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1.
Int J Mol Sci ; 23(9)2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35562951

RESUMO

Burkholderia cenocepacia is an opportunistic pathogen that can lead to severe infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Being an obligate aerobe, B. cenocepacia is unable to grow in the absence of oxygen. In this study, we show that the CF isolate B. cenocepacia H111 can survive in the absence of oxygen. Using a transposon sequencing (Tn-seq) approach, we identified 71 fitness determinants involved in anoxic survival, including a Crp-Fnr family transcriptional regulatory gene (anr2), genes coding for the sensor kinase RoxS and its response regulator RoxR, the sigma factor for flagella biosynthesis (FliA) and subunits of a cytochrome bd oxidase (CydA, CydB and the potentially novel subunit CydP). Individual knockouts of these fitness determinants significantly reduced anoxic survival, and inactivation of both anr copies is shown to be lethal under anoxic conditions. We also show that the two-component system RoxS/RoxR and FliA are important for virulence and swarming/swimming, respectively.


Assuntos
Infecções por Burkholderia , Burkholderia cenocepacia , Fibrose Cística , Burkholderia cenocepacia/fisiologia , Humanos , Hipóxia , Oxigênio , Virulência/genética
2.
Microbiologyopen ; 11(1): e1264, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35212475

RESUMO

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric ß-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/fisiologia , Burkholderia cenocepacia/fisiologia , DNA Bacteriano/fisiologia , Mimetismo Molecular/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidade , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Família Multigênica/genética , Virulência
3.
mSphere ; 5(4)2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669472

RESUMO

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation is an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2 and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant ΔBCAL3494 showed changes in biofilm structure and cell aggregation, while ΔBCAM0992 was less motile. B. cenocepacia wild-type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-molecule real-time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified, and qPCR experiments showed an upregulation of several genes, including biofilm- and motility-related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation, cell aggregation, and motility.IMPORTANCE CF patients diagnosed with Burkholderia cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multireplicon genome, and much remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to advance the biological understanding of B. cenocepacia cell adaptation via epigenetic regulation. In time, this might lead to novel approaches to tackle B. cenocepacia infections in CF patients.


Assuntos
Biofilmes/crescimento & desenvolvimento , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/fisiologia , Metilação de DNA , Epigênese Genética , Regulação Bacteriana da Expressão Gênica , Animais , Proteínas de Bactérias/genética , Larva/microbiologia , Metiltransferases/genética , Metiltransferases/metabolismo , Mariposas/microbiologia , Movimento , Virulência
4.
Cell Rep ; 31(9): 107721, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32492429

RESUMO

Burkholderia cenocepacia is an opportunistic bacterial pathogen that causes severe pulmonary infections in cystic fibrosis and chronic granulomatous disease patients. B. cenocepacia can survive inside infected macrophages within the B. cenocepacia-containing vacuole (BcCV) and to elicit a severe inflammatory response. By inactivating the host macrophage Rho GTPases, the bacterial effector TecA causes depolymerization of the cortical actin cytoskeleton. In this study, we find that B. cenocepacia induces the formation of large cytosolic F-actin clusters in infected macrophages. Cluster formation requires the nucleation-promoting factor WASH, the Arp2/3 complex, and TecA. Inactivation of Rho GTPases by bacterial toxins is necessary and sufficient to induce the formation of the cytosolic actin clusters. By hijacking WASH and Arp2/3 activity, B. cenocepacia disrupts interactions with the endolysosomal system, thereby delaying the maturation of the BcCV.


Assuntos
Citoesqueleto de Actina/metabolismo , Burkholderia cenocepacia/fisiologia , Proteínas dos Microfilamentos/metabolismo , Fagossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Células da Medula Óssea/citologia , Feminino , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Células RAW 264.7 , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genética , Proteínas rho de Ligação ao GTP/antagonistas & inibidores
5.
Int J Mol Sci ; 21(5)2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32131450

RESUMO

Biofilms are a multicellular way of life, where bacterial cells are close together and embedded in a hydrated macromolecular matrix which offers a number of advantages to the cells. Extracellular polysaccharides play an important role in matrix setup and maintenance. A water-insoluble polysaccharide was isolated and purified from the biofilm produced by Burkholderia cenocepacia strain H111, a cystic fibrosis pathogen. Its composition and glycosidic linkages were determined using Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS) on appropriate carbohydrate derivatives while its complete structure was unraveled by 1D and 2D NMR spectroscopy in deuterated sodium hydroxide (NaOD) aqueous solutions. All the collected data demonstrated the following repeating unit for the water-insoluble B. cenocepacia biofilm polysaccharide: [3)-α-d-Galp-(1→3)-α-d-Glcp-(1→3)-α-d-Galp-(1→3)-α-d-Manp-(1→]n Molecular modelling was used, coupled with NMR Nuclear Overhauser Effect (NOE) data, to obtain information about local structural motifs which could give hints about the polysaccharide insolubility. Both modelling and NMR data pointed at restricted dynamics of local conformations which were ascribed to the presence of inter-residue hydrogen bonds and to steric restrictions. In addition, the good correlation between NOE data and calculated interatomic distances by molecular dynamics simulations validated potential energy functions used for calculations.


Assuntos
Biofilmes , Burkholderia cenocepacia/metabolismo , Polissacarídeos Bacterianos/química , Burkholderia cenocepacia/fisiologia , Glicosídeos/análise , Interações Hidrofóbicas e Hidrofílicas , Polissacarídeos Bacterianos/metabolismo , Solubilidade
6.
Sci Rep ; 9(1): 16118, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31695169

RESUMO

The influence that Burkholderia cenocepacia adaptive evolution during long-term infection in cystic fibrosis (CF) patients has on cell wall morphology and mechanical properties is poorly understood despite their crucial role in cell physiology, persistent infection and pathogenesis. Cell wall morphology and physical properties of three B. cenocepacia isolates collected from a CF patient over a period of 3.5 years were compared using atomic force microscopy (AFM). These serial clonal variants include the first isolate retrieved from the patient and two late isolates obtained after three years of infection and before the patient's death with cepacia syndrome. A consistent and progressive decrease of cell height and a cell shape evolution during infection, from the typical rods to morphology closer to cocci, were observed. The images of cells grown in biofilms showed an identical cell size reduction pattern. Additionally, the apparent elasticity modulus significantly decreases from the early isolate to the last clonal variant retrieved from the patient but the intermediary highly antibiotic resistant clonal isolate showed the highest elasticity values. Concerning the adhesion of bacteria surface to the AFM tip, the first isolate was found to adhere better than the late isolates whose lipopolysaccharide (LPS) structure loss the O-antigen (OAg) during CF infection. The OAg is known to influence Gram-negative bacteria adhesion and be an important factor in B. cenocepacia adaptation to chronic infection. Results reinforce the concept of the occurrence of phenotypic heterogeneity and adaptive evolution, also at the level of cell size, form, envelope topography and physical properties during long-term infection.


Assuntos
Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/fisiologia , Parede Celular/química , Fibrose Cística/microbiologia , Fenômenos Biomecânicos , Burkholderia cenocepacia/química , Humanos , Pulmão/microbiologia , Microscopia de Força Atômica , Fenótipo
7.
BMC Genomics ; 20(1): 803, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31684866

RESUMO

BACKGROUND: Burkholderia cenocepacia is a human opportunistic pathogen causing devastating symptoms in patients suffering from immunodeficiency and cystic fibrosis. Out of the 303 B. cenocepacia strains with available genomes, the large majority were isolated from a clinical context. However, several isolates originate from other environmental sources ranging from aerosols to plant endosphere. Plants can represent reservoirs for human infections as some pathogens can survive and sometimes proliferate in the rhizosphere. We therefore investigated if B. cenocepacia had the same potential. RESULTS: We selected genome sequences from 31 different strains, representative of the diversity of ecological niches of B. cenocepacia, and conducted comparative genomic analyses in the aim of finding specific niche or host-related genetic determinants. Phylogenetic analyses and whole genome average nucleotide identity suggest that strains, registered as B. cenocepacia, belong to at least two different species. Core-genome analyses show that the clade enriched in environmental isolates lacks multiple key virulence factors, which are conserved in the sister clade where most clinical isolates fall, including the highly virulent ET12 lineage. Similarly, several plant associated genes display an opposite distribution between the two clades. Finally, we suggest that B. cenocepacia underwent a host jump from plants/environment to animals, as supported by the phylogenetic analysis. We eventually propose a name for the new species that lacks several genetic traits involved in human virulence. CONCLUSION: Regardless of the method used, our studies resulted in a disunited perspective of the B. cenocepacia species. Strains currently affiliated to this taxon belong to at least two distinct species, one having lost several determining animal virulence factors.


