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

Burkholderia cenocepacia differential gene expression during host-pathogen interactions and adaptation to the host environment

Members of the Burkholderia cepacia complex (Bcc) are important in medical, biotechnological and agricultural disciplines. These bacteria naturally occur in soil and water environments and have adapted to survive in association with plants and animals including humans. All Bcc species are opportunistic pathogens including Burkholderia cenocepacia that causes infections in cystic fibrosis and chronic granulomatous disease patients. The adaptation of B. cenocepacia to the host environment was assessed in a rat chronic respiratory infection model and compared to that of high cell-density in vitro-grown cultures using transcriptomics. The distribution of genes differentially expressed on chromosomes 1, 2 and 3 was relatively proportional to the size of each genomic element, whereas the proportion of plasmid-encoded genes differentially expressed was much higher relative to its size and most genes were induced in vivo. The majority of genes encoding known virulence factors, components of types II and III secretion systems and chromosome 2-encoded type IV secretion system were similarly expressed between in vitro and in vivo environments. Lower expression in vivo was detected for some genes encoding virulence factors controlled by the N&#172;-acyl-homoserine lactone dependent transcriptional regulator CepR and genes associated with flagellar motility, Flp type pilus formation and type VI secretion. Plasmid-encoded type IV secretion genes were markedly induced in vivo. Additional genes induced in vivo included several predicted to be involved in osmotic stress adaptation or intracellular survival, metal ion and nutrient transport, as well as those encoding outer membrane proteins. Genes identified in this study are potentially important for virulence during host-pathogen interactions and may be associated with survival and adaptation to the host environment during chronic lung infections

The Burkholderia cenocepacia LysR-Type Transcriptional Regulator ShvR Influences Expression of Quorum-Sensing, Protease, Type II Secretion, and afc Genes▿

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

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

Transcription of <i>cepIR</i> and <i>cciIR</i>.

<p>Transcription was monitored using promoter::<i>lux</i> fusions in LB ± OHL at 37°C. OHL was added at 8 h (arrow). (A) <i>cepI</i> (pCP300) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 17.5–28.5 h (p<0.05); significantly increased in K56-2ΔM1871 300 ρM OHL compared to that in K56-2 from 19.5–30 h p<0.001 and 37.5–44 h (p<0.01). (B) <i>cepR</i> (pRM432) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 4–12.5 h (p<0.001); significantly increased in K56-2ΔM1871 30 ρM OHL compared to that in K56-2 from 11.5–22.5 h (p<0.01); significantly increased in K56-2ΔM1871 300 ρM OHL compared to that in K56-2 from 8.5–25 h (p<0.001). (C) <i>cciIR</i> (pRM445) expression was: significantly decreased in K56-2ΔM1871 compared to that in K56-2 from 22.5–48 h (p<0.001).</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

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

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