Quorum-Sensing-Negative ( lasR ) Mutants of Pseudomonas aeruginosa Avoid Cell Lysis and Death

Abstract

In Pseudomonas aeruginosa, N-acylhomoserine lactone signals regulate the expression of several hundreds of genes, via the transcriptional regulator LasR and, in part, also via the subordinate regulator RhlR. This regulatory network termed quorum sensing contributes to the virulence of P. aeruginosa as a pathogen. The fact that two supposed PAO1 wild-type strains from strain collections were found to be defective for LasR function because of independent point mutations in the lasR gene led to the hypothesis that loss of quorum sensing might confer a selective advantage on P. aeruginosa under certain environmental conditions. A convenient plate assay for LasR function was devised, based on the observation that lasR mutants did not grow on adenosine as the sole carbon source because a key degradative enzyme, nucleoside hydrolase (Nuh), is positively controlled by LasR. The wild-type PAO1 and lasR mutants showed similar growth rates when incubated in nutrient yeast broth at pH 6.8 and 37°C with good aeration. However, after termination of growth during 30 to 54 h of incubation, when the pH rose to > 9, the lasR mutants were significantly more resistant to cell lysis and death than was the wild type. As a consequence, the lasR mutant-to-wild-type ratio increased about 10-fold in mixed cultures incubated for 54 h. In a PAO1 culture, five consecutive cycles of 48 h of incubation sufficed to enrich for about 10% of spontaneous mutants with a Nuh ؊ phenotype, and five of these mutants, which were functionally complemented by lasR ؉ , had mutations in lasR. The observation that, in buffered nutrient yeast broth, the wild type and lasR mutants exhibited similar low tendencies to undergo cell lysis and death suggests that alkaline stress may be a critical factor providing a selective survival advantage to lasR mutants.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…aeruginosa also requires a functional LasR QS system in order to grow on adenosine (343), which has been postulated to be a physiologically relevant carbon source during growth in vivo (241), although it should be mentioned that the relatively high frequency of lasR mutant isolates recovered from chronic CF patients (332) contradicts this assumption unless adenosine utilization has become LasR independent in these strains. In any event, it seems likely that inhibition of LasR during growth on adenosine would result in selective pressure to develop resistance to the inhibitor in order to maximize growth.…”

Quorum quenching quandary: resistance to antivirulence compounds

“…After conjugation, cells (100 ml) were added to 10 ml of oxygen-sensing minimal medium containing 0.1% (w/v) adenosine (Heurlier et al, 2005) supplemented with C-30, 75 mg ml À1 of tetracycline (to select P. aeruginosa transposon mutants) and 20 mg ml À1 of nalidixic acid (to kill the donor E. coli cells because P. aeruginosa is naturally resistant to this antibiotic).…”

“…For growth with C-30, overnight cultures in LuriaBertani (LB) medium were re-inoculated into 20 ml of oxygen-sensing minimal medium containing 0.1% (w/v) adenosine (Heurlier et al, 2005) with or without C-30, and the mixture was incubated with shaking (250 r.p.m. ); all cultures had the same initial turbidity at 600 nm.…”

“…In addition, furanone derivatives including C-30 have been predicted to bind LasR at the 3OC 12 -HSL binding site to inactivate LasR (Kim et al, 2008;Yang et al, 2009). Hence, growth on adenosine in minimal medium depends on a functional LasR QS pathway (Heurlier et al, 2005), and furanone probably inhibits this growth via LasR. Therefore, we reasoned that mutations that confer resistance to furanone C-30 will disrupt the ability of furanone to inhibit LasR-mediated QS in this strain, and these mutants may be identified easily based on our selection method (faster growth).…”

“…Our strategy was to utilize a minimal medium (so it resembles more closely clinical situations) using a growth compound whose assimilation requires QS; therefore, QS and growth were inhibited by the antivirulence compound C-30, which has become the gold standard for antivirulence compounds. Growth on adenosine by P. aeruginosa depends on the degradative enzyme, nucleoside hydrolase, which is positively controlled by LasR (Heurlier et al, 2005). LasR is the transcriptional regulator that mediates acyl-homoserine lactone-based QS in this strain via N-3-oxododecanoyl homoserine lactone (3OC 12 -HSL).…”