The Burkholderia cepacia Epidemic Strain Marker Is Part of a Novel Genomic Island Encoding Both Virulence and Metabolism-Associated Genes in Burkholderia cenocepacia

ABSTRACT The Burkholderia cepacia epidemic strain marker (BCESM) is a useful epidemiological marker for virulent B. cenocepacia strains that infect patients with cystic fibrosis. However, there was no evidence that the original marker, identified by random amplified polymorphic DNA fingerprinting, contributed to pathogenicity. Here we demonstrate that the BCESM is part of a novel genomic island encoding genes linked to both virulence and metabolism. The BCESM was present on a 31.7-kb low-GC-content island that encoded 35 predicted coding sequences (CDSs): an N-acyl homoserine lactone (AHL) synthase gene (cciI) and corresponding transcriptional regulator (cciR), representing the first time cell signaling genes have been found on a genomic island; fatty acid biosynthesis genes; an IS66 family transposase; transcriptional regulator CDSs; amino acid metabolism genes; and a group of hypothetical genes. Mutagenesis of the AHL synthase, amidase (amiI), and porin (opcI) genes on the island was carried out. Testing of the isogenic mutants in a rat model of chronic lung infection demonstrated that the amidase played a role in persistence, while the AHL synthase and porin were both involved in virulence. The island, designated the B. cenocepacia island (cci), is the first genomic island to be defined in the B. cepacia complex and its discovery validates the original epidemiological correlation of the BCESM with virulent CF strains. The features of the cci, which overlap both pathogenicity and metabolism, expand the concept of bacterial pathogenicity islands and illustrate the diversity of accessory functions that can be acquired by lateral gene transfer in bacteria.

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

Journal of Bacteriology ◽

10.1128/jb.01544-06 ◽

2007 ◽

Vol 189 (8) ◽

pp. 3006-3016 ◽

Cited By ~ 20

Author(s):

Rebecca J. Malott ◽

Pamela A. Sokol

Keyword(s):

Quorum Sensing ◽

Burkholderia Cepacia ◽

Regulatory Element ◽

Transcriptional Regulator ◽

Homoserine Lactone ◽

Burkholderia Vietnamiensis ◽

Sensing System ◽

Sensing Systems ◽

Quorum Sensing System ◽

Regulatory Organization

ABSTRACT 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.

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Distribution of Genes Encoding Putative Transmissibility Factors among Epidemic and Nonepidemic Strains ofBurkholderia cepacia from Cystic Fibrosis Patients in the United Kingdom

Journal of Clinical Microbiology ◽

10.1128/jcm.38.5.1763-1766.2000 ◽

2000 ◽

Vol 38 (5) ◽

pp. 1763-1766 ◽

Cited By ~ 55

Author(s):

Fiona E. Clode ◽

Mary E. Kaufmann ◽

Henry Malnick ◽

Tyrone L. Pitt

Keyword(s):

Cystic Fibrosis ◽

United Kingdom ◽

Genetic Markers ◽

Burkholderia Cepacia ◽

Insertion Sequence ◽

Random Amplified Polymorphic Dna ◽

Specific Marker ◽

Epidemic Strain ◽

The United Kingdom ◽

Rapd Pattern

In the last 15 years, Burkholderia cepacia has emerged as a significant pathogen in cystic fibrosis (CF) patients, mainly due to the severity of infection observed in a subset of patients and the fear of transmission of the organism to noncolonized patients. Although patients who deteriorate rapidly cannot be predicted by microbiological characteristics, three genetic markers have been described for strains that spread between patients. These are the cblA gene, encoding giant cable pili; a hybrid of two insertion sequences, IS1356 and IS402; and a 1.4-kb open reading frame known as the B. cepacia epidemic strain marker (BCESM). The latter two are of unknown function. An epidemic strain lineage was previously identified among CF patients in the United Kingdom that apparently had spread from North America and that was characterized by a specific random amplified polymorphic DNA (RAPD) pattern. We searched for the described genetic markers using specific PCR assays with 117 patient isolates of B. cepacia from 40 United Kingdom hospitals. Isolates were grouped according to genomovar and epidemic strain lineage RAPD pattern with a 10-base primer, P272. A total of 41 isolates from patients in 12 hospitals were classified as the epidemic strain, and 40 of these were distributed in genomovars IIIa (11 isolates), IIIb (1 isolate), and IIIc (28 isolates). All isolates of the epidemic strain were positive for the cblAgene and BCESM, but two lacked the insertion sequence hybrid. None of the 76 sporadic isolates contained cblA or the insertion sequence hybrid, but 11 of them were positive for BCESM. Nonepidemic isolates were distributed among genomovars I or IV (9), II (49), IIIa (11), IIIb (3), and IIIc (4). There were three clusters of cross-infection (one involving two patients and two involving three patients) with isolates of genomovar II. We conclude that in the United Kingdom, a single clonal lineage has spread between and within some hospitals providing care for CF patients. The presence of thecblA gene is the most specific marker for the epidemic strain. We recommend that all isolates of B. cepacia from CF patients should be screened by PCR to influence segregation and infection control strategies.

