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Identification of N‐acylhomoserine lactones in mucopuru respiratory secretions from cystic fibrosis patients

Department of Food Science, Cornell University, Итак, New York, United States
FEMS Microbiology Letters (Impact Factor: 2.72). 04/2005; 244(2):297-304. DOI: 10.1016/j.femsle.2005.01.055
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ABSTRACT Pseudomonas aeruginosa and species of the Burkholderia cepacia complex are the primary bacterial pathogens contributing to lung disease in patients with cystic fibrosis. Quorum sensing systems using N-acyl homoserine lactone (AHL) signal molecules are involved in the regulation of a number of virulence factors in these species. Extracts of mucopurulent respiratory secretions from 13 cystic fibrosis patients infected with P. aeruginosa and/or strains of the B. cepacia complex were fractionated using reverse-phase fast pressure liquid chromatography and analyzed for the presence of AHLs using a traI-luxCDABE-based reporter that responds to AHLs with acyl chains ranging between 4 and 12 carbons. Using this assay system, a broad range of AHLs were detected and identified despite being present at low concentrations in limited sample volumes. N-(3-oxo-dodecanoyl)-l-homoserine lactone, N-(3-oxo-decanoyl)-l-homoserine lactone and N-octanoyl-l-homoserine lactone (OHL) were the AHLs most frequently identified. OHL and N-decanoyl-l-homoserine lactone were detected in nanomolar concentrations compared to picomolar amounts of the 3-oxo-derivatives of the AHLs identified.

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    • "Detection of AHLs in freshly extracted spent media from B. cenocepacia K56-2 strains was performed using the bioluminescent reporter strain A. tumefaciens A136 (pCF218)(pMV26) (Sokol et al., 2003; Chambers et al., 2005) as previously described (Bernier et al., 2008). Briefly, overnight cultures from A. tumefaciens A136 (pCF218)(pMV26) were washed as described above and cells were resuspended in PBS and subsequently diluted 1:1500 (v/v) in LB. "
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    ABSTRACT: The phenylacetic acid degradation pathway of Burkholderia cenocepacia is active during cystic fibrosis-like conditions and is necessary for full pathogenicity of B. cenocepacia in nematode and rat infection models; however, the reasons for such requirements are unknown. Here, we show that the attenuated virulence of a phenylacetic acid catabolism mutant is due to quorum sensing inhibition. Unlike wild-type B. cenocepacia, a deletion mutant of the phenylacetyl-CoA monooxygenase complex (paaABCDE) released phenylacetic acid in the medium that favours infection in Caenorhabditis elegans. Addition of phenylacetic acid further decreased the pathogenicity of the paaABCDE, which cannot metabolize phenylacetic acid, but did not affect the wild-type, due to phenylacetic acid consumption. In line with reduced detection of acyl-homoserine lactones in spent medium, the paaABCDE exhibited transcriptional inhibition of the quorum sensing system cepIR. Phenotypes repressed in paaABCDE, protease activity and pathogenicity against C. elegans, increased with exogenous N-octanoyl-L-homoserine lactone. Thus, we demonstrate that the attenuated phenotype of B. cenocepacia paaABCDE is due to quorum sensing inhibition by release of phenylacetic acid, affecting N-octanoyl-L-homoserine lactone signalling. Further, we propose that active degradation of phenylacetic acid by B. cenocepacia during growth in cystic fibrosis-like conditions prevents accumulation of a quorum sensing inhibiting compound.
    Molecular Microbiology 08/2014; 94(3). DOI:10.1111/mmi.12771 · 5.03 Impact Factor
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    • "AHL have been detected from mucopurulent respiratory secretions (0Á5 pmol l À1 –66Á5 nmol l À1 ) and infected lung tissue (66 fmol g À1 –146 pmol g À1 ) (Favre-Bont e et al. 2002; Chambers et al. 2005). "
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    ABSTRACT: Aims The aim of this study was to use a sensitive method to screen and quantify 57 Vibrionaceae strains for the production of acyl-homoserine lactones (AHLs) and map the resulting AHL profiles onto a host phylogeny. Methods and Results We used a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) protocol to measure AHLs in spent media after bacterial growth. First, the presence/absence of AHLs (qualitative analysis) was measured to choose internal standard for subsequent quantitative AHL measurements. We screened 57 strains from three genera (Aliivibrio,Photobacterium and Vibrio) of the same family (i.e. Vibrionaceae). Our results show that about half of the isolates produced multiple AHLs, typically at 25–5000 nmol l−1. Conclusions This work shows that production of AHL quorum sensing signals is found widespread among Vibrionaceae bacteria and that closely related strains typically produce similar AHL profiles. Significance and Impact of the Study The AHL detection protocol presented in this study can be applied to a broad range of bacterial samples and may contribute to a wider mapping of AHL production in bacteria, for example, in clinically relevant strains.
    Journal of Applied Microbiology 05/2013; 115(3). DOI:10.1111/jam.12264 · 2.39 Impact Factor
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    • "The DcepI and DatsRDcepI mutants did not present any detectable AHL activity using this assay, even though they still contain cciI. These results were expected since the biosensor strain we used responds 1000 times better to C8-AHL than C6-AHL, which are mainly produced by CepI and CciI respectively (Chambers et al., 2005). Luciferase assays were performed using plasmids pCP300 (cepI::luxCDABE) and pRM445 (cciIR::luxCD- ABE) to determine whether cepI and/or the cciIR operon were differently expressed in WT and DatsR (Fig 4A and B). "
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    ABSTRACT: Burkholderia cenocepacia is commonly found in the environment and also as an important opportunistic pathogen infecting patients with cystic fibrosis. Successful infection by this bacterium requires coordinated expression of virulence factors, which is achieved through different quorum sensing (QS) regulatory systems. Biofilm formation and Type 6 secretion system (T6SS) expression in B. cenocepacia K56-2 are positively regulated by QS and negatively regulated by the sensor kinase hybrid AtsR. This study reveals that in addition to affecting biofilm and T6SS activity, the deletion of atsR in B. cenocepacia leads to overproduction of other QS-regulated virulence determinants including proteases and swarming motility. Expression of the QS genes, cepIR and cciIR, was upregulated in the ΔatsR mutant and resulted in early and increased N-acylhomoserine lactone (AHL) production, suggesting that AtsR plays a role in controlling the timing and fine-tuning of virulence gene expression by modulating QS signalling. Furthermore, a ΔatsRΔcepIΔcciI mutant could partially upregulate the same virulence determinants indicating that AtsR also modulates the expression of virulence genes by a second mechanism, independently of any AHL production. Together, our results strongly suggest that AtsR is a global virulence regulator in B. cenocepacia.
    Environmental Microbiology 07/2012; 15(2). DOI:10.1111/j.1462-2920.2012.02828.x · 6.24 Impact Factor
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