Active Efflux and Diffusion Are Involved in Transport of Pseudomonas aeruginosa Cell-to-Cell Signals

Pseudomonas aeruginosa remains a leading cause of both nosocomial infections in immunocompromised patients and chronic infections in cystic fibrosis patients (reviewed in references 9 and 57). P. aeruginosa virulence depends on cell-associated factors, including alginate and pili (5, 9), and secreted factors, including toxins, exotoxin A, and exoenzyme S (14, 28); proteases, elastase, alkaline protease, and LasA protease (13, 27, 47); and hemolysins, rhamnolipid, and phospholipase (25). Cell-to-cell signaling (quorum sensing) is required for expression of many P. aeruginosa virulence factors (see below) (6, 43). Intrinsic resistance of P. aeruginosa to many antibiotics and disinfectants also causes clinical problems (49). The intrinsic resistance is due to low outer membrane permeability and to multidrug efflux pumps that reduce the cellular level of antibiotics (reviewed in references 11 and 30). Three known P. aeruginosa multidrug efflux pumps are encoded by the mexAB-oprM, mexCD-oprJ, and mexEF-oprN operons, respectively (18, 45, 46). These pumps consist of a cytoplasmic membrane component of the resistance-nodulation-cell division (RND) family (39) thought to function as a proton antiport exporter (i.e., MexB), an outer membrane component thought to form channels (i.e., OprM), and a membrane fusion protein thought to link MexB and OprM (reviewed in reference 29). Bacterial RND pumps have also been shown to cause the efflux of many other organic compounds, including solvents and inhibitors (24, 29, 50). However, no natural products of P. aeruginosa have yet been shown to be subject to efflux via RND pumps. Quorum sensing (or autoinduction) is the controlled expression of specific genes in response to extracellular chemical signals produced by the bacteria themselves (6). Typically cells emit an N-acyl homoserine lactone signal called an autoinducer (AI), which is usually synthesized by a LuxI-type AI synthase, into the environment (6). At high cell densities, the AI reaches a threshold concentration and binds to a LuxR-type protein which is then able to activate target genes (6). To date, luxR-luxI-type quorum-sensing systems and their N-acyl homoserine lactone AIs have been found in many different gram-negative bacteria (6). In P. aeruginosa the las (lasR-lasI) (7, 37) and rhl (rhlR-rhlI) (33, 34) quorum-sensing systems direct the synthesis of two distinct AIs, N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) (formerly called PAI-1) (40) and N-butyryl homoserine lactone (C4-HSL) (formerly called PAI-2) (41, 59), respectively. LasR and 3OC12-HSL activate expression of several genes, including lasI itself (54), lasB (encoding elastase) (37), lasA (encoding LasA protease) (8), both xcp operons (xcpPQ and xcpR-Z, encoding the type II secretion apparatus [3]), and rhlR (20, 44). Recently, the las quorum-sensing system was shown to be involved in P. aeruginosa biofilm differentiation (4). C4-HSL and RhlR also activate expression of numerous genes, including rhlI itself (20), the rhamnolipid biosynthesis operon rhlAB (32, 42), lasB (1, 41, 42), and rpoS (encoding the stationary-phase sigma factor ςs) (20). Although many different N-acyl homoserine lactone AIs have been isolated from various gram-negative bacteria, to date, all differences are in the N-acyl side chain length (from C4 to C14) or degree of substitution (either 3-oxo, 3-hydroxy, saturated, or unsaturated) (6, 26, 48, 55). All AIs have been assumed to be freely diffusible in bacterial cells. This assumption is based on the fact that a radiolabeled Vibrio fischeri AI, N-3-oxo-hexanoyl homoserine lactone ([3H]3OC6-HSL), was shown to freely diffuse into and out of V. fischeri and Escherichia coli cells (15, 42). Here, we studied the uptake and efflux of 3OC12-HSL and C4-HSL by P. aeruginosa cells. Our results indicate that [3H]C4-HSL freely diffuses into and out of P. aeruginosa cells. In contrast, cellular concentrations of [3H]3OC12-HSL are higher than external levels. Our results show that the increased cellular level of [3H]3OC12-HSL is not due to association with LasR or RhlR and suggest that it is not due to active (inward) transport. We propose that the high cellular level of 3OC12-HSL is probably due to partitioning into the cell membranes. By use of P. aeruginosa Δ(mexAB-oprM) mutant cells or poisoned wild-type cells, we also show that 3OC12-HSL is subject to active efflux by the MexAB-OprM pump.