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Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

Microbiology ◽

10.1099/mic.0.022764-0 ◽

2009 ◽

Vol 155 (3) ◽

pp. 712-723 ◽

Cited By ~ 169

Author(s):

Valérie Dekimpe ◽

Eric Déziel

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Transcriptional Regulator ◽

Homoserine Lactone ◽

The Other ◽

Virulence Determinants ◽

Late Stationary Phase ◽

Regulatory Systems ◽

Specific Factors

Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C4-homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C12-HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.

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Evaluation of the electron transfer flavoprotein as an antibacterial target in Burkholderia cenocepacia

Canadian Journal of Microbiology ◽

10.1139/cjm-2017-0350 ◽

2017 ◽

Vol 63 (10) ◽

pp. 857-863 ◽

Cited By ~ 1

Author(s):

Maria S. Stietz ◽

Christina Lopez ◽

Osasumwen Osifo ◽

Marcelo E. Tolmasky ◽

Silvia T. Cardona

Keyword(s):

Electron Transfer ◽

Burkholderia Cepacia ◽

Multidrug Resistant ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Infection Model ◽

Electron Transfer Flavoprotein ◽

C Elegans

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.

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The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.05060-11 ◽

2011 ◽

Vol 10 (8) ◽

pp. 1034-1042 ◽

Cited By ~ 79

Author(s):

Rebecca A. Hall ◽

Kara J. Turner ◽

James Chaloupka ◽

Fabien Cottier ◽

Luisa De Sordi ◽

...

Keyword(s):

Candida Albicans ◽

Quorum Sensing ◽

Signaling Pathway ◽

Homoserine Lactone ◽

Burkholderia Cenocepacia ◽

Camp Signaling ◽

Mammalian Host ◽

Content Type ◽

Hyphal Development ◽

Natural Flora

ABSTRACTLiving as a commensal,Candida albicansmust adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects onC. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C12-homoserine lactone, a quorum-sensing molecule secreted byPseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of theCandidaadenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving theC. albicanshyphal repressor, Sfl1p. Deletion ofSFL1did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing inC. albicansis mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that theBurkholderia cenocepaciadiffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C12-homoserine lactone, may be used by other quorum-sensing molecules.

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Intracellular survival ofBurkholderia cepaciacomplex in phagocytic cells

Canadian Journal of Microbiology ◽

10.1139/cjm-2015-0316 ◽

2015 ◽

Vol 61 (9) ◽

pp. 607-615 ◽

Cited By ~ 23

Author(s):

Miguel A. Valvano

Keyword(s):

Burkholderia Cepacia ◽

Molecular Mechanisms ◽

Inflammatory Responses ◽

Genetic Manipulation ◽

Intracellular Survival ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Host Responses ◽

Current View ◽

Phagocytic Cells

Burkholderia cepacia complex (Bcc) species are a group of Gram-negative opportunistic pathogens that infect the airways of cystic fibrosis patients, and occasionally they infect other immunocompromised patients. Bcc bacteria display high-level multidrug resistance and chronically persist in the infected host while eliciting robust inflammatory responses. Studies using macrophages, neutrophils, and dendritic cells, combined with advances in the genetic manipulation of these bacteria, have increased our understanding of the molecular mechanisms of virulence in these pathogens and the molecular details of cell-host responses triggering inflammation. This article discusses our current view of the intracellular survival of Burkholderia cenocepacia within macrophages.

