RESUMO
Bordetella bronchiseptica chronically infects a wide range of mammals, and resides primarily in the nasal cavity of the infected host. Multiple virulence factors of Bordetella species have been studied in the context of lower respiratory tract infections, but relatively less is known about the bacterial life cycle in the nasal cavity. Evidences were discovered for Bvg intermediate (Bvg(i)) phase expression in vivo and that the major adhesin filamentous hemagglutinin plays a major role in the colonization of B. bronchiseptica in the unciliated olfactory epithelia of the nasal cavity.
Assuntos
Adesinas Bacterianas/metabolismo , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Regulação Bacteriana da Expressão Gênica , Cavidade Nasal/microbiologia , Fatores de Virulência de Bordetella/metabolismo , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/metabolismo , Epitélio/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Cavidade Nasal/citologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Virulência de Bordetella/genéticaRESUMO
We have previously reported that the respiratory pathogen Bordetella bronchiseptica can form biofilms in vitro. In this report, we demonstrate the disruption of B. bronchiseptica biofilms by rhamnolipids secreted from Pseudomonas aeruginosa. This suggests that biosurfactants such as rhamnolipids may be utilized as antimicrobial agents for removing Bordetella biofilms.
Assuntos
Biofilmes/efeitos dos fármacos , Bordetella bronchiseptica/efeitos dos fármacos , Glicolipídeos/farmacologia , Pseudomonas aeruginosa/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Bordetella bronchiseptica/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Glicolipídeos/isolamento & purificação , Coloração e Rotulagem , Tensoativos/isolamento & purificação , Tensoativos/farmacologiaRESUMO
We previously showed that the Bvg virulence control system regulates biofilm formation in Bordetella bronchiseptica (Y. Irie, S. Mattoo, and M. H. Yuk, J. Bacteriol. 186:5692-5698, 2004). Analyses of the extracellular components of B. bronchiseptica biofilm matrix revealed that the major sugar component in the matrix was xylose, and linkage analysis indicated a majority of it to be in a 4-linked polymeric form. The production of xylose was independent of Bvg regulation but instead was dependent on bacterial growth phase. In addition, N-acetyl-glucosamine in the matrix was found to be important for the initial development of the biofilm. These results suggest that B. bronchiseptica biofilm formation is growth phase dependent in addition to being regulated by the Bvg virulence system.
Assuntos
Biofilmes/crescimento & desenvolvimento , Bordetella/metabolismo , Regulação Bacteriana da Expressão Gênica , Xilose/biossíntese , Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Bordetella/genética , Bordetella/crescimento & desenvolvimento , Deleção de Genes , Polissacarídeos Bacterianos/biossíntese , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Bordetella bronchiseptica is a Gram-negative bacterium equipped with several colonization factors that allow it to establish a persistent infection of the murine respiratory tract. Previous studies indicate that B. bronchiseptica adenylate cyclase toxin (ACT) and the type III secretion system (TTSS) synergize to drive dendritic cells into an altered phenotype to down-regulate the host immune response. In this study, we examined the effects of B. bronchiseptica ACT and TTSS on murine bone marrow-derived macrophages. We demonstrate that ACT and TTSS are required for the inhibition of Ag-driven CD4+ T cell proliferation by bacteria-infected macrophages. We identify PGE2 as the mediator of this inhibition, and we show that ACT and the TTSS synergize to increase macrophage production of PGE2. We further demonstrate that B. bronchiseptica can modulate normal macrophage function and drive the immune response toward a Th17 phenotype classified by the significant production of IL-17. In this study, we show that B. bronchiseptica-infected macrophages can induce IL-17 production from naive CD4+ splenocytes, and that lung tissues from B. bronchiseptica-infected mice exhibit a strong Th17 immune response. ACT inhibited surface expression of CD40 and CD86, suppressed TNF-alpha production, and up-regulated IL-6 production. TTSS also synergized with ACT to up-regulate IL-10 and PGE2 secretion. These findings indicate that persistent colonization by B. bronchiseptica may rely on the ability of the bacteria to differentially modulate both macrophage and dendritic cell function leading to an altered adaptive immune response and subsequent bacterial colonization.
Assuntos
Bordetella bronchiseptica/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Inibidores do Crescimento/imunologia , Interleucina-17/biossíntese , Macrófagos/imunologia , Macrófagos/microbiologia , Toxina Adenilato Ciclase/farmacologia , Animais , Bordetella bronchiseptica/patogenicidade , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Dinoprostona/biossíntese , Feminino , Imunofenotipagem , Interleucina-17/metabolismo , Interleucina-17/fisiologia , Ativação Linfocitária/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Baço/citologia , Baço/imunologia , Baço/metabolismoRESUMO
Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-kappaB in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.
Assuntos
Bordetella bronchiseptica/metabolismo , Ativação de Macrófagos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Toxina Adenilato Ciclase/fisiologia , Animais , Bordetella bronchiseptica/imunologia , Células Cultivadas , Regulação para Baixo , Interleucina-10/biossíntese , Interleucina-6/biossíntese , Sistema de Sinalização das MAP Quinases , Camundongos , FosforilaçãoRESUMO
Chronic bacterial infection reflects a balance between the host immune response and bacterial factors that promote colonization and immune evasion. Bordetella bronchiseptica uses a type III secretion system (TTSS) to persist in the lower respiratory tract of mice. We hypothesize that colonization is facilitated by bacteria-driven modulation of dendritic cells (DCs), which leads to an immunosuppressive adaptive host response. Migration of DCs to the draining lymph nodes of the respiratory tract was significantly increased in mice infected with wild-type B. bronchiseptica compared with mice infected with TTSS mutant bacteria. Reduced colonization by TTSS-deficient bacteria was evident by 7 days after infection, whereas colonization by wild-type bacteria remained high. This decrease in colonization correlated with peak IFN-gamma production by restimulated splenocytes from infected animals. Wild-type bacteria also elicited peak IFN-gamma production on day 7, but the quantity was significantly lower than that elicited by TTSS mutant bacteria. Additionally, wild-type bacteria elicited higher levels of the immunosuppressive cytokine IL-10 compared with the TTSS mutant bacteria. B. bronchiseptica colonization in IL-10(-/-) mice was significantly reduced compared with infections in wild-type mice. These findings suggest that B. bronchiseptica use the TTSS to rapidly drive respiratory DCs to secondary lymphoid tissues where these APCs stimulate an immunosuppressive response characterized by increased IL-10 and decreased IFN-gamma production that favors bacterial persistence.
Assuntos
Infecções por Bordetella/imunologia , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Terapia de Imunossupressão , Animais , Proteínas de Bactérias/fisiologia , Infecções por Bordetella/metabolismo , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Células Dendríticas/citologia , Regulação para Baixo/imunologia , Interferon Tipo I/metabolismo , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismo , Infecções Respiratórias/microbiologia , Regulação para Cima/imunologiaRESUMO
Expression of innate immune genes such as beta-defensins is induced in airway epithelium by bacterial components via activation of NF-kappaB. We show here that live Gram-negative bacteria can similarly stimulate this pathway, resulting in upregulation of the beta-defensin tracheal antimicrobial peptide (TAP) in primary cultures of bovine tracheal epithelial cells (TECs), by a Toll-like receptor 4 (TLR4)-mediated pathway. The Gram-negative airway pathogen Bordetella bronchiseptica possesses a type III secretion system previously suggested to inhibit the nuclear translocation of NF-kappaB in a cell line by immunohistochemistry. We therefore hypothesized that this pathogen might interfere in the innate immune response of the epithelium. Exposure of TECs to wild-type B. bronchiseptica suppressed the activation of NF-kappaB and the subsequent induction of TAP mRNA levels, whereas a type III secretion-defective strain did not. These results suggest a mechanism for bacterial evasion of the innate immune response in the airway, which could allow for the observed persistent colonization of this pathogen.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bordetella bronchiseptica/patogenicidade , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Traqueia/microbiologia , beta-Defensinas/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Bovinos , Células Cultivadas , Células Epiteliais/microbiologia , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Receptor 4 Toll-Like , Receptores Toll-Like , Traqueia/citologia , Regulação para Cima , beta-Defensinas/genéticaRESUMO
Bordetella bronchiseptica utilizes a type III secretion system (TTSS) for induction of non-apoptotic cytotoxicity in host cells and modulation of host immunity. The identity of Bordetella TTSS effectors, however, has remained elusive. Here we report a genome-wide screen for TTSS effectors based on shared biophysical and functional characteristics of class I chaperones and their frequent colocalization with TTSS effectors. When applied to B. bronchiseptica, the screen identified the first TTSS chaperone-effector locus, btcA-bteA, and we experimentally confirmed its function. Expression of bteA is co-ordinated with expression of TTSS apparatus genes, BteA is secreted through the TTSS of B. bronchiseptica, it is required for cytotoxicity towards mammalian cells, and it is highly conserved in the human-adapted subspecies B. pertussis and B. parapertussis. Transfection of bteA into epithlieal cells results in rapid cell death, indicating that BteA alone is sufficient to induce potent cytotoxicity. Finally, an in vitro interaction between BteA and BtcA was demonstrated. The search for TTSS chaperones and effectors was then expanded to other bacterial genomes, including mammalian and insect pathogens, where we identified a large number of novel candidate chaperones and effectors. Although the majority of putative effectors are proteins of unknown function, several have similarities to eukaryotic protein domains or previously identified effectors from other species.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Técnicas Genéticas , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Western Blotting , Bordetella parapertussis/genética , Bordetella pertussis/genética , Morte Celular , Biologia Computacional , Sequência Conservada , Células Epiteliais/microbiologia , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Hemólise , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Filogenia , Ligação Proteica , Transporte Proteico , Homologia de Sequência de AminoácidosRESUMO
