RESUMO
The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription-PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell in vitro and with attenuated virulence in a murine model. This work presents data that support the role that the second messenger c-di-GMP plays in the pathogenesis of Bordetella.
Assuntos
Bordetella bronchiseptica , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Bordetella bronchiseptica/genética , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Sistemas de Secreção Tipo III/metabolismo , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory tract infections. It is a natural pathogen of a wide variety of mammals, including some used as laboratory models. This makes B. bronchiseptica an ideal organism to study pathogen-host interactions in order to unveil molecular mechanisms behind pathogenic processes. Even though genetic engineering is an essential tool in this area, there are just a few reports about genome manipulation techniques in this organism. In this article we describe an allelic exchange protocol based on double crossover recombination facilitated by the Bacillus subtilis sacB gene that can be applied for partial or complete gene knockouts, single-nucleotide mutations, or even introduction of coding sequences for transcriptional fusions. In contrast to previously employed techniques, this protocol renders genetically manipulated chromosomes without foreign DNA and enables the construction of successive genome manipulation using the same vector backbone. The entire procedure has been developed for fast and reliable manipulations with a total duration of 2 weeks. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Setting up strains Basic Protocol 2: Homologous recombination (first crossing-over) Alternate Protocol: B. bronchiseptica electroporation Basic Protocol 3: Screening for sucrose-sensitive clones Basic Protocol 4: Homologous recombination (second crossing-over) Basic Protocol 5: PCR screening of putative marker-exchange mutants Support Protocol: Electrocompetent cell preparation.
Assuntos
Bacillus subtilis/genética , Bordetella bronchiseptica/genética , Genes Bacterianos/genética , Engenharia Genética/métodos , Hexosiltransferases/genética , Técnicas Bacteriológicas/métodos , Técnicas de Inativação de Genes , Recombinação Homóloga , Reação em Cadeia da PolimeraseRESUMO
Bacteria can be motile and planktonic or, alternatively, sessile and participating in the biofilm mode of growth. The transition between these lifestyles can be regulated by a second messenger, cyclic dimeric GMP (c-di-GMP). High intracellular c-di-GMP concentration correlates with biofilm formation and motility inhibition in most bacteria, including Bordetella bronchiseptica, which causes respiratory tract infections in mammals and forms biofilms in infected mice. We previously described the diguanylate cyclase BdcA as involved in c-di-GMP synthesis and motility regulation in B. bronchiseptica; here, we further describe the mechanism whereby BdcA is able to regulate motility and biofilm formation. Amino acid replacement of GGDEF with GGAAF in BdcA is consistent with the conclusion that diguanylate cyclase activity is necessary for biofilm formation and motility regulation, although we were unable to confirm the stability of the mutant protein. In the absence of the bdcA gene, B. bronchiseptica showed enhanced motility, strengthening the hypothesis that BdcA regulates motility in B. bronchiseptica We showed that c-di-GMP-mediated motility inhibition involved regulation of flagellin expression, as high c-di-GMP levels achieved by expressing BdcA significantly reduced the level of flagellin protein. We also demonstrated that protein BB2109 is necessary for BdcA activity, motility inhibition, and biofilm formation. Finally, absence of the bdcA gene affected bacterial infection, implicating BdcA-regulated functions as important for bacterium-host interactions. This work supports the role of c-di-GMP in biofilm formation and motility regulation in B. bronchiseptica, as well as its impact on pathogenesis.IMPORTANCE Pathogenesis of Bordetella spp., like that of a number of other pathogens, involves biofilm formation. Biofilms increase tolerance to biotic and abiotic factors and are proposed as reservoirs of microbes for transmission to other organs (trachea, lungs) or other hosts. Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a second messenger that regulates transition between biofilm and planktonic lifestyles. In Bordetella bronchiseptica, high c-di-GMP levels inhibit motility and favor biofilm formation. In the present work, we characterized a B. bronchiseptica diguanylate cyclase, BdcA, which regulates motility and biofilm formation and affects the ability of B. bronchiseptica to colonize the murine respiratory tract. These results provide us with a better understanding of how B. bronchiseptica can infect a host.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Bordetella/metabolismo , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/enzimologia , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Infecções Respiratórias/microbiologia , Animais , Proteínas de Bactérias/genética , Infecções por Bordetella/genética , Bordetella bronchiseptica/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Movimento , Fósforo-Oxigênio Liases/genéticaRESUMO
Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-D-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2', O-acylated with C14:0(3-O-C10:0(3-OH) in C-3' and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4', both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane. Graphical Abstract.
