Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa

Abstract

Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase gene lasB and the rhamnosyltransferase genes rhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL and Pseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.Pseudomonas aeruginosa is an opportunistic pathogen which may cause pneumonia and bacteremia in immunocompromised hosts and is responsible for chronic destructive lung disease in patients suffering from cystic fibrosis. The pathogenicity of P. aeruginosa is attributable to an arsenal of virulence factors, some of which are cell associated (pili, nonpilus adhesins, lipopolysaccharide, and alginate) while others are secreted (proteases, rhamnolipids, exotoxin A, exoenzyme S, and pyocyanin). The production of many of these extracellular virulence factors is controlled by two cell-to-cell signaling systems, called las and rhl, which are both composed of a transcriptional regulator (LasR and RhlR, respectively) and an autoinducer synthase (LasI and RhlI, respectively). LasI and RhlI catalyze the last step in the synthesis of the cell-to-cell signaling molecules 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL) and C4-HSL, respectively; each of these molecules binds to, and activates, its corresponding transcriptional regulator. The systems are connected via a hierarchical cascade (19) and allow coordinated production of extracellular virulence factors, which occurs only when the bacterial cell density has reached a threshold (quorum). Recently, a novel signaling molecule, called PQS, for Pseudomonas quinolone signal (39), has been identified. Furthermore, the published genome sequence of PAO1 (53) has revealed a new modulator of cellto-cell signaling, termed QscR (4). This protein is homologous to both LasR and RhlR and seems to prevent premature transcription of quorum-sensing regulated genes.Besides its pathogenic capabilities, P. aeruginosa is well known for its intrinsic resistance to a wide range of antimicrobial agents and its ability to develop multidrug resistance following antibi...

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…Mutants of the nfxC type have been isolated in the clinic, but they are not very common. The most likely explanation for this is their reduced virulence (102,109,111), together with the fact that these mutations appear to be less effective at engendering antibiotic resistance in vivo than they are under laboratory conditions (77).…”

Multidrug resistant Acinetobacter baumannii--the role of AdeABC (RND family) efflux pump in resistance to antibiotics.

“…It has been suggested that the natural function of RND pumps might also include efflux of signal molecules required for cell-to-cell signaling (quorum sensing) [103]. Pseudomonas aeruginosa secretes a quinolone, designated PQS (Pseudomonas quinolone signal), which acts as quorum-sensing signal [95] and overexpression of MexEF-OprN was shown to affect PQS signalling [49]. This is a very interesting finding as quinolones constitute a widely used group of antimicrobials and are substrates for RND pumps.…”

Future research trends in the major chemical language of bacteria

“…Short chain AHLs ͑ϽC8͒ are believed to be able to freely diffuse across the cell envelope; whereas AHLs with longer acyl side chains may depend on transport systems (Kohler et al, 2001). Several LuxI family proteins have been studied at the biochemical and structural level revealing their precise mode of action (Pappas et al, 2004).…”