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

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection

Computer-Aided Identification of Recognized Drugs as Pseudomonas aeruginosa Quorum-Sensing Inhibitors▿ †

Attenuation of Pseudomonas aeruginosa virulence by the use of small-molecule quorum-sensing inhibitors (referred to as the antipathogenic drug principle) is likely to play a role in future treatment strategies for chronic infections. In this study, structure-based virtual screening was used in a search for putative quorum-sensing inhibitors from a database comprising approved drugs and natural compounds. The database was built from compounds which showed structural similarities to previously reported quorum-sensing inhibitors, the ligand of the P. aeruginosa quorum-sensing receptor LasR, and a quorum-sensing receptor agonist. Six top-ranking compounds, all recognized drugs, were identified and tested for quorum-sensing-inhibitory activity. Three compounds, salicylic acid, nifuroxazide, and chlorzoxazone, showed significant inhibition of quorum-sensing-regulated gene expression and related phenotypes in a dose-dependent manner. These results suggest that the identified compounds have the potential to be used as antipathogenic drugs. Furthermore, the results indicate that structure-based virtual screening is an efficient tool in the search for novel compounds to combat bacterial infections

Bursting the bubble on bacterial biofilms: a flow cell methodology

The flow cell biofilm system is an important and widely used tool for the in vitro cultivation and evaluation of bacterial biofilms under hydrodynamic conditions of flow. This paper provides an introduction to the background and use of such systems, accompanied by a detailed guide to the assembly of the apparatus including the description of new modifications which enhance its performance. As such, this is an essential guide for the novice biofilm researcher as well as providing valuable trouble-shooting techniques for even the most experienced laboratories. The adoption of a common and reliable methodology amongst researchers would enable findings to be shared and replicated amongst the biofilm research community, with the overall aim of advancing understanding and management of these complex and widespread bacterial communities

Fluorescence-based reporter for gauging cyclic Di-GMP levels in Pseudomonas aeruginosa

The increased tolerance toward the host immune system and antibiotics displayed by biofilm-forming Pseudomonas aeruginosa and other bacteria in chronic infections such as cystic fibrosis bronchopneumonia is of major concern. Targeting of biofilm formation is believed to be a key aspect in the development of novel antipathogenic drugs that can augment the effect of classic antibiotics by decreasing antimicrobial tolerance. The second messenger cyclic di-GMP is a positive regulator of biofilm formation, and cyclic di-GMP signaling is now regarded as a potential target for the development of antipathogenic compounds. Here we describe the development of fluorescent monitors that can gauge the cellular level of cyclic di-GMP in P. aeruginosa. We have created cyclic di-GMP level reporters by transcriptionally fusing the cyclic di-GMP-responsive cdrA promoter to genes encoding green fluorescent protein. We show that the reporter constructs give a fluorescent readout of the intracellular level of cyclic di-GMP in P. aeruginosa strains with different levels of cyclic di-GMP. Furthermore, we show that the reporters are able to detect increased turnover of cyclic di-GMP mediated by treatment of P. aeruginosa with the phosphodiesterase inducer nitric oxide. Considering that biofilm formation is a necessity for the subsequent development of a chronic infection and therefore a pathogenicity trait, the reporters display a significant potential for use in the identification of novel antipathogenic compounds targeting cyclic di-GMP signaling, as well as for use in research aiming at understanding the biofilm biology of P. aeruginosa

C-di-GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa

Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that controls the lifestyles of many bacteria. A high intracellular level of c-di-GMP induces a biofilm lifestyle, whereas a low intracellular level of c-di-GMP stimulates dispersal of biofilms and promotes a planktonic lifestyle. Here, we used the expression of different reporters to show that planktonic cells, biofilm cells, and cells dispersed from biofilms (DCells) had distinct intracellular c-di-GMP levels. Proteomics analysis showed that the low intracellular c-di-GMP level of DCells induced the expression of proteins required for the virulence and development of antimicrobial peptide resistance in Pseudomonas aeruginosa. In accordance with this, P. aeruginosa cells with low c-di-GMP levels were found to be more resistant to colistin than P. aeruginosa cells with high c-di-GMP levels. This finding contradicts the current dogma stating that dispersed cells are inevitably more susceptible to antibiotics than their sessile counterparts.Published versio

Duration of the chronic lung infection of CF patients harboring <i>P. aeruginosa</i> isolates producing both C4-HSL and 3-oxo-C12-HSL, either C4-HSL or 3-oxo-C12-HSL or none of the QS molecules and distribution of the rhamnolipid and elastase levels in CF <i>P. aeruginosa</i> isolates producing both, one or none of the QS molecules.

<p>*p = 0.0002.</p><p>**p = 0.016 (Mann-Whitney).</p>a<p>p = 0.008.</p>b<p>p<0.0001.</p>c<p>p<0.0001.</p>d<p>p<0.0001 (Mann-Whitney).</p>1<p>p = 0.01 (Mann-Whitney).</p

Mutations in the <i>rhlR</i> gene of <i>P. aeruginosa</i> isolates from CF patients.

<p>The nucleotide sequence alterations were identified by alignment with the PAO1 sequence. On top of the PAO1 sequence the nucleotide substitutions, insertions (ins) or deletions (Δ) are indicated. Under the amino acid sequence, frame shifts (fs), stop codons(*) or amino acid deletions (Δ) or changes are indicated in bold.</p

Mutations in the <i>lasR</i> gene (A) and <i>rhlR</i> gene (B) of <i>P. aeruginosa</i> isolates from CF patients.

<p>The nucleotide sequence alterations were identified by alignment with the PAO1 sequence. On top of the PAO1 sequence the nucleotide substitutions, insertions (ins) or deletions (Δ) are indicated. Under the amino acid sequence, frame shifts (fs), stop codons(*) or amino acid deletions (Δ) or changes are indicated in bold. The altered nucleotides are shown in yellow if the mutation was complemented with a plasmid containing the wild-type <i>lasR</i> or in grey if not complemented. Amino acid changes that have been previously shown to impair the LasR function <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010115#pone.0010115-Heurlier1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010115#pone.0010115-Kiratisin1" target="_blank">[38]</a> are marked in squares and amino acid changes that have been previously described in <i>in vitro</i> studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010115#pone.0010115-DArgenio1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010115#pone.0010115-Bottomley1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010115#pone.0010115-Kovach1" target="_blank">[48]</a> are encircled.</p

Distribution of <i>in vitro</i> rhamnolipid production in <i>P. aeruginosa</i> isolates from CF patients at different stages of lung infection.

<p>The box and whisker plots represent the median (thick line inside the box), 10, 25, 75 and 90 centiles of the <i>in vitro</i> rhamnolipid production (µg/ml) of <i>P. aeruginosa</i> isolates from intermittently colonized and chronically infected patients; Mann-Whitney test was used to investigate the significance of the difference between the groups.</p