RESUMO
The respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica employ a type III secretion system (T3SS) to inject a 69-kDa BteA effector protein into host cells. This effector is known to contain two functional domains, including an N-terminal lipid raft targeting (LRT) domain and a cytotoxic C-terminal domain that induces nonapoptotic and caspase-1-independent host cell death. However, the exact molecular mechanisms underlying the interaction of BteA with plasma membrane (PM) as well as its cytotoxic activity in the course of Bordetella infections remain poorly understood. Using a protein-lipid overlay assay and surface plasmon resonance, we show here that the recombinant LRT domain binds negatively charged membrane phospholipids. Specifically, we determined that the dissociation constants of the LRT domain-binding liposomes containing phosphatidylinositol 4,5-bisphosphate, phosphatidic acid, and phosphatidylserine were â¼450 nM, â¼490 nM, and â¼1.2 µM, respectively. Both phosphatidylserine and phosphatidylinositol 4,5-bisphosphate were required to target the LRT domain and/or full-length BteA to the PM of yeast cells. The membrane association further involved electrostatic and hydrophobic interactions of LRT and depended on a leucine residue in the L1 loop between the first two helices of the four-helix bundle. Importantly, charge-reversal substitutions within the L1 region disrupted PM localization of the BteA effector without hampering its cytotoxic activity during B. bronchiseptica infection of HeLa cells. The LRT-mediated targeting of BteA to the cytosolic leaflet of the PM of host cells is, therefore, dispensable for effector cytotoxicity.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Bactérias/genética , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/crescimento & desenvolvimento , Células HeLa , Humanos , Ligação Proteica , Domínios ProteicosRESUMO
Temperate phages are considered as natural vectors for gene transmission among bacteria due to the ability to integrate their genomes into a host chromosome, therefore, affect the fitness and phenotype of host bacteria. Many virulence genes of pathogenic bacteria were identified in temperate phage genomes, supporting the concept that temperate phages play important roles in increasing the bacterial pathogenicity through delivery of the virulence genes. However, little is known about the roles of temperate phages in attenuation of bacterial virulence. Here, we report a novel Bordetella bronchiseptica temperate phage, vB_BbrS_PHB09 (PHB09), which has a 42,129-bp dsDNA genome with a G+C content of 62.8%. Phylogenetic analysis based on large terminase subunit indicated that phage PHB09 represented a new member of the family Siphoviridae. The genome of PHB09 contains genes encoding lysogen-associated proteins, including integrase and cI protein. The integration site of PHB09 is specifically located within a pilin gene of B. bronchiseptica. Importantly, we found that the integration of phage PHB09 significantly decreased the virulence of parental strain B. bronchiseptica Bb01 in mice, most likely through disruption the expression of pilin gene. Moreover, a single shot of the prophage bearing B. bronchiseptica strain completely protected mice against lethal challenge with wild-type virulent B. bronchiseptica, indicating the vaccine potential of lysogenized strain. Our findings not only indicate the complicated roles of temperate phages in bacterial virulence other than simple delivery of virulent genes but also provide a potential strategy for developing bacterial vaccines.
Assuntos
Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/patogenicidade , Bordetella bronchiseptica/virologia , Lisogenia , Siphoviridae/fisiologia , Animais , Vacinas Bacterianas/imunologia , Infecções por Bordetella/prevenção & controle , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/imunologia , DNA Viral/genética , Feminino , Genoma Viral , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Prófagos/genética , Prófagos/fisiologia , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections in mammals, including humans, and are generally cultivated on Bordet-Gengou (BG) agar plates in laboratories. The medium requires animal blood as a supplement for better bacterial growth. However, using blood is problematic, as its constant supply is occasionally difficult because of the limited shelf-life. This study proposes modified BG agar plates supplemented with bovine serum albumin and fetal bovine serum as a simple and convenient medium that confers sufficient growth of bordetellae.
