Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Bjarnsholt, Thomas; Jensen, Peter Østrup; Jakobsen, Tim Holm; Phipps, Richard Kerry; Nielsen, Allan K.; Rybtke, Morten; Tolker‐Nielsen, Tim; Givskov, Michael; Høiby, Niels; Ciofu, Oana

doi:10.1371/journal.pone.0010115

Cited by 232 publications

(246 citation statements)

References 53 publications

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“…A recent transposon sequencing study of P. aeruginosa PA14 shows that mutations in type IV pilus associated genes increase fitness in the murine lung (43). Moreover, loss of both motility (44,45) and QS (46,47) functions are known to frequently evolve in P. aeruginosa chronic infections of the CF lung. Temperate phages, including those used here, can be present at very high densities in the CF lung [exceeding bacterial densities by orders of magnitude (13)], which taken together with our findings suggests that temperate phages could play an important role in CF lung infections by driving the evolution of these clinically important traits in P. aeruginosa.…”

Section: Resultsmentioning

confidence: 99%

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

Davies

James

Williams

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Temperate phages drive genomic diversification in bacterial pathogens. Phage-derived sequences are more common in pathogenic than nonpathogenic taxa and are associated with changes in pathogen virulence. High abundance and mobilization of temperate phages within hosts suggests that temperate phages could promote withinhost evolution of bacterial pathogens. However, their role in pathogen evolution has not been experimentally tested. We experimentally evolved replicate populations of Pseudomonas aeruginosa with or without a community of three temperate phages active in cystic fibrosis (CF) lung infections, including the transposable phage, ɸ4, which is closely related to phage D3112. Populations grew as freefloating biofilms in artificial sputum medium, mimicking sputum of CF lungs where P. aeruginosa is an important pathogen and undergoes evolutionary adaptation and diversification during chronic infection. Although bacterial populations adapted to the biofilm environment in both treatments, population genomic analysis revealed that phages altered both the trajectory and mode of evolution. Populations evolving with phages exhibited a greater degree of parallel evolution and faster selective sweeps than populations without phages. Phage ɸ4 integrated randomly into the bacterial chromosome, but integrations into motility-associated genes and regulators of quorum sensing systems essential for virulence were selected in parallel, strongly suggesting that these insertional inactivation mutations were adaptive. Temperate phages, and in particular transposable phages, are therefore likely to facilitate adaptive evolution of bacterial pathogens within hosts.Pseudomonas aeruginosa | cystic fibrosis | mobile genetic element | experimental evolution | bacteriophage C omparative genomics suggests that temperate phages play an important role in the evolution and genomic diversification of bacterial pathogens (1). Bacterial genomes often contain a range of intact and remnant prophage elements (1-3), and ecologically important bacterial traits are believed to be phage-derived (e.g., phage-derived bacteriocins) (4). Phage-related sequences are observed more frequently in pathogenic than nonpathogenic strains (5), and prophage acquisition can be associated with changes in pathogen virulence (6, 7). Prophages can directly contribute accessory gene functions (1, 8) or disrupt bacterial genes by insertional inactivation. Of particular note are the transposable class of temperate phages (also known as mutator phages), including D3112 of Pseudomonas aeruginosa (9, 10), which integrate throughout the chromosome disrupting existing genes and increasing the supply of mutations available to selection. Recent reports of high rates of phage mobilization within hosts (11) and high temperate phage abundance in humans (12), including at sites of chronic infection where phage particles have been observed to exceed bacterial host densities by 10-to 100-fold (13), suggests that temperate phages could play an important role in driving within-host...

