Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N-acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde, and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also cause mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR-specific inhibitory activities over a range of concentrations below that causing toxicity. ortho-Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho-vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho-vanillin as a small molecule QS modulator against RhlR. IMPORTANCE:Quorum sensing (QS) regulates many aspects of bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in Pseudomonas aeruginosa. Potent and selective QS inhibitors remain relatively scarce. However, natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes, and related derivatives, in LasR and RhlR. We demonstrate that ortho-vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for P. aeruginosa QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities.

ABSTRACTThe opportunistic bacteriumPseudomonas aeruginosauses the LasR-I quorum sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically,lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism allowing thelasR-null mutants to persist under tobramycin selection. We hypothesized that the effects of inactivatinglasRon tobramycin resistance might be dependent on the presence or absence of other gene mutations in that strain, a phenomenon known as epistasis. To test this hypothesis, we inactivatedlasRin several highly tobramycin-resistant isolates from long-term evolution experiments. We show that the effects of ΔlasRon tobramycin resistance are strain dependent, which is due to a single mutation in thefusA1gene encoding the translation elongation factor EF-G1A (G61A nucleotide substitution). ThefusA1G61A mutation confers a strong selective advantage to ΔlasRmutants under tobramycin treatment. The effects offusA1G61A on ΔlasR-dependent tobramycin resistance are dependent on the MexXY efflux pump and the MexXY regulator ArmZ. ThefusA1mutation also modulates ΔlasRmutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results provide a possible explanation for the emergence oflasR-null mutants in clinical isolates and illustrate the importance of epistatic gene interactions in the evolution of quorum sensing.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum-sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism that permits the emergence of lasR-null mutants under tobramycin selection. We hypothesized that some other genetic mutations that emerge in these isolates might modulate the effects of lasR-null mutations on antibiotic resistance. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. In some of these isolates, inactivating lasR further increased resistance, compared with decreasing resistance of the wild-type ancestor. These strain-dependent effects were due to a G61A nucleotide polymorphism in the fusA1 gene encoding amino acid substitution A21T in the translation elongation factor EF-G1A. The EF-G1A mutational effects required the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulated ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results identify a gene mutation that can reverse the direction of the antibiotic selection of lasR mutants, a phenomenon known as sign epistasis, and provide a possible explanation for the emergence of lasR-null mutants in clinical isolates. IMPORTANCE One of the most common mutations in Pseudomonas aeruginosa clinical isolates is in the quorum sensing lasR gene. In laboratory strains, lasR disruption decreases resistance to the clinical antibiotic tobramycin. To understand how lasR mutations emerge in tobramycin-treated patients, we mutated lasR in highly tobramycin-resistant laboratory strains and determined the effects on resistance. Disrupting lasR enhanced the resistance of some strains. These strains had a single amino acid substitution in the translation factor EF-G1A. The EF-G1A mutation reversed the selective effects of tobramycin on lasR mutants. These results illustrate how adaptive mutations can lead to the emergence of new traits in a population and are relevant to understanding how genetic diversity contributes to the progression of disease during chronic infections.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum sensing system to increase resistance to the aminioglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism allowing the lasR-null mutants to persist under tobramycin selection. We hypothesized that the effects of inactivating lasR on tobramycin resistance might be dependent on the presence or absence of other gene mutations in that strain, a phenomenon known as epistasis. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. We show that the effects of ΔlasR on tobramycin resistance are strain dependent. The effects can be attributed to a point mutation in the gene encoding the translation elongation factor fusA1 (G61A nucleotide substitution), which confers a strong selective advantage to lasR-null PA14 under tobramycin selection. This fusA1 G61A mutation results in increased activity of the MexXY efflux pump and expression of the mexXY regulator ArmZ. The fusA1 mutation can also modulate ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results demonstrate the importance of epistatic gene interactions on antibiotic susceptibility of lasR-null mutants. These results support of the idea that gene interactions might play a significant role in the evolution of quorum sensing in P. aeruginosa.

The bacterium Pseudomonas aeruginosa causes serious disease in immunocompromised patients and is a model for studying quorum sensing, a cell-cell signaling system that becomes activated at a certain population or "quorum." The P. aeruginosa master quorum sensing regulator is LasR, which drives transcription of dozens of genes including those coding for virulence and antibiotic resistance. Paradoxically, lasR-null mutants are common in infections of tobramycin-treated patients, presenting a challenge to ongoing efforts to develop anti-LasR therapeutics. To understand how these mutants might arise, we performed a laboratory evolution experiment. We grew P. aeruginosa populations with sublethal tobramycin. Every day, we transferred the population to fresh medium and every three days we increased the tobramycin concentration. Variants from these populations became highly tobramycin resistant. Surprisingly, inactivating LasR in some of the variants further increased resistance, which was the opposite effect as that observed in the ancestral strain where LasR inactivation decreased tobramycin resistance. This phenomenon is known as sign epistasis. The evolved variants with the altered LasR phenotype all encoded a point mutation in the translation elongation factor fusA1. fusA1frequently incurs mutations as a mechanism of adaptation to tobramycin in antibiotic-treated patient infections. We showed that the fusA1 mutation reversed the effect of LasR on antibiotic resistance, and thus is responsible for sign epistatic effects on LasR. Our results suggest that antibiotic selection might drive the accumulation of mutations that reverse the effect of LasR mutants on tobramycin resistance. These results could possibly explain how lasR mutants emerge in infected patients and have implications for developing novel therapeutics to treat antibiotic-resistant infections.