Assuntos
Produtos Biológicos , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella parapertussis/crescimento & desenvolvimento , Bordetella pertussis/crescimento & desenvolvimento , Meios de Cultura/química , Soroalbumina Bovina/análise , Ágar , Soroalbumina Bovina/químicaRESUMO
Background: Why resistance to specific antibiotics emerges and spreads rapidly in some bacteria confronting these drugs but not others remains a mystery. Resistance to erythromycin in the respiratory pathogens Staphylococcus aureus and Streptococcus pneumoniae emerged rapidly and increased problematically. However, resistance is uncommon amongst the classic Bordetella species despite infections being treated with this macrolide for decades. Objectives: We examined whether the apparent progenitor of the classic Bordetella spp., Bordetella bronchiseptica, is able to rapidly generate de novo resistance to antibiotics and, if so, why such resistance might not persist and propagate. Methods: Independent strains of B. bronchiseptica resistant to erythromycin were generated in vitro by successively passaging them in increasing subinhibitory concentrations of this macrolide. Resistant mutants obtained were evaluated for their capacity to infect mice, and for other virulence properties including adherence, cytotoxicity and induction of cytokines. Results: B. bronchiseptica rapidly developed stable and persistent antibiotic resistance de novo. Unlike the previously reported trade-off in fitness, multiple independent resistant mutants were not defective in their rates of growth in vitro but were consistently defective in colonizing mice and lost a variety of virulence phenotypes. These changes rendered them avirulent but phenotypically similar to the previously described growth phase associated with the ability to survive in soil, water and/or other extra-mammalian environments. Conclusions: These observations raise the possibility that antibiotic resistance in some organisms results in trade-offs that are not quantifiable in routine measures of general fitness such as growth in vitro, but are pronounced in various aspects of infection in the natural host.
Assuntos
Antibacterianos/farmacologia , Infecções por Bordetella/microbiologia , Infecções por Bordetella/patologia , Bordetella bronchiseptica/efeitos dos fármacos , Bordetella bronchiseptica/patogenicidade , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Animais , Aderência Bacteriana , Toxinas Bacterianas/metabolismo , Bordetella bronchiseptica/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos , Mutação , Seleção Genética , Inoculações Seriadas , VirulênciaRESUMO
Many of the pathogenic species of the genus Bordetella have an absolute requirement for nicotinic acid (NA) for laboratory growth. These Gram-negative bacteria also harbor a gene cluster homologous to the nic cluster of Pseudomonas putida which is involved in the aerobic degradation of NA and its transcriptional control. We report here that BpsR, a negative regulator of biofilm formation and Bps polysaccharide production, controls the growth of Bordetella bronchiseptica by repressing the expression of nic genes. The severe growth defect of the ΔbpsR strain in Stainer-Scholte medium was restored by supplementation with NA, which also functioned as an inducer of nic genes at low micromolar concentrations that are usually present in animals and humans. Purified BpsR protein bound to the nic promoter region, and its DNA binding activity was inhibited by 6-hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradative pathway. Reporter assays with the isogenic mutant derivative of the wild-type (WT) strain harboring deletion in nicA, which encodes a putative nicotinic acid hydroxylase responsible for conversion of NA to 6-HNA, showed that 6-HNA is the actual inducer of the nic genes in the bacterial cell. Gene expression profiling further showed that BpsR dually activated and repressed the expression of genes associated with pathogenesis, transcriptional regulation, metabolism, and other cellular processes. We discuss the implications of these findings with respect to the selection of pyridines such as NA and quinolinic acid for optimum bacterial growth depending on the ecological niche.IMPORTANCE BpsR, the previously described regulator of biofilm formation and Bps polysaccharide production, controls Bordetella bronchiseptica growth by regulating the expression of genes involved in the degradation of nicotinic acid (NA). 6-Hydroxynicotinic acid (6-HNA), the first metabolite of the NA degradation pathway prevented BpsR from binding to DNA and was the actual in vivo inducer. We hypothesize that BpsR enables Bordetella bacteria to efficiently and selectively utilize NA for their survival depending on the environment in which they reside. The results reported herein lay the foundation for future investigations of how BpsR and the alteration of its activity by NA orchestrate the control of Bordetella growth, metabolism, biofilm formation, and pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/metabolismo , Regulação Bacteriana da Expressão Gênica , Niacina/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Bordetella bronchiseptica/genética , Deleção de Genes , Genes Reguladores , Transcrição GênicaRESUMO
