**5. Conclusions**

The MexAB-OprM efflux pump spans the complete cell wall of *P. aeruginosa*, crossing the peptidoglycan in the periplasmic compartment. Here we analyzed the possible specific interaction of the MexA and OprM proteins with the PG purified from *E. coli* or *P. aeruginosa* revealing, when proteins are purified in DDM, no interaction for OprM and an interaction of MexA only with the PG of *E. coli* that is three times thicker in vivo than the one of *P. aeruginosa*. Nevertheless, previous association of the two proteins before analysis reveals a stabilization of the complex–PG interaction in both cases. This is further reinforced with the PG of *P. aeruginosa* after the exchange of DDM detergent for C12E8 for the two proteins. Even if no covalent binding is expected, this synergistic interaction could correspond to a required step of the assembly mechanism, suggesting the PG to be an important actor of the process. It could help to stabilize MexA to keep it in the proper quaternary structure in order to increase the speed of the pump formation with OprM present in the opposite membrane.

**Supplementary Materials:** Supplementary Materials can be found at https://www.mdpi.com/1422 -0067/22/10/5328/s1. Figure S1: Pull-down experiments performed with the PG extracted from *P. aeruginosa* and with the MexA and OprM proteins mixed at two different ratios, 2:1 and 3:1., Figure S2: Pull-down experiments performed with the PG extracted from *P. aeruginosa* and with the MexA and OprM modified proteins, OprMΔCter corresponding to a truncation of 13 residues at its C-terminus.

**Author Contributions:** Conceptualization, G.P. and I.B.; Funding acquisition, I.B.; Investigation, M.M., M.L., M.S., D.M.-L. and G.P.; Methodology, D.M.-L., G.P. and I.B.; Project administration, D.M.-L., G.P. and I.B.; Supervision, G.P. and I.B.; Validation, D.M.-L., G.P. and I.B.; Writing—original draft, I.B.; Writing—review and editing, D.M.-L. and G.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Agence Nationale de la Recherche, gran<sup>t</sup> numbers ANR-17-CE11-0028-02 and ANR-12-BSV8-0010. MM was supported by ANR-12-BSV8-0010. M.L. was supported by a ministerial doctoral funding.

**Acknowledgments:** We thank Maria Antónia Sousa Correia da Silva for her participation in the pull-down experiments during her mobility training in IB laboratory.

**Conflicts of Interest:** The authors declare no conflict of interest.
