**1. Introduction**

2

The cell wall of Gram-negative bacteria comprises three main layers: the outer membrane lipid bilayer coated by lipopolysaccharides at the exterior; the inner membrane that meets the cytoplasm; and in between the periplasmic matrix where peptidoglycan (PG) confers the mechanical rigidity of the cell wall. Membrane transporters such as efflux pumps span these layers and expulse a wide variety of drugs leading to natural and acquired antimicrobial resistances. Resistance Nodulation-cell Division (RND) family is one of the six families of efflux pumps [1,2], acting as a three-component complex made of an inner-membrane transporter (RND), a periplasmic membrane fusion protein (MFP), and an outer-membrane factor channel (OMF). We focus our interest on the efflux pump MexAB-OprM from *Pseudomonas aeruginosa (P. aeruginosa)*, an opportunistic Gram-negative bacterium responsible of severe nosocomial infections and having developed resistance to several families of antibiotics. It has been recently classified as high priority by WHO in antimicrobial research [3] and belongs to the group ESKAPE named by the initials of the pathogens for which there is an urgen<sup>t</sup> need for new treatments. The constitutive RND efflux pumps MexAB-OprM from *P. aeruginosa* and AcrAB-TolC from *Escherichia coli* (*E. coli)* have been extensively studied by different approaches making them archetypal models for the structural and functional comprehension of the efflux pump mechanism. The structure of each protein forming the pump has been solved by X-ray crystallography [4–12] and the whole assembly was recently determined by cryo-electron microscopy (cryo-EM) [13–16].

**Citation:** Ma, M.; Lustig, M.; Salem, M.; Mengin-Lecreulx, D.; Phan, G.; Broutin, I. MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of *Escherichia coli* and *Pseudomonas aeruginosa*. *Int. J. Mol. Sci.* **2021**, *22*, 5328. https://doi.org/ 10.3390/ijms22105328

Academic Editor: Masoud Jelokhani-Niaraki

Received: 14 April 2021 Accepted: 12 May 2021 Published: 18 May 2021

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The comparison of these structures from several homologous pumps, together with in vitro measurements of transport through proteoliposomes [17,18], in cellulo determination of the minimal inhibitory concentration (MIC) of antibiotics for mutant strains [19,20], and the extensive modeling and molecular dynamic calculations [21,22], have led to a more precise understanding of the efflux pathways [23–28] and the conformational modifications [14,29,30] necessary to expulse the drugs out of the cell. We know now that the RND protein works as homotrimer with a proton motive force via proton relay amino acids localized in the membranous domain. This proton transfer is combined with the movement of the RND periplasmic domains where each monomer adopts alternatively Loose, Tight, and Close or Open conformations [31], to capture the drug at different entries and push it toward the RND funnel domain [16,32]. The drug then crosses the periplasm by a tunnel formed by the hexameric MFP proteins and is finally expulsed out of the cell through the OMF channel. Even if some hypotheses have been given on the final opening mechanism of the OMF [15,33,34], it is still not clear how and when the MFP and OMF interact. In vitro and in absence of its outer membrane partner, the MFP seems to be unstructured or too flexible to adopt a defined shape around the RND as illustrated by the recent structure of TriABC solved by cryo-EM [35]. In cellulo the tripartite pump must traverse the PG layer to convey the molecules, however, little is known about the relationship between the PG and the pumps. Is the PG a passive mesh made of long glycan chains crosslinked by short peptides or does it make specific interactions with the efflux pump components? If yes, which components are involved in PG recognition? In order to answer these questions, we purified the PG from *E. coli* and *P. aeruginosa* and performed pull-down assays to analyze their interaction with purified OprM and MexA proteins. Here we show that the presence of PG stabilizes the MexA-OprM binary complex formation, highlighting the PG as an important new actor of the assembly of the RND efflux pumps.
