AbstractThe striking feature of the ubiquitous protein EfTu (Thermo unstable ribosomal Elongation factor) is its moonlighting (multifunctional) activity. Beyond its function at the ribosomal level it should be exported to the bacterial surface and act as an environmental sensor. In Bacillus cereus, and other cutaneous bacteria, it serves as a Substance P (SP) receptor and is essential for bacterial adaptation to the host. However, the modus operandi of EfTu as a bacterial sensor remains to be investigated. Studies realized by confocal and transmission electron microscopy revealed that, in the absence of an exogenous signal, EfTu is not exposed on the bacterial surface but is recruited under the effect of SP. In addition, SP acts as a transcriptional regulator of the tuf gene encoding for EfTu. As observed using gadolinium chloride, an inhibitor of membrane mechanosensitive channels (Msc), Msc control EfTu export and subsequently the bacterial response to SP both in terms of cytotoxicity and biofilm formation activity. Microscale thermophoresis revealed that in response to SP, EfTu can form homopolymers. This event should occur after EfTu export and, as shown by proteo-liposome reconstruction studies, SP appears to promote EfTu polymers association to the membrane, leading subsequently to the bacterial response. Molecular modeling suggests that this mechanism should involve EfTu unfolding and insertion into the bacterial cytoplasmic membrane, presumably through formation of homopolymers. This study is unraveling the original mechanism action of EfTu as a bacterial sensor but also reveals that this protein should have a broader role, including in eukaryotes.