gingivalis growth in vitro without the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants of P. gingivalis in the feoB2 (PG_1294) gene that are resistant to killing by inhibitory L. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. Specific L. delbrueckii strains or their products may be effective in the treatment and prevention of P. gingivalis-associated periodontal disease.IMPORTANCEP. gingivalis is implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production in L. delbrueckii could be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.
Assuntos
Proteínas de Bactérias/metabolismo , Lactobacillus delbrueckii/fisiologia , Porphyromonas gingivalis/fisiologia , Piruvato Oxidase/metabolismo , Lactobacillus delbrueckii/enzimologia , Probióticos/química , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Iogurte/microbiologiaRESUMO
Cellular pigmentation is an important virulence factor of the oral pathogen Porphyromonas gingivalis Pigmentation has been associated with many bacterial functions, including but not limited to colonization, maintaining a local anaerobic environment by binding oxygen molecules, and defense against reactive oxygen species (ROS) produced by immune cells. Pigmentation-associated loci identified to date have involved lipopolysaccharide, fimbriae, and heme acquisition and processing. We utilized a transposon mutant library of P. gingivalis strain ATCC 33277 and screened for pigmentation-defective colonies using massively parallel sequencing of the transposon junctions (Tn-seq) to identify genes involved in pigmentation. Transposon insertions at 235 separate sites, located in 67 genes and 15 intergenic regions, resulted in altered pigmentation: 7 of the genes had previously been shown to be involved in pigmentation, while 75 genes and intergenic regions had not. To further confirm identification, we generated a smaller transposon mutant library in P. gingivalis strain W83 and identified pigment mutations in several of the same loci as those identified in the screen in ATCC 33277 but also eight that were not identified in the ATCC 33277 screen. PGN_0361/PG_0264, a putative glycosyltransferase gene located between two tRNA synthetase genes and adjacent to a miniature inverted-repeat transposable element, was identified in the Tn-seq screen and then verified through targeted deletion and complementation. Deletion mutations in PGN_0361/PG_0264 glycosyltransferase abolish pigmentation, modulate gingipain protease activity, and alter lipopolysaccharide. The mechanisms of involvement in pigmentation for other loci identified in this study remain to be determined, but our screen provides the most complete survey of genes involved in pigmentation to date.IMPORTANCEP. gingivalis has been implicated in the onset and progression of periodontal disease. One important virulence factor is the bacterium's ability to produce pigment. Using a transposon library, we were able to identify both known and novel genes involved in pigmentation of P. gingivalis We identified a glycosyltransferase, previously not associated with pigmentation, that is required for pigmentation and determined its mechanism of involvement. A better understanding of the genes involved in pigmentation may lead to new insights into the complex mechanisms involved in this important virulence characteristic and could facilitate development of novel therapeutics.
Assuntos
Elementos de DNA Transponíveis/genética , Glicosiltransferases/metabolismo , Pigmentos Biológicos/metabolismo , Porphyromonas gingivalis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Bacterianos , Glicosiltransferases/genética , Mutação , Técnicas de Amplificação de Ácido Nucleico , Pigmentos Biológicos/genética , Porphyromonas gingivalis/genéticaRESUMO
Porphyromonas gingivalis is the major causative agent of periodontitis, and it may also be involved in the development of systemic diseases (atherosclerosis, rheumatoid arthritis). P. gingivalis is found on and within oral and gingival epithelial cells following binding to surface components of host cells, which serve as receptors for the bacterium. Evidence is presented in this study that shows that transglutaminase 2 (TG2) plays a critical role in the adherence of P. gingivalis to host cells. Studies of confocal microscopy indicate colocalization of P. gingivalis with TG2 on the surface of HEp-2 epithelial cells, with clusters of TG2 seen at bacterial attachment sites. By silencing the expression of TG2 with siRNA in HEp-2 cells, P. gingivalis association was greatly diminished. The bacterium does not bind well to a mouse fibroblast cell line that produces low amounts of surface TG2, but binding can be restored by introduction of TG2 expressed on a plasmid. TG2 can form very tight complexes with fibronectin (FN), and the complementary binding sites of the two proteins are known. A synthetic peptide that mimics the main FN-binding sequence of TG2 blocks the formation of TG2-FN complexes and is highly effective in inhibiting adherence of P. gingivalis to host cells. These findings provide evidence of a role for cell-surface TG2 in bacterial attachment and subsequent internalization.
