Reciprocal regulation by the CepIR and CciIR quorum sensing systems in Burkholderia cenocepacia

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia </it>belongs to a group of closely related organisms called the <it>B. cepacia </it>complex (Bcc) which are important opportunistic human pathogens. <it>B. cenocepacia </it>utilizes a mechanism of cell-cell communication called quorum sensing to control gene expression including genes involved in virulence. The <it>B. cenocepacia </it>quorum sensing network includes the CepIR and CciIR regulatory systems.</p> <p>Results</p> <p>Global gene expression profiles during growth in stationary phase were generated using microarrays of <it>B. cenocepacia cepR</it>, <it>cciR </it>and <it>cepRcciIR </it>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 <it>cepRcciIR </it>mutant suggested that CepR is positioned upstream of CciR in the quorum sensing hierarchy in <it>B. cenocepacia</it>. 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::<it>lux </it>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.</p> <p>Conclusion</p> <p>For the first time, the influence of CciIR on global gene regulation in <it>B. cenocepacia </it>has been elucidated. Novel genes under the control of the CepIR and CciIR quorum sensing systems in <it>B. cenocepacia </it>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 <it>B. cenocepacia </it>strains carrying the cenocepacia island.</p

Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates

Burkholderia cepacia complex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc species B. multivorans were highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made by B. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients

Quorum-sensing in burkolderia cenocepacia and burkolderia vietnamiensis

Bibliography: p. 183-204Some pages are in colour

Expression of the bviIR and cepIR Quorum-Sensing Systems of Burkholderia vietnamiensis

Burkholderia vietnamiensis has both the cepIR quorum-sensing system that is widely distributed among the Burkholderia cepacia complex (BCC) and the bviIR system. Comparison of the expression of cepI, cepR, bviI, and bviR-luxCDABE fusions in B. vietnamiensis G4 and the G4 cepR and bviR mutants determined that the expression of bviI requires both a functional cognate regulator, BviR, and functional CepR. The cepIR system, however, is not regulated by BviR. Unlike the cepIR genes in other BCC species, the cepIR genes are not autoregulated in G4. N-Acyl-homoserine lactone (AHL) production profiles in G4 cepI, cepR, bviI, and bviR mutants confirmed the regulatory organization of the G4 quorum-sensing systems. The regulatory network in strain PC259 is similar to that in G4, except that CepR positively regulates cepI and negatively regulates cepR. AHL production and the bviI expression levels in seven B. vietnamiensis isolates were compared. All strains produced N-octanoyl-homoserine lactone and N-hexanoyl-homoserine lactone; however, only one of four clinical strains but all three environmental strains produced the BviI synthase product, N-decanoyl-homoserine lactone (DHL). The three strains that did not produce DHL expressed bviR but not bviI. Heterologous expression of bviR restored DHL production in these strains. The bviIR loci of the non-DHL-producing strains were sequenced to confirm that bviR encodes a functional transcriptional regulator. Lack of expression of G4 bviI in these three strains indicated that an additional regulatory element may be involved in the regulation of bviIR expression in certain strains of B. vietnamiensis

Characterization of the cciIR Quorum-Sensing System in Burkholderia cenocepacia

Several transmissible Burkholderia cenocepacia strains that infect multiple cystic fibrosis patients contain a genomic island designated as the cenocepacia island (cci). The cci contains a predicted N-acylhomoserine lactone (AHL) synthase gene, cciI, and a predicted response regulator gene, cciR. AHL production profiles indicated that CciI catalyzes the synthesis of N-hexanoyl-l-homoserine lactone and minor amounts of N-octanoyl-l-homoserine lactone. The cciI and cciR genes were found to be cotranscribed by reverse transcription-PCR analysis, and the expression of a cciIR::luxCDABE fusion in a cciR mutant suggested that the cciIR system negatively regulates its own expression. B. cenocepacia strains also have a cepIR quorum-sensing system. Expression of cepI::luxCDABE or cepR::luxCDABE fusions in a cciR mutant showed that CciR negatively regulates cepI but does not regulate cepR. Expression of the cciIR::luxCDABE fusion in a cepR mutant indicated that functional CepR is required for cciIR expression. Phylogenetic analysis suggested that the cciIR system was acquired by horizontal gene transfer from a distantly related organism and subsequently incorporated into the ancestral cepIR regulatory network. Mutations in cciI, cciR, cepI cciI, and cepR cciR were constructed in B. cenocepacia K56-2. The cciI mutant had greater protease activity and less swarming motility than the parent strain. The cciR mutant had less protease activity than the parent strain. The phenotypes of the cepI cciI and cepR cciR mutants were similar to cepI or cepR mutants, with less protease activity and swarming motility than the parent strain

A Burkholderia cenocepacia Orphan LuxR Homolog Is Involved in Quorum-Sensing Regulation▿ †

Burkholderia cenocepacia utilizes quorum sensing to control gene expression, including the expression of genes involved in virulence. In addition to CepR and CciR, a third LuxR homolog, CepR2, was found to regulate gene expression and virulence factor production. All B. cenocepacia strains examined contained this orphan LuxR homolog, which was not associated with an adjacent N-acyl-homoserine lactone synthase gene. Expression of cepR2 was negatively autoregulated and was negatively regulated by CciR in strain K56-2. Microarray analysis and quantitative reverse transcription-PCR determined that CepR2 did not influence expression of cepIR or cciIR. However, in strain K56-2, CepR2 negatively regulated expression of several known quorum-sensing-controlled genes, including genes encoding zinc metalloproteases. CepR2 exerted positive and negative regulation on genes on three chromosomes, including strong negative regulation of a gene cluster located adjacent to cepR2. In strain H111, which lacks the CciIR quorum-sensing system, CepR2 positively regulated pyochelin production by controlling transcription of one of the operons required for the biosynthesis of the siderophore in an N-acyl-homoserine lactone-independent manner. CepR2 activation of a luxI promoter was demonstrated in a heterologous Escherichia coli host, providing further evidence that CepR2 can function in the absence of signaling molecules. This study demonstrates that the orphan LuxR homolog CepR2 contributes to the quorum-sensing regulatory network in two distinct strains of B. cenocepacia

Quorum-Sensing Mutations Affect Attachment and Stability of Burkholderia cenocepacia Biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients