Gram-negative bacteria use a variety of self-produced autoinducers such as acylated homoserine lactones as a language for quorum sensing (QS) within and between bacterial species. Several bacterial species synthesize specific acylated homoserine lactones (acyl-HSLs) by means of a LuxI-type enzyme, and respond to cognate acyl-HSL by using a LuxR-type intracellular receptor [1, 2]. It is considered that the selection of bacterial languages is necessary to regulate gene expression and thus it leads to a growth advantage in several environments.
The opportunistic bacterium P. aeruginosa is widespread in various environments and utilizes two acyl-HSL signaling molecules, N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), and N-butanoyl-L-homoserine lactone (C4-HSL), and two receptor proteins, LasR and RhlR, respectively . 3-oxo-C12-HSL binds to LasR and activates LasR function. The 3-oxo-C12-HSL-LasR complex regulates many genes, including the rhl system [4–6]. Furthermore, P. aeruginosa uses a third signal, Pseudomonas quinolone signal (PQS) and the PqsR receptor protein . Expression of many virulence factors is regulated by QS in P. aeruginosa[4–6, 8, 9]. Accordingly, a specific response to an autoinducer is important to determine the virulence of P. aeruginosa.
Analysis of the crystal structures of the N-terminal half of the P. aeruginosa full-length LasR or the crystal structure of A. tumefaciences full-length TraR, which is a homolog of P. aeruginosa LasR, in a complex with its cognate autoinducer has been performed [6, 10]. These structural analyses indicated that the N-terminal half of the full length LuxR-type protein includes the dimerization domain and the acyl-HSL binding domain [6, 10]. These reports indicated that the ligand binds to the N-terminal half of the full-length LuxR-type protein at an enclosed cavity far from the N-terminal dimerization region. It has been suggested that the acyl side-chain length of acyl-HSLs is not the main factor that determines the specificity of receptor protein binding [6, 10]. It is considered that the binding model for the acyl-HSL-LuxR transcriptional protein family is common among Gram-negative bacteria [6, 10]. However, it was shown that the responses to acyl-HSLs in P. aeruginosa are specific [4, 11]. We hypothesize that there is an unidentified signal selection mechanism for the selection of acyl-HSLs according to the binding affinity of LasR in P. aeruginosa.
Resistance-nodulation-division (RND)-type efflux pumps are one type of antibiotic efflux system. RND-type efflux pumps are commonly found in gram-negative bacteria. RND family transporters catalyze the active efflux of many antibiotics and chemotherapeutic agents. They consist of an inner-membrane component belonging to the RND superfamily of secondary transporters, a channel-forming outer membrane factor (OMF), and a periplasmic membrane fusion protein (MFP) to achieve the direct extrusion of substrates across the two membranes of gram-negative bacteria .
The major P. aeruginosa RND-type efflux pump, MexAB-OprM provides the bacterium natural resistance to a broad spectrum of antibiotics and is not just for antimicrobial resistance . On the other hand, it was reported that MexAB-OprM participates in the efflux of acyl-HSLs from P. aeruginosa[13, 14]. These reports indicated that P. aeruginosa cells are not freely permeable to 3-oxo-C12-HSL in contrast to C4-HSL. Instead, it was shown that MexAB-OprM is involved in the active efflux of 3-oxo-C12-HSL [13, 14]. Furthermore, a MexAB-OprM deletion mutant has a decreased capacity to invade or transmigrate across MDCK cells . It was considered that QS-regulated virulence factors are affected by the MexAB-OprM efflux pump activity.
In this study, we hypothesized that MexAB-OprM of P. aeruginosa might function in the selection of acyl-HSLs, and we provide evidence to support this hypothesis. To examine the QS responses to several exogenous acyl-HSLs in P. aeruginosa, herein we focused on the las system because this system controls the rhl system and the PQS system hierarchically in P. aeruginosa[2, 5, 7]. These studies indicate that MexAB-OprM prevents the access of exogenous 3-oxo-acyl-HSLs to LasR, and thus LasR binds specifically to 3-oxo-C12-HSL. The results demonstrate a new QS regulation mechanism via the efflux system MexAB-OprM in P. aeruginosa.