Efflux-pumps systems: constitutive and inducible expression

Efflux pumps play an important role in antibiotic resistance. These pumps may be specific for a substrate or may extrude a broad range of compounds including dyes, detergents, fatty acids and antibiotics of multiple classes structurally unrelated. Thus, it is probable that efflux pumps were created so that harmful substances could be transported out of the bacterial cell, thus, allowing for survival.

Based primarily on amino acid sequence identity, on the energy source required to drive export and on substrate specificities, efflux pumps have been categorized in five superfamilies including (i) the ATP-binding cassette family, (ii) the small multidrug resistance family, (iii) the major facilitator superfamily, (iv) the resistance-nodulation-division (RND) family, and (v) the multidrug and toxic compound extrusion family. In P. aeruginosa, genome sequence analysis has revealed the presence of efflux systems from all five superfamilies, being the RND family the most prevalent with 12 different systems identified.

Figure 1.2. Location of RND-type efflux pumps components across the outer and inner membrane in P. aeruginosa. MFP: membrane fusion protein, RND:

transporter protein, and OMF: outer membrane factor.

The RND-type efflux pumps are secondary active transporters that derive the energy required for compound extrusion by proton motive force and are typically organized as a tripartite consisting of a periplasmic membrane fusion protein, a transporter protein in the inner membrane and an outer membrane factor.

Within this complex, the inner membrane protein captures the substrates from either, the phospholipid bilayer of the inner membrane of the bacterial cell envelope or the cytoplasm, and transports them into the extracellular medium via the OMP, being the cooperation between these proteins mediated by the periplasmic protein (Figure 1.2.) [Lister PD et al, 2009; Li XZ et al, 2015].

The genes coding for the RND efflux pumps components are organized into operons in the Pseudomonas aeruginosa chromosome. Not all of them code for an outer membrane factor and, thus, the tripartite efflux pump is completed by taking this protein from a different efflux pump system (e.g. MexXY). As well, some of them harbor an adjacent regulatory gene transcribed in the same orientation or divergently from the operon and whose products act repressing or activating the operon expression (Figure 1.3. and Table 1.1.) [Lister PD et al, 2009].

Figure 1.3. RND efflux operons in P. aeruginosa. Operons encoding the 10 RND pumps (excluding the 2 metal cation transporters) are represented. Color scheme:

green, transcriptional regulator; purple, membrane fusion protein; light blue, RND transporter; dark blue, OMP; and orange, protein with unknown function.

Most RND efflux systems in P. aeruginosa exhibit broad substrate specificity and recognize many structurally dissimilar compounds (Table 1.1). Of all systems, MexAB-OprM and MexXY contribute to its intrinsic antibiotic resistance as all the others are not expressed in WT strains.

Table 1.1. Substrates for the RND efflux systems of P. aeruginosa Efflux system Substrates

Antibiotics Additional compounds

MexAB-OprM β-lactams (not imipenem), β-lactamase inhibitors, fluoroquinolones (FQ), chloramphenicol, macrolides, novobiocin, tetracyclines, trimethoprim, sulfonamides

Biocides, detergents, dyes, homserin lactones, aromatic hydrocarbons

MexXY-OprM/Opm-^a Penicillins (not carbenicillin and sulbenicillin) , cephalosporins (not ceftazidime), meropenem, FQ, aminoglycosides (AMG), tetracyclines, macrolides, chloramphenicol MexCD-OprJ Penicillins, cephalosporins (not ceftazidime),

meropenem, FQ, chloramphenicol, macrolides, novobiocin tetracyclines, trimethoprim

Biocides, detergents, dyes, aromatic hydrocarbons

MexEF-OprN FQ, cloranphenicol, trimethoprim Biocides, aromatic hydrocarbons MexJK-OprM/OpmH Tetracyclines, erythromycin Biocides

MexGHI-OpmD FQ Vanadium

MexVW-OprM FQ, tetracyclines, chloramphenicol, erythromycin

MexPQ-OpmE FQ, tetracyclines, chloramphenicol, macrolides

MexMN-OprM Chloramphenicol, thiamphenicol

TriABC-OpmH Triclosan

a MexXY may utilize OpmB, OpmG, OpmH and/or OmpI as OMFs.

1.3.3.1. Constitutive expression of MexAB-OprM

MexAB-OprM was the first RND multidrug efflux system to be described in P. aeruginosa [Poole K et al, 1993; Li XZ et al, 1995]. As shown in Table 1.1., this pump is able to export antibiotic compounds from different families and exhibits the broadest substrate profile for the β-lactam class including carboxypenicillins, aztreonam, cefotaxime, ceftazidime and meropenem.

