Pamela A. Sokol

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.

Burkholderia cenocepaciais an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates,l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence inB. cenocepacia.

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.

Burkholderia cenocepacia is a significant opportunistic pathogen in individuals with cystic fibrosis. ShvR, a LysR-type transcriptional regulator, has previously been shown to influence colony morphology, biofilm formation, virulence in plant and animal infection models, and some quorum-sensing-dependent phenotypes. In the present study, it was shown that ShvR negatively regulates its own expression, as is typical for LysR-type regulators. The production of quorum-sensing signal molecules was detected earlier in growth in the shvR mutant than in the wild type, and ShvR repressed expression of the quorum-sensing regulatory genes cepIR and cciIR . Microarray analysis and transcriptional fusions revealed that ShvR regulated over 1,000 genes, including the zinc metalloproteases zmpA and zmpB . The shvR mutant displayed increased gene expression of the type II secretion system and significantly increased protease and lipase activities. Both ShvR and CepR influence expression of a 24-kb genomic region adjacent to shvR that includes the afcA and afcC operons, required for the production of an antifungal agent; however, the reduction in expression was substantially greater in the shvR mutant than in the cepR mutant. Only the shvR mutation resulted in reduced antifungal activity against Rhizoctonia solani. ShvR, but not CepR, was shown to directly regulate expression of the afcA and afcC promoters. In summary, ShvR was determined to have a significant influence on the expression of quorum-sensing, protease, lipase, type II secretion, and afc genes.

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.

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 .

Background: Bacteria use N-acyl homoserine lactone (AHL) molecules to regulate the expression of genes in a density-dependent manner. Several biosensors have been developed and engineered to detect the presence of all types of AHLs. Results: In this study, we describe the usefulness of a traI-luxCDABE-based biosensor to quickly detect AHLs from previously characterized mutants of Burkholderia cenocepacia and Pseudomonas aeruginosa in both liquid and soft-agar co-culture assays in a high-throughput manner. The technique uses a co-culture system where the strain producing the AHLs is grown simultaneously with the reporter strain. Use of this assay in liquid co-culture allows the measurement of AHL activity in real time over growth. We tested this assay with Burkholderia cenocepacia and Pseudomonas aeruginosa but it should be applicable to a broad range of gram negative species that produce AHLs. Conclusion: The co-culture assays described enable the detection of AHL production in both P. aeruginosa and B. cenocepacia and should be applicable to AHL analysis in other bacterial species. The high-throughput adaptation of the liquid co-culture assay could facilitate the screening of large libraries for the identification of mutants or compounds that block the synthesis or activity of AHLs.

Burkholderia cenocepacia strain K56-2 typically has rough colony morphology on agar medium; however, shiny colony variants (shv) can appear spontaneously. These shv all had a minimum of 50% reduction in biomass formation and were generally avirulent in an alfalfa seedling infection model. Three shv—K56-2 S15, K56-2 S76, and K56-2 S86—were analyzed for virulence in a chronic agar bead model of respiratory infection and, although all shv were able to establish chronic infection, they produced significantly less lung histopathology than the rough K56-2. Transmission electron microscopy revealed that an extracellular matrix surrounding bacterial cells was absent or reduced in the shv compared to the rough wild type. Transposon mutagenesis was performed on the rough wild-type strain and a mutant with an insertion upstream of ORF BCAS0225, coding for a putative LysR-type regulator, exhibited shiny colony morphology, reduced biofilm production, increased N -acyl homoserine lactone production, and avirulence in alfalfa. The rough parental colony morphotype, biofilm formation, and virulence in alfalfa were restored by providing BCAS0225 in trans in the BCAS0225::pGSVTp- luxCDABF mutant. Introduction of BCAS0225 restored the rough morphotype in several shv which were determined to have spontaneous mutations in this gene. In the present study, we show that the conversion from rough wild type to shv in B. cenocepacia correlates with reduced biofilm formation and virulence, and we determined that BCAS0225 is one gene involved in the regulation of these phenotypes.

The genus Burkholderia not only contains the primary pathogens Burkholderia pseudomallei and Burkholderia mallei but also several species that have emerged as opportunistic pathogens in persons suffering from cystic fibrosis or chronic granulomatous disease and immunocompromised individuals. Burkholderia species utilize quorum-sensing (QS) systems that rely on N-acyl-homoserine lactone (AHL) signal molecules to express virulence factors and other functions in a population-density-dependent manner. Most Burkholderia species employ the CepIR QS system, which relies on N-octanoyl-homoserine lactone. However, some strains harbour multiple QS systems and produce numerous AHLs. QS systems have been demonstrated to be essential for full virulence in various infection models and, thus, these regulatory systems represent attractive targets for the development of novel therapeutics.

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 .