Objective: We constructed Bordetella bronchiseptica QH0814 hcp mutant to characterize its pathogenicity.[Methods] Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD50) and the infection capacity. Methods: Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QH0814Δhcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose (LD50) and the infection capacity. Results: There was no significant variation of the growth rate between the mutant and the parental strain. Compared with the parental strain, the adherence ability of the mutant did not change remarkably. However, the invasion ability decreased significantly. Mice lethal test showed that the LD50 of the mutant was higher than that of the parental strain. Correspondingly, the bacterial colonization of the mutant in mice blood, lung and liver was much less than that of the parental strain. Conclusion: The knocking-out of the hcp gene had no influence on bacterial growth, but it could attenuate significantly the invasion and colonization of the bacterium. Therefore, the gene may play a role in the pathogenesis of Bordetella bronchiseptica.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Bordetella/veterinária , Bordetella bronchiseptica/metabolismo , Doenças dos Suínos/microbiologia , Animais , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Camundongos , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , Suínos , VirulênciaRESUMO
BACKGROUND: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. RESULTS: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. CONCLUSIONS: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.
Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/crescimento & desenvolvimento , Actinobacillus pleuropneumoniae/metabolismo , Biofilmes/crescimento & desenvolvimento , NAD/deficiência , Acetilglucosamina/metabolismo , Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/isolamento & purificação , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Biofilmes/efeitos dos fármacos , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/metabolismo , Meios de Cultura , Desoxirribonuclease I/farmacologia , Endopeptidase K/farmacologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Hibridização in Situ Fluorescente , Microscopia Confocal , Niacinamida/análogos & derivados , Niacinamida/deficiência , Mononucleotídeo de Nicotinamida/deficiência , Pasteurella multocida/crescimento & desenvolvimento , Pasteurella multocida/metabolismo , Piridinas/metabolismo , Compostos de Piridínio , Especificidade da Espécie , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Células-Tronco , Streptococcus suis/crescimento & desenvolvimento , Streptococcus suis/metabolismo , Suínos , Doenças dos Suínos/microbiologiaRESUMO
Bordetella bronchiseptica, an aerobic Gram-negative bacterium, is capable of colonizing the respiratory tract of diverse animals and chronically persists inside the hosts by forming biofilm. Most known virulence factors in Bordetella species are regulated by the BvgAS two-component transduction system. The Bvg-activated proteins play a critical role during host infection. OmpQ is an outer membrane porin protein which is expressed under BvgAS control. Here, we studied the contribution of OmpQ to the biofilm formation process by B. bronchiseptica. We found that the lack of expression of OmpQ did not affect the growth kinetics and final biomass of B. bronchiseptica under planktonic growth conditions. The ΔompQ mutant strain displayed no differences in attachment level and in early steps of biofilm formation. However, deletion of the ompQ gene attenuated the ability of B. bronchiseptica to form a mature biofilm. Analysis of ompQ gene expression during the biofilm formation process by B. bronchiseptica showed a dynamic expression pattern, with an increase of biofilm culture at 48âh. Moreover, we demonstrated that the addition of serum anti-OmpQ had the potential to reduce the biofilm biomass formation in a dose-dependent manner. In conclusion, we showed for the first time, to the best of our knowledge, evidence of the contribution of OmpQ to a process of importance for B. bronchiseptica pathobiology. Our results indicate that OmpQ plays a role during the biofilm development process, particularly at later stages of development, and that this porin could be a potential target for strategies of biofilm formation inhibition.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Biofilmes/crescimento & desenvolvimento , Bordetella bronchiseptica , Porinas/genética , Fatores de Virulência de Bordetella/genética , Proteínas de Bactérias/genética , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genéticaRESUMO