Assuntos
Aderência Bacteriana , Proteínas de Ligação ao GTP/metabolismo , Interações Hospedeiro-Patógeno , Porphyromonas gingivalis/fisiologia , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação ao GTP/genética , Inativação Gênica , Humanos , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Células NIH 3T3 , Porphyromonas gingivalis/enzimologia , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/genéticaRESUMO
The oral cavity contains distinct microenvironments that serve as oral barriers, such as the non-shedding surface of the teeth (e.g., enamel), the epithelial mucosa and gingival tissue (attached gingiva) where microbial communities coexist. The interactions and balances between these communities are responsible for oral tissue homeostasis or dysbiosis, that ultimately dictate health or disease. Disruption of this equilibrium can lead to chronic inflammation and permanent tissue damage in the case of chronic periodontitis. There are currently no experimental tissue models able to mimic the structural, physical, and metabolic conditions present in the human oral gingival tissue to support the long-term investigation of host-pathogens imbalances. Herein, the authors report an in vitro 3D anatomical gingival tissue model, fabricated from silk biopolymer by casting a replica mold of an adult human mandibular gingiva to recreate a tooth-gum unit. The model is based on human primary cultures that recapitulate physiological tissue organization, as well as a native oxygen gradient within the gingival pocket to support human subgingival plaque microbiome with a physiologically relevant level of microbial diversity up to 24 h. The modulation of inflammatory markers in the presence of oral microbiome indicates the humanized functional response of this model and establishes a new set of tools to investigate host-pathogen imbalances in gingivitis and periodontal diseases.
Assuntos
Gengivite , Microbiota , Doenças Periodontais , Adulto , Humanos , Gengiva , Bolsa GengivalRESUMO
BACKGROUND: Porphyromonas gingivalis is a Gram-negative anaerobic bacterium associated with periodontal disease onset and progression. Genetic tools for the manipulation of bacterial genomes allow for in-depth mechanistic studies of metabolism, physiology, interspecies and host-pathogen interactions. Analysis of the essential genes, protein-coding sequences necessary for survival of P. gingivalis by transposon mutagenesis has not previously been attempted due to the limitations of available transposon systems for the organism. We adapted a Mariner transposon system for mutagenesis of P. gingivalis and created an insertion mutant library. By analyzing the location of insertions using massively-parallel sequencing technology we used this mutant library to define genes essential for P. gingivalis survival under in vitro conditions. RESULTS: In mutagenesis experiments we identified 463 genes in P. gingivalis strain ATCC 33277 that are putatively essential for viability in vitro. Comparing the 463 P. gingivalis essential genes with previous essential gene studies, 364 of the 463 are homologues to essential genes in other species; 339 are shared with more than one other species. Twenty-five genes are known to be essential in P. gingivalis and B. thetaiotaomicron only. Significant enrichment of essential genes within Cluster of Orthologous Groups 'D' (cell division), 'I' (lipid transport and metabolism) and 'J' (translation/ribosome) were identified. Previously, the P. gingivalis core genome was shown to encode 1,476 proteins out of a possible 1,909; 434 of 463 essential genes are contained within the core genome. Thus, for the species P. gingivalis twenty-two, seventy-seven and twenty-three percent of the genome respectively are devoted to essential, core and accessory functions. CONCLUSIONS: A Mariner transposon system can be adapted to create mutant libraries in P. gingivalis amenable to analysis by next-generation sequencing technologies. In silico analysis of genes essential for in vitro growth demonstrates that although the majority are homologous across bacterial species as a whole, species and strain-specific subsets are apparent. Understanding the putative essential genes of P. gingivalis will provide insights into metabolic pathways and niche adaptations as well as clinical therapeutic strategies.
