Quorum sensing and virulence of Pseudomonas aeruginosa during lung infection of cystic fibrosis patients

Abstract

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.

Figure 1. Distribution of in vitro rhamnolipid production in P. aeruginosa isolates from CF patients at different stages of lung infection.

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

Figure 2. Distribution of rhamnolipid production in mucoid and nonmucoid isolates.

Box and whisker plots represent the median (thick line inside the box), 10, 25, 75 and 90 centiles of the in vitro rhamnolipid production (µg/ml) of mucoid and non-mucoid P. aeruginosa isolates from chronically infected CF patients. Mann-Whitney test was used to investigate the significance of the difference between the groups.

Figure 3. Distribution of rhamnolipid production in isolates belonging to different clones.

Box and whisker plots represent the median (thick line inside the box), 10, 25, 75 and 90 centiles of the in vitro rhamnolipid production (µg/ml) of P. aeruginosa isolates belonging to different clones included in the study. The clones DK1 and DK2 are the two dominating clones in Denmark. Clone NO is a dominating clone in Norway and the non-clonal isolates have no clonal relationship. Mann-Whitney test was used to investigate the significance of the difference between the groups.

Figure 4. Distribution of rhamnolipid production in isolates with or without mutations in the QS regulatory genes.

Box and whisker plots represent the median (thick line inside the box), 10, 25, 75 and 90 centiles of the in vitro rhamnolipid production (µg/ml) of P. aeruginosa isolates (A) with (rhlR−) or without mutations (rhlR+) in the regulatory gene rhlR; (B) with (lasR−) or without mutations (lasR+) in the regulatory gene lasR Mann-Whitney test was used to investigate the significance of the difference between the groups.

Figure 5. Mutations in the lasR gene (A) and rhlR gene (B) of P. aeruginosa isolates from CF patients.

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 lasR or in grey if not complemented. Amino acid changes that have been previously shown to impair the LasR function , are marked in squares and amino acid changes that have been previously described in in vitro studies , , are encircled.

Figure 6. Mutations in the rhlR gene of P. aeruginosa isolates from CF patients.

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.

Figure 7. Association between occurrence of mutations in the QS genes and the mutability of the isolates.

Box and whisker plots represent the median (thick line inside the box), 10, 25, 75 and 90 centiles of the mutation frequency measured by the occurrence of spontaneous antibiotic resistance in isolates with mutations in both QS regulatory genes (lasR−rhlR−), only in one of the regulatory genes (lasR−rhlR+) or (lasR+rhlR−) or without mutations in either of the regulatory genes (lasR+rhlR+). Mann-Whitney test was used to investigate the significance of the differences between the groups.