Bordetella bronchiseptica is a Gram-negative bacterium responsible for respiratory diseases in many mammalian hosts, including humans. This pathogen has been shown as able to persist inside the host cells, even in the phagosomes that are acidified to pH 4.5-5.0 after bacterial infection. Here we evaluated the resistance of B. bronchiseptica to survive under acidic conditions. In particular we analyzed the bacterial capacity to develop the mechanism known as acid tolerance response (ATR). Our studies were mainly focused on the avirulent phase of the bacteria since this phenotypic phase was reported to be more resistant to environmental stress conditions than the virulent phase. Results from B. bronchiseptica in virulent phase were also included for comparison purposes. In fact, for B. bronchiseptica 9.73 bacteria in virulent phase we observed that the viability of bacteria does not decrease significantly when grown at pH as low as 4.5, but it is affected when the pH of the medium was equal to or less than 4.0. After acid-adaptation at pH 5.5 for several hours, the survival rate of B. bronchiseptica 9.73 at lethal pH 4.0 for 6h was increased. Interestingly, the avirulent phase mediated by the two-component BvgAS system conferred further resistance to lethal acid challenge and a marked increase in the magnitude of the expressed ATR. The ATR for this avirulent phase seems to be associated with changes in LPS and surface protein profiles. 2D-gel electrophoresis revealed at least 25 polypeptides differentially expressed, 17 of which were only expressed or over-expressed under acid conditions. Using MALDI-TOF mass spectrometry, 10 of these differentially expressed polypeptides were identified.
Assuntos
Bordetella bronchiseptica/fisiologia , Tolerância a Medicamentos , Adaptação Fisiológica , Bordetella bronchiseptica/efeitos dos fármacos , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Concentração de Íons de Hidrogênio , Viabilidade Microbiana , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição , VirulênciaRESUMO
Analyses of bacterial genes expressed in response to the host environment provide clues to understanding the host-pathogen interactions that lead to the establishment of infection. In this study, a novel method named In Vivo Expressed-Tag ImmunoPrecipitation (IVET-PI) was developed for detecting genes expressed in bacteria that are recovered in a small numbers from host tissues. IVET-IP was designed to overcome some drawbacks of previous similar methods. We applied IVET-IP to Bordetella bronchiseptica colonizing rat trachea and identified 173 genes that were expressed in the bacteria over the entire course of an infection. These gene products included two transcriptional factors that are involved in the expression of filamentous hemagglutinin, adenylate cyclase toxin, and major virulence factors for the bordetellae. We consider that this method might provide novel insight into the course of Bordetella infection.
Assuntos
Infecções por Bordetella/veterinária , Bordetella bronchiseptica/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Imunoprecipitação/métodos , Traqueia/microbiologia , Animais , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/genética , Feminino , Ratos WistarRESUMO
The type III secretion system (T3SS) is a sophisticated protein secretion machinery that delivers bacterial virulence proteins into host cells. A needle-tip protein, Bsp22 , is one of the secreted substrates of the T3SS and plays an essential role in the full function of the T3SS in Bordetella bronchiseptica. In this study, we found that BB1618 functions as a chaperone for Bsp22 . The deletion of BB1618 resulted in a dramatic impairment of Bsp22 secretion into the culture supernatants and Bsp22 stability in the bacterial cytosol. In contrast, the secretion of other type III secreted proteins was not affected by the BB1618 mutation. Furthermore, the BB1618 mutant strain could not induce cytotoxicity and displayed the same phenotypes as the Bsp22 mutant strain. An immunoprecipitation assay demonstrated that BB1618 interacts with Bsp22 , but not with BopB and BopD . Thus, we identified BB1618 as a specific type III chaperone for Bsp22 . Therefore, we propose that BB1618 be renamed Btc22 for the Bordetella type III chaperone for Bsp22 .