Assuntos
Biblioteca Gênica , Genes Essenciais/genética , Porphyromonas gingivalis/genética , Sequência de Bases , Mapeamento Cromossômico , Biologia Computacional , Primers do DNA/genética , Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Mutagênese/genética , Reação em Cadeia da PolimeraseRESUMO
The virulence of the dental caries pathogen Streptococcus mutans relies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced by S. mutans participate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressed gbpB antisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation of gbpB by the two-component system VicRK was investigated, and phenotypic analysis of a vicK mutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrose-dependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulon/fisiologia , Streptococcus mutans/metabolismo , Proteínas de Bactérias/genética , Regulação para Baixo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , RNA Bacteriano , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/genética , Streptococcus mutans/fisiologia , Sacarose/farmacologiaRESUMO
Porphyromonas gingivalis, a Gram-negative oral anaerobe, is strongly associated with chronic adult periodontitis, and it utilizes FimA fimbriae to persistently colonize and evade host defenses in the periodontal crevice. The FimA-related gene cluster (the fim gene cluster) is positively regulated by the FimS-FimR two-component system. In this study, comparative analyses between fimbriate type strain ATCC 33277 and fimbria-deficient strain W83 revealed differences in their fimS loci, which encode FimS histidine kinase. Using a reciprocal gene exchange system, we established that FimS from W83 is malfunctional. Complementation analysis with chimeric fimS constructs revealed that W83 FimS has a defective kinase domain due to a truncated conserved G3 box motif that provides an ATP-binding pocket. The introduction of the functional fimS from 33277 restored the production, but not polymerization, of endogenous FimA subunits in W83. Further analyses with a fimA-exchanged W83 isogenic strain showed that even the fimbria-deficient W83 retains the ability to polymerize FimA from 33277, indicating the assembly of mature FimA by a primary structure-dependent mechanism. It also was shown that the substantial expression of 33277-type FimA fimbriae in the W83 derivative requires the introduction and expression of the functional 33277 fimS. These findings indicate that FimSR is the unique and universal regulatory system that activates the fim gene cluster in a fimA genotype-independent manner.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Porphyromonas gingivalis/fisiologia , Proteínas Quinases/metabolismo , Proteínas de Bactérias/genética , Teste de Complementação Genética , Histidina Quinase , Porphyromonas gingivalis/enzimologia , Porphyromonas gingivalis/metabolismo , Proteínas Quinases/genética , Deleção de Sequência , Transdução de SinaisRESUMO
Porphyromonas gingivalis, a Gram-negative oral anaerobe, is associated with periodontal diseases that, in some form, affect up to 80% of the U.S. population. The organism is highly proteolytic, and noncatalytic adhesin domains of the major proteases, gingipains, are involved in bacterium-host interactions. Recently, we showed that gingipain adhesin peptide A44 hijacks the host's clathrin-dependent endocytosis system, allowing the peptide and whole bacteria to be internalized by epithelial cells. In the present study, we found by cell fractionation assays and confocal microscopy that peptide A44 translocated to host mitochondria. Cell viability assays and quantitative real-time PCR showed that the peptide interacted with the cell death machinery by triggering upregulation of antiapoptotic factors bcl-2 and bcl-XL and prevented staurosporine-induced apoptosis for up to 12 h. We confirmed these findings with Western blot analyses of caspase-9 activation in time course experiments with staurosporine. Finally, we verified a similar antiapoptotic effect for P. gingivalis, showing for the first time that the organism manipulated mitochondrial functions during the first hours of infection, thus resisting host cell clearance by apoptosis of infected cells. This mechanism may enable the bacteria to persist in the protected cellular environment until the next step in pathogenesis, progression or resolution of infection.
