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

A Unique Regulator Contributes to Quorum Sensing and Virulence in Burkholderia cenocepacia

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

Genetic organization of the BCAM1871 locus.

<p>BCAM1871 is located downstream from <i>cepI</i> with 52 bp separating <i>cepI</i> and BCAM1871 which were determined to be co-transcribed (solid line) using RT-PCR. Size (bp) of each ORF is indicated. The <i>cepI</i> transcription start site is located 28 bp upstream of the ATG start codon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037611#pone.0037611-Weingart1" target="_blank">[18]</a>. One promoter (arrow) was identified for the <i>cepI</i>-BCAM1871 operon, and this was located upstream of <i>cepI</i> and included in the <i>cepI</i> promoter::<i>lux</i> fusion (pCP300). Nomenclature used to describe K56-dI2 and K56-2ΔBCAM1871 mutants.</p

Bacterial persistence and inflammation in a rat chronic respiratory infection model.

<p>At seven days postinfection rat lungs were harvested and used for (A) quantitative bacteriology by plating lung homogenates and determining the number of colony forming units or (B) quantitative histopathology analysis of hematoxylin and eosin stained lung sections. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037611#s2" target="_blank">Results</a> are displayed using scatter plots with mean values represented by the horizontal bars. P values indicate significant differences in lungs infected with K56-2ΔM1871 compared to that in K56-2.</p

Effects of BCAM1871 on transcription, phenotype and virulence of <i>B. cenocepacia</i>.

<p>BCAM1871 positively (+) influences transcription of several genes invoved in altering phenotypes that contribute to pathogenesis in rat and nematode infection models.</p

Transcription of <i>shvR</i> and <i>afcA</i>.

<p>Transcription was monitored using promoter::<i>lux</i> fusions in LB at 37°C. (A) <i>shvR</i> expression was significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 7–13 h and 28.5–38.5 h (p<0.001). (B) <i>afcA</i> expression was significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 26–44 h (p<0.001).</p

Protease activity and transcription of <i>zmpA</i> and <i>zmpB</i>.

<p>Protease activity was determined using D-BHI with 1.5% skim milk agar plates after 40 h incubation at 37°C, ±2500 ρM OHL. (A) Significantly different compared to: a, K56-2; b, K56-dI2. (B) Significantly different compared to: a, K56-2 (pUCP28T); b, appropriate parent strain K56-2ΔM1871 (pUCP28T) or K56-dI2 (pUCP28T). All p values <0.001. Transcription was monitored using promoter::<i>lux</i> fusions in LB ± OHL at 37°C. OHL was added at 8 h (arrow). (C) <i>zmpA</i> (pBS13) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 14–14.5, 16–22 and 27.5–37.5 h (p<0.05). (D) <i>zmpB</i> (pBS9) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 11.5–48 h (p<0.05).</p

Influence of BCAM1871 on AHL activity.

<p>AHL activity was monitored using <i>A. tumefaciens</i> A136 (pCF218) (pMV26) in a real-time liquid co-culture assay. (A) Significantly decreased in K56-2ΔM1871 (pUCP28T) compared to that in K56-2 (pUCP28T) from 16–30 h. Significantly increased in K56-2ΔM1871 (p28T-M1871) compared to that in K56-2ΔM1871 (pUCP28T) from 18–30 h. Significantly decreased in K56-dI2 (pUCP28T) compared to that in K56-2ΔM1871 (pUCP28T) from 16–30 h. (B) Significantly increased in K56-2 (p28T-M1871) compared to that in K56-2 (pUCP28T) from 2–10 h and 18–30 h. Significantly increased in K56-2 (pSLS250) compared to that in K56-2 (p28T-M1871) from 4–25 h. (C) Significantly increased in K56-2ΔM1871 (pSLS250) compared to that in K56-2ΔM1871 (pUCP28T) from 2–30 h. No significant difference in K56-dI2 (pUCP28T) compared to that in K56-dI2 (p28T-M1871). All p values<0.001.</p

Oligonucleotide primers used in this study.

a<p>Restriction enzyme sites underlined.</p

Nematode survival and transcription of <i>aidA</i>.

<p>(A) Nematode survival was monitored by analyzing response to touch after feeding on strains indicated. *, significantly decreased in K56-2ΔM1871 (pUCP28T) or K56-dI2 (pUCP28T) compared to that in K56-2 (pUCP28T) (p<0.0001). §,significantly increased in K56-2ΔM1871 (p28T-M1871) compared to that in K56-2ΔM1871 (pUCP28T) (p<0.0001). Values are the means ± standard error of results from plates containing at least 25 worms per plate and are representative of results from at least two individual trials. (B) Transcription was monitored using promoter::<i>lux</i> fusions in LB ± OHL at 37°C. OHL was added at 8 h (arrow). <i>aidA</i> expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 29.5–42.5 h (p<0.05) (most timepoints); significantly increased in K56-2ΔM1871 300 ρM OHL compared to that in K56-2 from 30–41 h (p<0.001).</p