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Section: Resultsmentioning

confidence: 99%

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

Davies

James

Williams

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…8) and is even shared by LuxR. Furthermore, one LasR mutation observed in two different studies of clinical P. aeruginosa isolates is C203R, correlated with loss of LasR function as measured by elastase activity (3,6). These clinical findings further support an important role for Cys 203 .…”

Section: Discussionmentioning

confidence: 99%

“…The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that establishes chronic infections in immunocompromised patients, with persistent pulmonary colonization in individuals with cystic fibrosis (1)(2)(3). Like many bacteria, P. aeruginosa employs quorumsensing (QS) 4 machinery to communicate local population density through the exchange of self-produced signaling molecules (4).…”

mentioning

confidence: 99%

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

Kafle¹,

Amoh²,

Reaves³

et al. 2016

Journal of Biological Chemistry

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The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys 79 in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys 201 -Cys 203 disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with ␤-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that establishes chronic infections in immunocompromised patients, with persistent pulmonary colonization in individuals with cystic fibrosis (1-3). Like many bacteria, P. aeruginosa employs quorumsensing (QS) 4 machinery to communicate local population density through the exchange of self-produced signaling molecules (4). QS contributes to the development of acute infections by coordinating the production of multiple virulence factors, including elastase and pyocyanin, and the formation of biofilms (5-7). QS in P. aeruginosa is largely mediated by N-acyl L-homoserine lactone autoinducer molecules, biosynthesized by LuxI-type synthases, and detected by LuxR-type regulator proteins (8). The two major systems responsive to acylhomoserine lactones (AHLs) in P. aeruginosa are LasI/LasR and RhlI/RhlR. LasI and RhlI are LuxI-type synthases, whereas LasR and RhlR are LuxR-type regulatory proteins. These AHL-dependent systems are also closely connected to signaling governed by 2-alkyl-4-quinolones in P. aeruginosa (9).The regulator protein LasR, with its cognate synthase LasI, functions at the top of the P. aeruginosa quorum-sensing hierarchy (10). Like other LuxR-type proteins, this transcription factor is composed of two domains (11). The amino-terminal ligand binding domain (LBD) specifically recognizes its autoinducer, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C 12 -HSL), promoting protein folding and dimerization, whereas the DNA binding domain (DBD) recognizes target sites to activate downstream gene expression. There has been significant interest in understanding LasR, as its inhibition represents a...

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“…This process is mediated by a switch from planktonic to sessile forms, and attachment to organic tissues or abiotic surfaces, and involves virulence factors such as alginate, elastase, LPS and rhamnolipids (Bjarnsholt et al, 2010). Colonization as biofilms enables bacterial communication and coordination of virulence factor expression via cell-density-dependent (quorum sensing, QS) regulation, which employs the las and rhl encoded systems.…”

Section: Introductionmentioning

confidence: 99%

Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica

Davis

Brown

2016

Journal of Medical Microbiology

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Respiratory infections are common causes of morbidity and mortality worldwide. We sought to assess the multiple antibiotic resistance (MAR) index, fitness and virulence potential in Pseudomonas aeruginosa from patients with lower respiratory tract infections. Isolates were assessed for antimicrobial susceptibility, in vitro competitive fitness, and pigment, elastase and rhamnolipid production. Oxidative stress tolerance was determined on both planktonic and biofilm cells, and virulence potential was tested in a plant model. Mean MAR index for isolates was 0.34 (range 0.17-0.50). Whilst isolates exhibited good biofilm formation in the presence of ciprofloxacin, there was no significant difference in biofilm production over the concentration range assessed. Several drug-resistant strains were out-competed by a susceptible strain even in the presence of antibiotic. H 2 O 2 exerted a greater oxidative stress than tert-butylhydroperoxide and, as expected, biofilms were more resistant than planktonic cells. Whilst most (81 %) isolates were pigmented there was no significant difference between pigmented and non-pigmented isolates when elastolytic activity was compared (P.0.05). More than half of the isolates produced the quorum sensing mediator rhamnolipid and infection of the plant model by bacteria occurred whether elastase or rhamnolipid was present or absent. These data suggest that non-pigmented strains of P. aeruginosa might pose an equally significant microbiological threat as pigmented strains even though pigment production appeared to be strongly associated with elastase expression. Whilst dual expression of elastase and rhamnolipid by these bacteria would cause severe tissue damage (as seen in the plant model), non-production of either does not prevent bacteria from causing serious infection.

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Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Cited by 232 publications

References 53 publications

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

Temperate phages both mediate and drive adaptive evolution in pathogen biofilms

Molecular Insights into the Impact of Oxidative Stress on the Quorum-Sensing Regulator Protein LasR

Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica

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