Assuntos
Adesinas Bacterianas/metabolismo , Apoptose , Cisteína Endopeptidases/metabolismo , Células Epiteliais/microbiologia , Mitocôndrias/metabolismo , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/patogenicidade , Western Blotting , Caspase 9/metabolismo , Fracionamento Celular , Linhagem Celular , Sobrevivência Celular , Cisteína Endopeptidases Gingipaínas , Humanos , Microscopia Confocal , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Regulação para Cima , Proteína bcl-X/biossínteseRESUMO
Hydrogen sulfide (H(2)S), a volatile sulfur compound, is implicated as a cause of inflammation, especially when it is produced by bacteria colonizing gastrointestinal organs. However, it is unclear if H(2)S produced by periodontal pathogens affects the inflammatory responses mediated by oral/gingival epithelial cells. Therefore, the aims of this study were (1) to compare the in vitro production of H(2)S among 14 strains of oral bacteria and (2) to evaluate the effects of H(2)S on inflammatory response induced in host oral/gingival epithelial cells. Porphyromonas gingivalis (Pg) produced the most H(2)S in culture, which, in turn, resulted in the promotion of proinflammatory cytokine IL-8 from both gingival and oral epithelial cells. The up-regulation of IL-8 expression was reproduced by the exogenously applied H(2)S. Furthermore, the mutant strains of Pg that do not produce major soluble virulent factors, i.e. gingipains, still showed the production of H(2)S, as well as the promotion of epithelial IL-8 production, which was abrogated by H(2)S scavenging reagents. These results demonstrated that Pg produces a concentration of H(2)S capable of up-regulating IL-8 expression induced in gingival and oral epithelial cells, revealing a possible mechanism that may promote the inflammation in periodontal disease.
Assuntos
Gengiva/imunologia , Gengivite/imunologia , Sulfeto de Hidrogênio/metabolismo , Interleucina-8/biossíntese , Porphyromonas gingivalis/metabolismo , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Gengiva/microbiologia , Gengivite/microbiologia , HumanosRESUMO
Porphyromonas gingivalis, a Gram-negative oral anaerobe, is associated with periodontitis, a disease that in some form affects up to 80% of the adult population in the USA. The organism interacts with gingival epithelium and surrounding tissue, and in this study we analysed interactions initiated by P. gingivalis and by a peptide derived from the adhesin domain of arg-gingipain A, a member of a family of surface cysteine proteinases. Recombinant peptide A44 blocked adherence of bacteria to host cell monolayers, and bound to components of the cell membrane fraction. In pull-down assays A44 associated with proteins involved in a clathrin-dependent endocytosis pathway. Inhibitor studies confirmed a role for clathrin, and confocal microscopy demonstrated that both A44-coated beads and intact bacteria colocalized with GFP-clathrin in host cells. Finally, we used siRNA to determine whether clathrin or caveolin-1 was involved in association of peptide and intact bacteria with host cells. Again, the results of these assays indicated that association of both A44 and P. gingivalis depended on the presence of clathrin, and support a working model in which A44 initiates a clathrin-dependent pathway that potentially leads to internalization of peptide or bacteria by host epithelial cells.
Assuntos
Adesinas Bacterianas/metabolismo , Clatrina/metabolismo , Cisteína Endopeptidases/metabolismo , Endocitose , Porphyromonas gingivalis/fisiologia , Adulto , Aderência Bacteriana , Membrana Celular/metabolismo , Células Epiteliais/microbiologia , Inativação Gênica , Cisteína Endopeptidases Gingipaínas , Humanos , Microscopia Confocal , Ligação ProteicaRESUMO
Background: Bacteria use two-component signal transduction systems (among others) to perceive and respond to environmental changes. Within the genus Porphyromonas, we observed degeneration of these systems, as exemplified by the loss of RprX, the sensor kinase partner of the RprY. Objective: The purpose of this study was to investigate modulation of RprY function by acetylation. Design: The transcriptional activity of the rprY-pat genes were measured by RT-PCR and 5'-RACE. The acetylation of RprY were detected by western blotting. Electromobility shift and in vitro ChIP assays were used to measure the DNA binding activity of RprY. The expression of RprY target genes was measured by qRT-PCR. Effects of acetylation on phosphorylation of RprY were measured by Phos-tag gels. Results: The rprY gene is cotranscribed with pat. RprY is acetylated in vivo, and autoacetylated in vitro in a reaction that is enhanced by Pat; the CobB sirtuin deacetylates RprY. Acetylation reduced the DNA binding of RprY. Induced oxidative stress decreased production of RprY in vivo, increased its acetylation and increased expression of nqrA. Conclusions: We propose that to compensate for the loss of RprX, P. gingivalis has evolved a novel mechanism to inactivate RprY through acetylation.