Assuntos
Adaptação Fisiológica/genética , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/fisiologia , Interações Hospedeiro-Patógeno/genética , Plantas/microbiologia , Burkholderia cenocepacia/patogenicidade , Evolução Molecular , Humanos , Filogenia , Virulência
8.
J Immunol ; 201(7): 2016-2027, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30120123

RESUMO

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.


Assuntos
Infecções por Burkholderia/imunologia , Burkholderia cenocepacia/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/imunologia , Mutação/genética , Neutrófilos/imunologia , Pneumonia/imunologia , Adolescente , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Criança , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Homeostase , Humanos , Imunidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Infiltração de Neutrófilos , Espécies Reativas de Oxigênio/metabolismo
9.
Biomed Res Int ; 2018: 4271560, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29850514

RESUMO

Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.


Assuntos
Burkholderia cenocepacia/fisiologia , Macrófagos/microbiologia , Pinocitose , Animais , Sistemas de Secreção Bacterianos , Feminino , Macrófagos/ultraestrutura , Camundongos Endogâmicos BALB C
10.
Int J Mol Sci ; 18(11)2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-29135920

RESUMO

Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656T. Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts.


Assuntos
Burkholderia cenocepacia/fisiologia , Microbiologia Ambiental , Aptidão Genética , Infecções Respiratórias/microbiologia , Adaptação Fisiológica , Animais , Carga Bacteriana , Biofilmes , Burkholderia cenocepacia/patogenicidade , Doença Crônica , Células Clonais , Contagem de Colônia Microbiana , Estimativa de Kaplan-Meier , Larva/microbiologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Técnica de Amplificação ao Acaso de DNA Polimórfico , Inoculações Seriadas , Virulência
11.
Can J Microbiol ; 63(10): 857-863, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28817787

RESUMO

There are hundreds of essential genes in multidrug-resistant bacterial genomes, but only a few of their products are exploited as antibacterial targets. An example is the electron transfer flavoprotein (ETF), which is required for growth and viability in Burkholderia cenocepacia. Here, we evaluated ETF as an antibiotic target for Burkholderia cepacia complex (Bcc). Depletion of the bacterial ETF during infection of Caenorhabditis elegans significantly extended survival of the nematodes, proving that ETF is essential for survival of B. cenocepacia in this host model. In spite of the arrest in respiration in ETF mutants, the inhibition of etf expression did not increase the formation of persister cells, when treated with high doses of ciprofloxacin or meropenem. To test if etf translation could be inhibited by RNA interference, antisense oligonucleotides that target the etfBA operon were synthesized. One antisense oligonucleotide was effective in inhibiting etfB translation in vitro but not in vivo, highlighting the challenge of reduced membrane permeability for the design of drugs against B. cenocepacia. This work contributes to the validation of ETF of B. cenocepacia as a target for antibacterial therapy and demonstrates the utility of a C. elegans liquid killing assay to validate gene essentiality in an in vivo infection model.


Assuntos
Burkholderia cenocepacia/genética , Caenorhabditis elegans/microbiologia , Flavoproteínas Transferidoras de Elétrons/genética , Animais , Antibacterianos/farmacologia , Burkholderia cenocepacia/fisiologia , Caenorhabditis elegans/fisiologia , Permeabilidade da Membrana Celular , Ciprofloxacina/farmacologia , Flavoproteínas Transferidoras de Elétrons/metabolismo , Meropeném , Mutação , Oligonucleotídeos Antissenso/genética , Interferência de RNA , Tienamicinas/farmacologia
12.
PLoS One ; 12(3): e0172723, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28248999

RESUMO

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.