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Disruption of Quorum Sensing and Virulence inBurkholderia cenocepaciaby a Structural Analogue of thecis-2-Dodecenoic Acid Signal

Applied and Environmental Microbiology ◽

10.1128/aem.00105-19 ◽

2019 ◽

Vol 85 (8) ◽

Cited By ~ 4

Author(s):

Chaoyu Cui ◽

Shihao Song ◽

Chunxi Yang ◽

Xiuyun Sun ◽

Yutong Huang ◽

...

Keyword(s):

Quorum Sensing ◽

Bacterial Infections ◽

Opportunistic Pathogen ◽

Homoserine Lactone ◽

Burkholderia Cenocepacia ◽

Enoic Acid ◽

Acyl Homoserine Lactone ◽

Structural Analogue ◽

Content Type ◽

Signal Production

ABSTRACTQuorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities.Burkholderia cenocepaciaemploys a molecular mechanism in which thecis-2-dodecenoic acid (namedBurkholderiadiffusiblesignalfactor [BDSF]) QS system regulatesN-acyl homoserine lactone (AHL) signal production and virulence by modulating intracellular levels of cyclic diguanosine monophosphate (c-di-GMP). Thus, inhibition of BDSF signaling may offer a non-antibiotic-based therapeutic strategy against BDSF-regulated bacterial infections. In this study, we report the synthesis of small-molecule mimics of the BDSF signal and evaluate their ability to inhibit BDSF QS signaling inB. cenocepacia. A novel structural analogue of BDSF, 14-Me-C16:Δ2(cis-14-methylpentadec-2-enoic acid), was observed to inhibit BDSF production and impair BDSF-regulated phenotypes inB. cenocepacia, including motility, biofilm formation, and virulence, while it did not inhibit the growth rate of this pathogen. 14-Me-C16:Δ2also reduced AHL signal production. Genetic and biochemical analyses showed that 14-Me-C16:Δ2inhibited the production of the BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Notably, 14-Me-C16:Δ2attenuated BDSF-regulated phenotypes in variousBurkholderiaspecies. These findings suggest that 14-Me-C16:Δ2could potentially be developed as a new therapeutic agent against pathogenicBurkholderiaspecies by interfering with their QS signaling.IMPORTANCEBurkholderia cenocepaciais an important opportunistic pathogen which can cause life-threatening infections in susceptible individuals, particularly in cystic fibrosis and immunocompromised patients. It usually employs two types of quorum sensing (QS) systems, including thecis-2-dodecenoic acid (BDSF) system andN-acyl homoserine lactone (AHL) system, to regulate virulence. In this study, we have designed and identified an unsaturated fatty acid compound (cis-14-methylpentadec-2-enoic acid [14-Me-C16:Δ2]) that is capable of interfering withB. cenocepaciaQS signaling and virulence. We demonstrate that 14-Me-C16:Δ2reduced BDSF and AHL signal production inB. cenocepacia. It also impaired QS-regulated phenotypes in variousBurkholderiaspecies. These results suggest that 14-Me-C16:Δ2could interfere with QS signaling in manyBurkholderiaspecies and might be developed as a new antibacterial agent.

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In Vitro Activity of a Novel Glycopolymer against Biofilms of Burkholderia cepacia Complex Cystic Fibrosis Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00498-19 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 1

Author(s):

Vidya P. Narayanaswamy ◽

Andrew P. Duncan ◽

John J. LiPuma ◽

William P. Wiesmann ◽

Shenda M. Baker ◽

...

Keyword(s):

Cystic Fibrosis ◽

Burkholderia Cepacia ◽

Laser Scanning Microscopy ◽

Burkholderia Cenocepacia ◽

Burkholderia Cepacia Complex ◽

Confocal Laser ◽

Content Type ◽

Biofilm Removal ◽

Increased Risk

ABSTRACT Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia gladioli, Burkholderia dolosa, Burkholderia vietnamiensis, and B. cepacia. These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro. Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 μg/ml PAAG following a 1-h treatment (P < 0.01). A significant reduction in biofilm thickness was observed following a 10-min treatment of Bcc biofilms with PAAG compared to that with vehicle control (P < 0.001) in TCP, MBEC, and CLSM analyses. PAAG also rapidly permeabilizes bacteria within the first 10 min of treatment. Glycopolymers, such as PAAG, are a new class of large-molecule therapeutics that support the treatment of recalcitrant Bcc biofilm.

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