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/fisiologia , Bordetella bronchiseptica/metabolismo , Bordetella bronchiseptica/patogenicidade , Chaperonas Moleculares/metabolismo , Animais , Proteínas de Bactérias/genética , Bordetella bronchiseptica/crescimento & desenvolvimento , Linhagem Celular , Eritrócitos/microbiologia , Células HeLa , Hemólise , Humanos , Imunoprecipitação , Chaperonas Moleculares/genética , Transporte Proteico , Coelhos , VirulênciaRESUMO
Growth phase-dependent gene regulation has recently been demonstrated to occur in Bordetella pertussis, with many transcripts, including known virulence factors, significantly decreasing during the transition from logarithmic to stationary-phase growth. Given that B. pertussis is thought to have derived from a Bordetella bronchiseptica-like ancestor, we hypothesized that growth phase-dependent gene regulation would also occur in B. bronchiseptica. Microarray analysis revealed and quantitative real-time PCR (qRT-PCR) confirmed that growth phase-dependent gene regulation occurs in B. bronchiseptica, resulting in prominent temporal shifts in global gene expression. Two virulence phenotypes associated with these gene expression changes were tested. We found that growth-dependent increases in expression of some type III secretion system (TTSS) genes led to a growth phase-dependent increase in a TTSS-dependent function, cytotoxicity. Although the transcription of genes encoding adhesins previously shown to mediate adherence was decreased in late-log and stationary phases, we found that the adherence of B. bronchiseptica did not decrease in these later phases of growth. Microarray analysis revealed and qRT-PCR confirmed that growth phase-dependent gene regulation occurred in both Bvg(+) and Bvg(-) phase-locked mutants, indicating that growth phase-dependent gene regulation in B. bronchiseptica can function independently from the BvgAS regulatory system.
Assuntos
Bordetella bronchiseptica/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Animais , Aderência Bacteriana , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Linhagem Celular , Contagem de Colônia Microbiana , Macrófagos/microbiologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of Bordetella bronchiseptica in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the Bordetella bfeA enterobactin catechol xenosiderophore receptor gene, neither a bfeA mutant nor a bfeR regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a tonB mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The B. bronchiseptica genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of (55)Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates B. bronchiseptica iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.
Assuntos
Bordetella bronchiseptica/efeitos dos fármacos , Bordetella bronchiseptica/metabolismo , Ferro/metabolismo , Norepinefrina/farmacologia , Transferrina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/crescimento & desenvolvimento , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Ácidos Hidroxâmicos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Simpatomiméticos/farmacologiaRESUMO
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
Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.
Assuntos
Bordetella bronchiseptica/metabolismo , Hemina/metabolismo , Hemoglobinas/metabolismo , Mioglobina/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Bordetella bronchiseptica/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ferro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , CachaloteRESUMO
Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptica. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaccharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/metabolismo , Polissacarídeos Bacterianos/metabolismo , Infecções Respiratórias/microbiologia , Animais , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/ultraestrutura , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Cavidade Nasal/microbiologia , Cavidade Nasal/ultraestruturaRESUMO
The genus Bordetella includes a group of closely related mammalian pathogens that cause a variety of respiratory diseases in a long list of animals (B. bronchiseptica) and whooping cough in humans (B. pertussis and B. parapertussis). While past research has examined how these pathogens are eliminated from the lower respiratory tract, the host factors that control and/or clear the bordetellae from the upper respiratory tract remain unclear. We hypothesized that immunoglobulin A (IgA), the predominant mucosal antibody isotype, would have a protective role against these mucosal pathogens. IgA(-/-) mice were indistinguishable from wild-type mice in their control and clearance of B. pertussis or B. parapertussis, suggesting that IgA is not crucial to immunity to these organisms. However, naïve and convalescent IgA(-/-) mice were defective in reducing the numbers of B. bronchiseptica in the upper respiratory tract compared to wild-type controls. Passively transferred serum from convalescent IgA(-/-) mice was not as effective as serum from convalescent wild-type mice in clearing this pathogen from the tracheae of naive recipient mice. IgA induced by B. bronchiseptica infection predominantly recognized lipopolysaccharide-containing O-antigen, and antibodies against O-antigen were important to bacterial clearance from the trachea. Since an IgA response contributes to the control of B. bronchiseptica infection of the upper respiratory tract, immunization strategies aimed at inducing B. bronchiseptica-specific IgA may be beneficial to preventing the spread of this bacterium among domestic animal populations.
