Co-regulation of {beta}-lactam resistance, alginate production and quorum sensing in Pseudomonas aeruginosa

Abstract

Development of β-lactam resistance, production of alginate and modulation of virulence factor expression that alters host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. In this study, we propose that a co-regulatory network exists between these mechanisms. We compared the promoter activities of ampR, algT/U, lasR, lasI, rhlR, rhlI and lasA genes, representing the β-lactam antibiotic resistance master regulatory gene, the alginate switch operon, the las and rhl quorum-sensing (QS) genes, and the LasA staphylolytic protease, respectively. Four isogenic P. aeruginosa strains, the prototypic Alg(-) PAO1, Alg(-) PAOampR, the mucoid Alg(+) PAOmucA22 (Alg(+) PDO300) and Alg(+) PAOmucA22ampR (Alg(+) PDOampR) were used. We found that in the presence of AmpR regulator and β-lactam antibiotic, the extracytoplasmic function sigma factor AlgT/U positively regulated P(ampR), whereas AmpR negatively regulated P(algT/U). On the basis of this finding we suggest the presence of a negative feedback loop to limit algT/U expression. In addition, the functional AlgT/U caused a significant decrease in the expression of QS genes, whereas loss of ampR only resulted in increased P(lasI) and P(lasR) transcription. The upregulation of the las QS system is likely to be responsible for the increased lasA promoter and the LasA protease activities in Alg(-) PAOampR and Alg(+) PDOampR. The enhanced expression of virulence factors in the ampR strains correlated with a higher rate of Caenorhabditis elegans paralysis. Hence, this study shows that the loss of ampR results in increased virulence, and is indicative of the existence of a co-regulatory network between β-lactam resistance, alginate production, QS and virulence factor production, with AmpR playing a central role.

Fig. 1.

β-Lactamase expression in the Alg⁺ PDOampR mutant. Assays were performed using the parent strain Alg⁺ PDO300, the mutant Alg⁺ PDOampR and Alg⁺ PDOampR (pAmpR) in the absence (−) and presence (+) of an inducer. The plasmid pAmpR carries the wild-type ampR gene on a broad-host-range low-copy-number plasmid pME6030 (Heeb et al., 2000). Fresh cultures of OD₆₀₀ 0.6–0.8 were induced with 100 μg benzylpenicillin ml⁻¹ for 3 h before harvesting. Assays were performed on sonicated lysate using nitrocefin as a chromogenic substrate. Assays were performed in triplicate. One Miller unit of β-lactamase is defined as 1 nmol nitrocefin hydrolysed min⁻¹ (μg protein)⁻¹.

Fig. 2.

The effects of the ampR mutation on algT/U transcription. The promoter fusion P_algT/U-lacZ was introduced into Alg⁻ PAO1, Alg⁻ PAOampR, Alg⁺ PDO300 and Alg⁺ PDOampR. Induction was carried out using 500 μg benzylpenicillin ml⁻¹ and the β-galactosidase activity was determined in Miller units after 30 min incubation. The basal level of expression was detected in the promoterless lacZ vector, pLP170.

Fig. 3.

The effects of the ampR mutation on QS las and rhl gene transcription. The alteration in the transcription of the QS systems in Alg⁻ PAO1 (hatched bars), Alg⁻ PAOampR (grey bars), Alg⁺ PDO300 (white bars) and Alg⁺ PDOampR (black bars) was monitored using four transcriptional fusions, P_lasI-lacZ, P_lasR-lacZ, P_rhlI-lacZ and P_rhlR-lacZ. The promoterless lacZ vector has a low basal level of activity of <20 Miller units.

Fig. 4.

Kinetics of the paralysis of C. elegans by P. aeruginosa Alg⁻ PAO1 wild-type strain (—), Alg⁻ PAOampR (– ^{. .} –), complemented Alg⁻ PAOampR(pAmpR) (– ^. –), Alg⁺ PDO300 (-^.-), Alg⁺ PDOampR (--) and complemented Alg⁺ PDOampR(pAmpR) (^…). L4 stage larval hermaphrodite Bristol N2 C. elegans were placed on each brain heart infusion agar plate containing a bacterial lawn and scored for dead worms by microscopic examination. E. coli OP50 (— —) was used as a negative control. Values are the mean±sd of triplicate analyses. Results were statistically significant (P<0.05 for PAO1 vs PAOΔampR at 2 h, and PDO300 and PDOampR at 4 h).