RESUMO
We present an overview of how members of the oral microbiota respond to their environment by regulating gene expression through two-component signal transduction systems (TCSs) to support conditions compatible with homeostasis in oral biofilms or drive the equilibrium toward dysbiosis in response to environmental changes. Using studies on the sub-gingival Gram-negative anaerobe Porphyromonas gingivalis and Gram-positive streptococci as examples, we focus on the molecular mechanisms involved in activation of TCS and species specificities of TCS regulons.
RESUMO
The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines.
Assuntos
Proteínas de Bactérias/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Porphyromonas gingivalis/imunologia , Animais , Antibiose , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citocinas/análise , Citocinas/metabolismo , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Escherichia coli/genética , Feminino , Fusobacterium/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-1/análise , Interleucina-1/metabolismo , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Porphyromonas gingivalis/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
Porphyromonas gingivalis is a keystone pathogen in the development and progression of periodontal disease. Obstacles to the development of saturated transposon libraries have previously limited transposon mutant-based screens as well as essential gene studies. We have developed a system for efficient transposon mutagenesis of P. gingivalis using a modified mariner transposon. Tn-seq is a technique that allows for quantitative assessment of individual mutants within a transposon mutant library by sequencing the transposon-genome junctions and then compiling mutant presence by mapping to a base genome. Using Tn-seq, it is possible to quickly define all the insertional mutants in a library and thus identify nonessential genes under the conditions in which the library was produced. Identification of fitness of individual mutants under specific conditions can be performed by exposing the library to selective pressures.
Assuntos
Elementos de DNA Transponíveis/genética , Biblioteca Gênica , Genes Essenciais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidade , Fatores de Virulência/genética , Mapeamento Cromossômico , Meios de Cultura , Mutagênese Insercional , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Estatística como AssuntoRESUMO
The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis) that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.
Assuntos
Vacinas contra a AIDS/imunologia , Vetores Genéticos/genética , Tolerância Imunológica , Mucosa Bucal/imunologia , Streptococcus mitis/genética , Linfócitos T/imunologia , Vacinas Sintéticas/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Antivirais/imunologia , Feminino , Vetores Genéticos/administração & dosagem , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Streptococcus mitis/imunologia , Vacinas Sintéticas/genéticaRESUMO
BACKGROUND: Repetitive regions of DNA and transposable elements have been found to constitute large percentages of eukaryotic and prokaryotic genomes. Such elements are known to be involved in transcriptional regulation, host-pathogen interactions and genome evolution. RESULTS: We identified a minisatellite contained within a miniature inverted-repeat transposable element (MITE) in Porphyromonas gingivalis. The P. gingivalis minisatellite and associated MITE, named 'BrickBuilt', comprises a tandemly repeating twenty-three nucleotide DNA sequence lacking spacer regions between repeats, and with flanking 'leader' and 'tail' subunits that include small inverted-repeat ends. Forms of the BrickBuilt MITE are found 19 times in the genome of P. gingivalis strain ATCC 33277, and also multiple times within the strains W83, TDC60, HG66 and JCVI SC001. BrickBuilt is always located intergenically ranging between 49 and 591 nucleotides from the nearest upstream and downstream coding sequences. Segments of BrickBuilt contain promoter elements with bidirectional transcription capabilities. CONCLUSIONS: We performed a bioinformatic analysis of BrickBuilt utilizing existing whole genome sequencing, microarray and RNAseq data, as well as performing in vitro promoter probe assays to determine potential roles, mechanisms and regulation of the expression of these elements and their affect on surrounding loci. The multiplicity, localization and limited host range nature of MITEs and MITE-like elements in P. gingivalis suggest that these elements may play an important role in facilitating genome evolution as well as modulating the transcriptional regulatory system.