Assuntos
Biofilmes/efeitos dos fármacos , Infecções por Burkholderia/tratamento farmacológico , Burkholderia cenocepacia/fisiologia , Econazol/farmacologia , Miconazol/farmacologia , Pneumonia Bacteriana/tratamento farmacológico , Tobramicina/farmacologia , Células A549 , Animais , Biofilmes/crescimento & desenvolvimento , Infecções por Burkholderia/metabolismo , Infecções por Burkholderia/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada/métodos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/patologia
13.
Genome Res ; 27(4): 650-662, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28325850

RESUMO

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.


Assuntos
Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/genética , Fibrose Cística/microbiologia , Fenótipo , Polimorfismo Genético , Adolescente , Animais , Biofilmes , Infecções por Burkholderia/complicações , Burkholderia cenocepacia/isolamento & purificação , Burkholderia cenocepacia/patogenicidade , Burkholderia cenocepacia/fisiologia , Criança , Pré-Escolar , Fibrose Cística/complicações , Genótipo , Humanos , Pulmão/microbiologia , Mariposas/microbiologia , Virulência , Adulto Jovem
14.
Can J Microbiol ; 63(5): 427-438, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28178425

RESUMO

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.


Assuntos
Aspergillus fumigatus , Burkholderia cenocepacia/efeitos dos fármacos , Fibrose Cística , Escarro/química , Antifúngicos/metabolismo , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Sequência de Bases , Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/fisiologia , Meios de Cultura/síntese química , Fibrose Cística/microbiologia , Humanos , Fenilacetatos/metabolismo , Fenilacetatos/farmacologia , Fenilalanina/metabolismo , Pseudomonas aeruginosa/genética
15.
Cell Microbiol ; 19(4)2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27684048

RESUMO

Chronic lung disease caused by persistent bacterial infections is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). CF pathogens acquire antibiotic resistance, overcome host defenses, and impose uncontrolled inflammation that ultimately may cause permanent damage of lungs' airways. Among the multiple CF-associated pathogens, Burkholderia cenocepacia and other Burkholderia cepacia complex bacteria have become prominent contributors of disease progression. Here, we demonstrate that BcaA, a trimeric autotransporter adhesin (TAA) from the epidemic strain B. cenocepacia K56-2, is a tumor necrosis factor receptor 1-interacting protein able to regulate components of the tumor necrosis factor signaling pathway and ultimately leading to a significant production of the proinflammatory cytokine IL-8. Notably, this study is the first to demonstrate that a protein belonging to the TAA family is involved in the induction of the inflammatory response during B. cenocepacia infections, contributing to the success of the pathogen. Moreover, our results reinforce the relevance of the TAA BcaA as a multifunctional protein with a major role in B. cenocepacia virulence.


Assuntos
Adesinas Bacterianas/química , Infecções por Burkholderia/microbiologia , Burkholderia cenocepacia/fisiologia , Pneumonia/microbiologia , Receptores Tipo I de Fatores de Necrose Tumoral/química , Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Ligação Proteica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais
16.
Autophagy ; 12(11): 2026-2037, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27541364

RESUMO

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.


Assuntos
Autofagia/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/patologia , Regulação da Expressão Gênica , Macrófagos/metabolismo , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Animais , Antagomirs/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Burkholderia cenocepacia/fisiologia , Células Cultivadas , Fibrose Cística/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Homozigoto , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Células NIH 3T3
17.
Int J Antimicrob Agents ; 47(6): 446-50, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27179816

RESUMO

Chronic lung infection with bacteria from the Burkholderia cepacia complex (BCC), and in particular B. cenocepacia, is associated with significant morbidity and mortality in patients with cystic fibrosis (CF). B. cenocepacia can spread from person to person and exhibits intrinsic broad-spectrum antibiotic resistance. Recently, atmospheric pressure non-thermal plasmas (APNTPs) have gained increasing attention as a novel approach to the prevention and treatment of a variety of hospital-acquired infections. In this study, we evaluated an in-house-designed kHz-driven plasma source for the treatment of biofilms of a number of clinical CF B. cenocepacia isolates. The results demonstrated that APNTP is an effective and efficient tool for the eradication of B. cenocepacia biofilms but that efficacy is highly variable across different isolates. Determination of phenotypic differences between isolates in an attempt to understand variability in plasma tolerance revealed that isolates which are highly tolerant to APNTP typically produce biofilms of greater biomass than their more sensitive counterparts. This indicates a potential role for biofilm matrix components in biofilm tolerance to APNTP exposure. Furthermore, significant isolate-dependent differences in catalase activity in planktonic bacteria positively correlated with phenotypic resistance to APNTP by isolates grown in biofilms.


Assuntos
Pressão Atmosférica , Biofilmes/efeitos dos fármacos , Burkholderia cenocepacia/efeitos dos fármacos , Burkholderia cenocepacia/fisiologia , Desinfetantes/farmacologia , Tolerância a Medicamentos , Gases em Plasma/farmacologia
18.
Sci Rep ; 6: 21140, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26879174

RESUMO

Previous studies have demonstrated that extracellular glutathione reduces the ability of the Cystic Fibrosis pathogen Burkholderia cenocepacia to infect primary or immortalized epithelial respiratory cells. We report here that the adhesion and invasion ability of B. cenocepacia is limited also by thiol-oxidizing and disulphide-reducing agents and by protein disulfide isomerase (PDI) inhibitors. PDI inhibitors also reduce the proinflammatory response elicited by cells in response to Burkholderia. These findings indicate that a membrane-associated PDI catalyzes thiol/disulphide exchange reactions which favor bacterial infection. The combined use of selective PDI inhibitors, RNA silencing and specific antibodies identified ERp57 as a major PDI involved in the interaction between B. cenocepacia and epithelial cells. This study contributes to the elucidation of the Burkholderia pathogenic mechanisms by showing that this microorganism exploits a membrane-associated host protein to infect epithelial cells and identifies ERp57 as a putative pharmacological target for the treatment of Burkholderia lung infections.


Assuntos
Burkholderia cenocepacia/fisiologia , Dissulfetos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Mucosa Respiratória/metabolismo , Mucosa Respiratória/microbiologia , Burkholderia cenocepacia/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia , Mediadores da Inflamação/metabolismo , Modelos Biológicos , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética
19.
Appl Environ Microbiol ; 82(3): 843-56, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26590274

RESUMO

Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate p-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated in vivo and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.


Assuntos
Biofilmes/crescimento & desenvolvimento , Burkholderia cenocepacia/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Proteínas de Bactérias/genética , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Burkholderia cenocepacia/patogenicidade , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Humanos , Larva/microbiologia , Macrófagos/microbiologia , Camundongos , Mariposas/microbiologia , Fosforilação , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/genética , Células RAW 264.7 , Virulência
20.
J Water Health ; 13(3): 773-6, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26322762

RESUMO

Clinical cystic fibrosis (CF) Pseudomonas aeruginosa (n=6) and Burkholderia cenocepacia (n=4) were inoculated in marine brines from the Dead Sea and the Atlantic Ocean and their survival was monitored over a 1 month duration. In Dead Sea samples, all P. aeruginosa and B. cenocepacia isolates were non-detectable by culture following 24 h incubation, including the non-selective enrichment samples. In the Atlantic Ocean brine, over a 1 month period, mean P. aeruginosa counts decreased by only 0.25 log10 units and mean B. cenocepacia counts decreased by approximately 4 log10 units (10,000 cfu/ml). This study demonstrated that Dead Sea brine exerted a lethal effect within 24 h on planktonic P. aeruginosa and B. cenocepacia. Thus, the Dead Sea effectively purges these organisms from its environment on a daily basis.


Assuntos
Burkholderia cenocepacia/fisiologia , Fibrose Cística/microbiologia , Pseudomonas aeruginosa/fisiologia , Água do Mar/microbiologia , Humanos , Viabilidade Microbiana , Oceanos e Mares , Microbiologia da Água
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