Bordetella species utilize the BvgAS (Bordetella virulence gene) two-component signal transduction system to sense the environment and regulate gene expression among at least three phases: a virulent Bvg+ phase, a nonvirulent Bvg- phase, and an intermediate Bvgi phase. Genes expressed in the Bvg+ phase encode known virulence factors, including adhesins such as filamentous hemagglutinin (FHA) and fimbriae, as well as toxins such as the bifunctional adenylate cyclase/hemolysin (ACY). Previous studies showed that in the Bvgi phase, FHA and fimbriae continue to be expressed, but ACY expression is significantly downregulated. In this report, we determine that Bordetella bronchiseptica can form biofilms in vitro and that the generation of biofilm is maximal in the Bvgi phase. We show that FHA is required for maximal biofilm formation and that fimbriae may also contribute to this phenotype. However, expression of ACY inhibits biofilm formation, most likely via interactions with FHA. Therefore, the coordinated regulation of adhesins and ACY expression leads to maximal biofilm formation in the Bvgi phase in B. bronchiseptica.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/fisiologia , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/fisiologia , Virulência/genética , Adaptação Fisiológica , Toxina Adenilato Ciclase/genética , Toxina Adenilato Ciclase/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/citologia , Bordetella bronchiseptica/patogenicidade , Hemaglutininas/genética , Hemaglutininas/metabolismo , Niacina/metabolismo , Regulon , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/metabolismoRESUMO
The BvgAS virulence control system regulates the expression of type III secretion genes in Bordetella subspecies that infect humans and other mammals. We have identified five open reading frames, btrS, btrU, btrX, btrW and btrV, that are activated by BvgAS and encode regulatory factors that control type III secretion at the levels of transcription, protein expression and secretion. The btrS gene product bears sequence similarity to ECF (extracytoplasmic function) sigma factors and is required for transcription of the bsc locus. btrU, btrW and btrV encode proteins predicted to contain PP2C-like Ser phosphatase, HPK (His protein kinase)-like Ser kinase and STAS anti-sigma factor antagonist domains, respectively, which are characteristic of Gram-positive partner switching proteins in Bacillus subtilis. BtrU and BtrW are required for secretion of proteins that are exported by the bsc type III secretion system, whereas BtrV is specifically required for protein synthesis and/or stability. Bordetella species have thus evolved a unique cascade to differentially regulate type III secretion that combines a canonical phosphorelay system with an ECF sigma factor and a set of proteins with domain signatures that define partner switchers, which were traditionally thought to function only in Gram-positive bacteria. The presence of multiple layers and mechanisms of regulation most likely reflects the need to integrate multiple signals in controlling type III secretion. The bsc and btr loci are nearly identical between broad-host-range and human-specific Bordetella. Comparative analysis of Bordetella subspecies revealed that, whereas bsc and btr loci were transcribed in all subspecies, only broad-host-range strains expressed a functional type III secretion system in vitro. The block in type III secretion is post-transcriptional in human-adapted strains, and signal recognition appears to be a point of divergence between subspecies.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella/genética , Bordetella/metabolismo , Fatores de Virulência de Bordetella/genética , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reguladores , Fosfoproteínas Fosfatases/genética , Filogenia , Proteínas Quinases/genética , Transporte Proteico , Homologia de Sequência de Aminoácidos , Fator sigma/antagonistas & inibidores , Fator sigma/genética , Transdução de Sinais , Transcrição Gênica , Fatores de Virulência de Bordetella/metabolismoRESUMO
Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.
Assuntos
Toxina Adenilato Ciclase/fisiologia , Infecções por Bordetella/imunologia , Bordetella bronchiseptica/imunologia , Células Dendríticas/imunologia , Animais , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-2 , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/fisiologia , Antígenos CD40/biossíntese , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Tolerância Imunológica , Interleucina-12/biossíntese , Interleucina-12/genética , Interleucina-12/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , FagocitoseRESUMO
The genomes of three closely related bordetellae are currently being sequenced, thus providing an opportunity for comparative genomic approaches driven by an understanding of the comparative biology of these three bacteria. Although the other strains being sequenced are well studied, the strain of Bordetella parapertussis chosen for sequencing is a recent human clinical isolate (strain 12822) that has yet to be characterized in detail. This investigation reports the first phenotypic characterization of this strain, which will likely become the prototype for this species in comparison with the prototype strains of B. pertussis (Tohama I), B. bronchiseptica (RB50), and other isolates of B. parapertussis. Multiple in vitro and in vivo assays distinguished each species. B. parapertussis was more similar to B. bronchiseptica than to B. pertussis in many assays, including in BvgS signaling characteristics, presence of urease activity, regulation of urease expression by BvgAS, virulence in the respiratory tracts of immunocompromised mice, induction of anti-Bordetella antibodies, and serum antimicrobial resistance. In other assays, B. parapertussis was distinct from all other species (in pigment production) or more similar to B. pertussis (by lack of motility and cytotoxicity to a macrophage-like cell line). These results begin to provide phenotypes that can be related to genetic differences identified in the genomic sequences of bordetellae.