RESUMO
Porphyromonas gingivalis is a Gram-negative oral anaerobe which is strongly associated with periodontal disease. Environmental changes in the gingival sulcus trigger the growth of P. gingivalis and a concurrent shift from periodontal health to disease. Bacteria adjust their physiology in response to environmental changes and gene regulation by two-component phospho-relay systems is one mechanism by which such adjustments are effected. In P. gingivalis RprY is an orphan response regulator and previously we showed that the RprY regulon included genes associated with oxidative stress and sodium metabolism. The goals of the present study were to identify environmental signals that induce rprY and clarify the role of the regulator in the stress response. In Escherichia coli an RprY-LacZ fusion protein was induced in sodium- depleted medium and a P. gingivalis rprY mutant was unable to grow in similar medium. By several approaches we established that sodium depletion induced up-regulation of genes involved in oxidative stress. In addition, we demonstrated that RprY interacted directly with the promoters of several molecular chaperones. Further, both genetic and transcription data suggest that the regulator acts as a repressor. We conclude that RprY is one of the regulators that controls stress responses in P. gingivalis, possibly by acting as a repressor since an rprY mutant showed a superstress reponse in sodium-depleted medium which we propose inhibited growth.
Assuntos
Proteínas de Bactérias/metabolismo , Estresse Oxidativo , Porphyromonas gingivalis/fisiologia , Cloreto de Sódio/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Osmorregulação , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Ativação Transcricional , TranscriptomaRESUMO
Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with infection of the periodontia. The organism has a small number of two-component signal transduction systems, and after comparing genome sequences of strains W83 and ATCC 33277 we discovered that the latter was mutant in histidine kinase (PGN_0752), while the cognate response regulator (PGN_0753) remained intact. Microarray-based transcriptional profiling and ChIP-seq assays were carried out with an ATCC 33277 transconjugant containing the functional histidine kinase from strain W83 (PG0719). The data showed that the regulon of this signal transduction system contained genes that were involved in hemin acquisition, including gingipains, at least three transport systems, as well as being self-regulated. Direct regulation by the response regulator was confirmed by electrophoretic mobility shift assays. In addition, the system appears to be activated by hemin and the regulator acts as both an activator and repressor.
Assuntos
Proteínas de Bactérias/genética , Infecções por Bacteroidaceae/microbiologia , Hemina/metabolismo , Interações Hospedeiro-Patógeno , Porphyromonas gingivalis/fisiologia , Proteínas Quinases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Infecções por Bacteroidaceae/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Histidina Quinase , Humanos , Ferro/metabolismo , Dados de Sequência Molecular , Mutação , Porphyromonas gingivalis/genética , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Porphyromonas gingivalis is a Gram-negative oral anaerobe associated with chronic adult periodontitis. Its ecological niche is the gingival crevice, where the organism adapts to the challenges of the infectious process such as host defence and bacterial products. Bacterial responses to environmental changes are partly regulated by two-component signal transduction systems. Several intact systems were annotated in the genome of P. gingivalis, as well as an orphan regulator encoding a homologue of RprY, a response regulator from Bacteroides fragilis. With the goal of defining the environmental cues that activate RprY in P. gingivalis, we used several strategies to identify its regulon. Results from gene expression and DNA-protein binding assays identified target genes that were either involved in transport functions or associated with oxidative stress, and indicated that RprY can act as an activator and a repressor. RprY positively activated the primary sodium pump, NADH : ubiquinone oxidoreductase (NQR), and RprY protein also interacted with the promoter regions of nqrA genes from B. fragilis and Vibrio cholerae. Given that gingival bleeding and infiltration of host defence cells are symptoms of periodontal infection, iron products released from blood and reactive oxygen species from polymorphonuclear leucocytes may be potential inducers of the RprY regulon.
Assuntos
Porphyromonas gingivalis/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Bactérias/biossíntese , Bacteroides fragilis/genética , Sequência de Bases , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Bacteriano/biossíntese , RNA Mensageiro/biossíntese , Regulon , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Vibrio cholerae/genéticaRESUMO
Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity ( approximately 61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased auto-aggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.