Assuntos
Bordetella bronchiseptica/genética , Glicosiltransferases/genética , Lipídeo A/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bordetella bronchiseptica/química , Bordetella bronchiseptica/metabolismo , Difosfatos/química , Glucosamina/química , Glicosiltransferases/química , Lipídeo A/análise , Lipídeo A/genética , Mutação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Biofilm formation is important for infection by many pathogens. Bordetella bronchiseptica causes respiratory tract infections in mammals and forms biofilm structures in nasal epithelium of infected mice. We previously demonstrated that cyclic di-GMP is involved in biofilm formation in B. bronchiseptica. In the present work, based on their previously reported function in Pseudomonas fluorescens, we identified three genes in the B. bronchiseptica genome likely involved in c-di-GMP-dependent biofilm formation: brtA, lapD and lapG. Genetic analysis confirmed a role for BrtA, LapD and LapG in biofilm formation using microtiter plate assays, as well as scanning electron and fluorescent microscopy to analyze the phenotypes of mutants lacking these proteins. In vitro and in vivo studies showed that the protease LapG of B. bronchiseptica cleaves the N-terminal domain of BrtA, as well as the LapA protein of P. fluorescens, indicating functional conservation between these species. Furthermore, while BrtA and LapG appear to have little or no impact on colonization in a mouse model of infection, a B. bronchiseptica strain lacking the LapG protease has a significantly higher rate of inducing a severe disease outcome compared to the wild type. These findings support a role for c-di-GMP acting through BrtA/LapD/LapG to modulate biofilm formation, as well as impact pathogenesis, by B. bronchiseptica.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/fisiologia , GMP Cíclico/análogos & derivados , Animais , Proteínas de Bactérias/genética , Western Blotting , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/metabolismo , GMP Cíclico/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Patógeno , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Mutação , Pseudomonas fluorescens/genética , Infecções Respiratórias/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
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 causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services.
Assuntos
Bordetella bronchiseptica/genética , Flagelos/genética , Técnicas de Amplificação de Ácido Nucleico/economia , Testes Genéticos , Testes Laboratoriais/análise , Infecções por Bordetella/diagnóstico , Reação em Cadeia da PolimeraseRESUMO
Bordetella bronchiseptica causes acute and chronic respiratory infections in diverse animal species and occasionally in humans. In this study, we described the establishment of a simple, sensitive and cost-efficient loop-mediated isothermal amplification (LAMP) assay for the detection of B. bronchiseptica. A set of primers towards a 235 bp region within the flagellum gene of B. bronchiseptica was designed with online software.. The specificity of the LAMP assay was examined by using 6 porcine pathogens and 100 nasal swabs collected from healthy pigs and suspect infected pigs. The results indicated that positive reactions were confirmed for all B. bronchiseptica and no cross-reactivity was observed from other non-B. bronchiseptica. In sensitivity evaluations, the technique successfully detected a serial dilutions of extracted B. bronchiseptica DNA with a detection limit of 9 copies, which was 10 times more sensitive than that of PCR. Compared with conventional PCR, the higher sensitivity of LAMP method and no need for the complex instrumentation make this LAMP assay a promising alternative for the diagnosis of B. bronchiseptica in rural areas and developing countries where there lacks of complex laboratory services. (AU)
Assuntos
Bordetella bronchiseptica/genética , Flagelos/genética , Técnicas de Amplificação de Ácido Nucleico/economia , Testes Genéticos , Testes Laboratoriais/análise , Infecções por Bordetella/diagnóstico , Reação em Cadeia da PolimeraseRESUMO
The signalling molecule bis-(3'-5')-cyclic-dimeric guanosine monophosphate (c-di-GMP) is a central regulator of diverse cellular functions, including motility, biofilm formation, cell cycle progression and virulence, in bacteria. Multiple diguanylate cyclase and phosphodiesterase-domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) modulate the levels of the second messenger c-di-GMP to transmit signals and obtain such specific cellular responses. In the genus Bordetella this c-di-GMP network is poorly studied. In this work, we evaluated the expression of two phenotypes in Bordetella bronchiseptica regulated by c-di-GMP, biofilm formation and motility, under the influence of ectopic expression of Pseudomonas aeruginosa proteins with EAL or GGDEF domains that regulates the c-di-GMP level. In agreement with previous reports for other bacteria, we observed that B. bronchiseptica is able to form biofilm and reduce its motility only when GGDEF domain protein is expressed. Moreover we identify a GGDEF domain protein (BB3576) with diguanylate cyclase activity that participates in motility and biofilm regulation in B. bronchiseptica. These results demonstrate for the first time, to our knowledge, the presence of c-di-GMP regulatory signalling in B. bronchiseptica.
Assuntos
Biofilmes , Bordetella bronchiseptica/citologia , Bordetella bronchiseptica/metabolismo , GMP Cíclico/metabolismo , Transdução de Sinais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bordetella bronchiseptica/química , Bordetella bronchiseptica/genética , Regulação Bacteriana da Expressão Gênica , Estrutura Terciária de ProteínaRESUMO
The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains.