RESUMO
Quorum sensing (QS) contributes to the virulence of Pseudomonas aeruginosa and Burkholderia cepacia complex lung infections. P. aeruginosa QS mutants are frequently isolated from patients with cystic fibrosis. The objective of this study was to determine whether similar adaptations occur over time in B. cepacia complex isolates. Forty-five Burkholderia multivorans and Burkholderia cenocepacia sequential isolates from patients with cystic fibrosis were analyzed for N-acyl-homoserine lactone activity. All but one isolate produced N-acyl-homoserine lactones. The B. cenocepacia N-acyl-homoserine lactone-negative isolate contained mutations in cepR and cciR. Growth competition assays were performed that compared B. cenocepacia clinical and laboratory defined wild-type and QS mutants. Survival of the laboratory wild-type and QS mutants varied, dependent on the mutation. The clinical wild-type isolate demonstrated a growth advantage over its QS mutant. These data suggest that there is a selective advantage for strains with QS systems and that QS mutations do not occur at a high frequency in B. cepacia complex isolates.
Assuntos
Infecções por Burkholderia/microbiologia , Complexo Burkholderia cepacia/fisiologia , Fibrose Cística/complicações , Percepção de Quorum/fisiologia , Acil-Butirolactonas/metabolismo , Infecções por Burkholderia/complicações , Complexo Burkholderia cepacia/genética , Complexo Burkholderia cepacia/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , MutaçãoRESUMO
BACKGROUND: Burkholderia cenocepacia belongs to a group of closely related organisms called the B. cepacia complex (Bcc) which are important opportunistic human pathogens. B. cenocepacia utilizes a mechanism of cell-cell communication called quorum sensing to control gene expression including genes involved in virulence. The B. cenocepacia quorum sensing network includes the CepIR and CciIR regulatory systems. RESULTS: Global gene expression profiles during growth in stationary phase were generated using microarrays of B. cenocepacia cepR, cciR and cepRcciIR mutants. This is the first time CciR was shown to be a global regulator of quorum sensing gene expression. CepR was primarily responsible for positive regulation of gene expression while CciR generally exerted negative gene regulation. Many of the genes that were regulated by both quorum sensing systems were reciprocally regulated by CepR and CciR. Microarray analysis of the cepRcciIR mutant suggested that CepR is positioned upstream of CciR in the quorum sensing hierarchy in B. cenocepacia. A comparison of CepIR-regulated genes identified in previous studies and in the current study showed a substantial amount of overlap validating the microarray approach. Several novel quorum sensing-controlled genes were confirmed using qRT-PCR or promoter::lux fusions. CepR and CciR inversely regulated flagellar-associated genes, the nematocidal protein AidA and a large gene cluster on Chromosome 3. CepR and CciR also regulated genes required for iron transport, synthesis of extracellular enzymes and surface appendages, resistance to oxidative stress, and phage-related genes. CONCLUSION: For the first time, the influence of CciIR on global gene regulation in B. cenocepacia has been elucidated. Novel genes under the control of the CepIR and CciIR quorum sensing systems in B. cenocepacia have been identified. The two quorum sensing systems exert reciprocal regulation of many genes likely enabling fine-tuned control of quorum sensing gene expression in B. cenocepacia strains carrying the cenocepacia island.
Assuntos
Burkholderia cepacia/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Análise por Conglomerados , DNA Bacteriano/genética , Ligases/genética , Análise de Sequência com Séries de Oligonucleotídeos , ÓperonRESUMO
Burkholderia cenocepacia causes chronic and life-threatening respiratory infections in immunocompromized people. The B. cenocepacia N-acyl-homoserine lactone (AHL)-dependent quorum sensing system relies on the production of AHLs by the synthases CepI and CciI while CepR, CciR and CepR2 control expression of many genes important for pathogenesis. Downstream from, and co-transcribed with cepI, lies BCAM1871 encoding a hypothetical protein that was uncharacterized prior to this study. Orthologs of B. cenocepacia BCAM1871 are uniquely found in Burkholderia spp and are conserved in their genomic locations in pathogenic Burkholderia. We observed significant effects on AHL activity upon mutation or overexpression of BCAM1871, although these effects were more subtle than those observed for CepI indicating BCAM1871 acts as an enhancer of AHL activity. Transcription of cepI, cepR and cciIR was significantly reduced in the BCAM1871 mutant. Swimming and swarming motilities as well as transcription of fliC, encoding flagellin, were significantly reduced in the BCAM1871 mutant. Protease activity and transcription of zmpA and zmpB, encoding extracellular zinc metalloproteases, were undetectable in the BCAM1871 mutant indicating a more significant effect of mutating BCAM1871 than cepI. Exogenous addition of OHL restored cepI, cepR and fliC transcription but had no effect on motility, protease activity or zmpA or zmpB transcription suggesting AHL-independent effects. The BCAM1871 mutant exhibited significantly reduced virulence in rat chronic respiratory and nematode infection models. Gene expression and phenotypic assays as well as vertebrate and invertebrate infection models showed that BCAM1871 significantly contributes to pathogenesis in B. cenocepacia.