Assuntos
Infecções por Bordetella/imunologia , Infecções por Bordetella/microbiologia , Bordetella/imunologia , Imunoglobulina A/fisiologia , Animais , Formação de Anticorpos/genética , Infecções por Bordetella/genética , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/imunologia , Bordetella parapertussis/crescimento & desenvolvimento , Bordetella parapertussis/imunologia , Bordetella pertussis/crescimento & desenvolvimento , Bordetella pertussis/imunologia , Imunidade Inata/genética , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imunoglobulina A/genética , Pneumopatias/imunologia , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos O/imunologia , Infecções Respiratórias/genética , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Traqueia/imunologia , Traqueia/microbiologiaRESUMO
We examine the effects of aquatic toxicity on Daphnia magna, the antimicrobial activity of new anionic lysine-derivative surfactants, and the influence of different-sized counterions associated with the surfactants. Surfactants with Tris and Lithium had less of a toxic effect on Daphnia, while all surfactants proved highly active against yeasts and the gram-negative bacteria Bordetella bronchiseptica. Counterion size was found to have no effect on aquatic toxicity or antimicrobial activity.
Assuntos
Anti-Infecciosos/farmacologia , Bordetella bronchiseptica/crescimento & desenvolvimento , Daphnia/crescimento & desenvolvimento , Lisina/farmacologia , Tensoativos/farmacologia , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/toxicidade , Lisina/síntese química , Lisina/toxicidade , Tensoativos/síntese química , Tensoativos/toxicidadeRESUMO
Several species of pathogenic microorganisms have developed strategies to survive and persist in vital organs which are normally maintained as sterile by the generation of strong immune responses. Here, we report an immunomodulation involving the Bordetella bronchiseptica type III secretion system (TTSS) which contributes to bacterial survival in the lower respiratory tract of the host. The prolonged persistence of B. bronchiseptica that was observed in gamma interferon (IFN-gamma)-/- mice indicates that the efficient clearance of bacteria from the lower respiratory tract requires not only B cells and antibodies but also IFN-gamma production. Our data also suggest that interleukin-10 (IL-10)-producing splenocytes are generated early during infection and that IL-10 inhibits IFN-gamma-producing cells and delays the clearance of B. bronchiseptica from the lungs. The TTSS of B. bronchiseptica inhibits the generation of IFN-gamma-producing splenocytes and is required for long-term bacterial persistence in the lower respiratory tract in wild-type mice. This suggests that a mechanism involving the modulation of IFN-gamma production by the TTSS facilitates B. bronchiseptica survival in the lower respiratory tract.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/metabolismo , Infecções por Bordetella/imunologia , Bordetella bronchiseptica/patogenicidade , Interferon gama/antagonistas & inibidores , Animais , Anticorpos Antibacterianos/sangue , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/imunologia , Contagem de Colônia Microbiana , Humanos , Interferon gama/deficiência , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-10/metabolismo , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/imunologiaRESUMO
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.