This system is expressed constitutively in cells grown under standard laboratory conditions [Poole K & Srikumar R, 2001] and laboratory-constructed MexAB-OprM knockout mutants have been shown to be hypersensitive to its substrates [Li XZ et al, 1995; Masuda N et al, 1999; Morita Y et al, 2001]. In WT P. aeruginosa strains, MexAB-OprM expression is growth-phase-dependent, reaching its maximum in late log-phase/early stationary phase. This dependency led to the suggestion that MexAB-OprM expression could be regulated by the

quorum sensing (QS) system (cell to cell communication) and, in this sense, it has been demonstrated that N-butyryl-L-homoserin-lactones enhance its expression.

All three components of this efflux pump are encoded within the same operon (Figure 1.3.), which additionally harbors a regulatory protein (MexR) located directly upstream but transcribed divergently from MexA-MexB-OprM coding genes. MexR belongs to the MarR family member and is the major regulator of this efflux pump system. It binds as a stable homodimer to two sites within the mexR-mexA intergenic region overlapping the promoters for mexR and mexAB-oprM and, thus, repressing their expression. Recently, it has been demonstrated that MexR repressor capacity depends on its redox state as, within the stable homodimer, MexR-Cys residues form intermonomer disulfide bonds whose oxidation eventually lead to its dissociation from the promoter DNA [Chen H et al, 2008; Chen H et al, 2010]. MexR activity has been found to be additionally controlled by armR encoded product, as it binds to MexR diminishing its repressor activity [Daigle DM et al, 2007; Wilke MS et al, 2008]. Finally, MexAB-OprM expression is controlled by nalD, which encodes a TetR family repressor-like protein that binds to a second promoter upstream of mexA-mexB-oprM [Morita Y et al, 2006a]. Also of note, it has been shown that oprM expression can occur independently of mexA-mexB, through an alternative weak promoter within mexB [Zhao Q et al, 1998], which ensures sufficient levels of this OMP to other P. aeruginosa efflux systems (MexXY, MexJK, MexVW and MexMN) even when mexA-mexB-oprM expression is compromised.

Mutation-driven overexpression of this efflux system will be discussed later in section 1.8.

1.3.3.2. Inducible expression of MexXY

The MexXY efflux system was discovered several years later, in 1999, being the fourth efflux system to be identified in P. aeruginosa PAO1 [Aires JR et al, 1999; Mine T et al, 1999]. It is able to extrude a wide variety of substrates (Table 1.1.) and, of note, is the only efflux pump encoded in P. aeruginosa chromosome with the ability to mediate aminoglycoside resistance.

MexXY expression is induced when bacterial cells are grown in the presence of sub-inhibitory concentrations of some of its antibiotic substrates such as tetracycline, erythromycin or aminoglycosides. Additionally, P. aeruginosa PAO1 mutants lacking this efflux system are hypersusceptible to its substrates which suggests that it contributes to the intrinsic antibiotic resistance to these agents [Aires JR et al, 1999; Masuda N et al, 2000].

Genetically, the operon coding for MexXY lacks an outer membrane factor (Figure 1.3.).

Therefore, it takes the OMF protein from other operons to complete the tripartite system.

Mainly, OprM completes the tripartite system but other porins such as OpmB, OpmG, OpmH

2002]. Located upstream but transcribed divergently from mexX-mexY, is encountered mexZ which encodes a protein that belongs to the TetR family of transcriptional regulators and negatively regulates its expression (Figure 1.3.). Similar to MexR (section 1.3.3.1.), MexZ binds as a homodimer to an inverted repeated sequence within the intergenic region mexZ-mexX, overlapping the putative mexX-mexY promoter [Matsuo Y et al, 2004] and repressing its expression.

In contrast to other drug-inducible multidrug efflux systems, MexXY inducers do not alter MexZ and mexZ-mexX interactions. Instead, induction has been shown to be dependent on drug-ribosome interactions and to occur, although in a lesser extent, even in the mexZ mutant [Jeannot K et al, 2005]. Therefore, these data suggest an alternative biological role for the MexXY system beyond antibiotics efflux. Multiple pathways participate in the regulation of mexX-mexY induction. Although ribosome disruption has been shown to impact the expression of a myriad of genes, by using a transposon insertion mutant library PA5471 was found to be not only drug-inducible but also required for mexX-mexY induced expression [Morita Y et al, 2006b]. Later on, it was demonstrates that the antimicrobial-inducible PA5471 gene product has interacts with the repressor MexZ and, thus, interfere with its DNA binding activity [Yamamoto M et al, 2009].

More recently, it has been also demonstrated the involvement of parR, a gene coding for the response regulator of the two-component regulatory system ParR-ParS, in promoting either induced or constitutive mexX-mexY upregulation. In addition, this gene was demonstrated to be also implicated in OprD porin downregulation and in lipopolysaccharide (LPS) modification in a MexZ-independent manner [Muller C et al, 2011].

Mutation-driven overexpression of this efflux system will be discussed later in section 1.8.