Structural basis for maintenance of bacterial outer membrane lipid asymmetry

Abstract

The Gram-negative bacterial outer membrane (OM) is a unique bilayer that forms an efficient permeation barrier to protect the cell from noxious compounds . The defining characteristic of the OM is lipid asymmetry, with phospholipids comprising the inner leaflet and lipopolysaccharides comprising the outer leaflet . This asymmetry is maintained by the Mla pathway, a six-component system that is widespread in Gram-negative bacteria and is thought to mediate retrograde transport of misplaced phospholipids from the outer leaflet of the OM to the cytoplasmic membrane . The OM lipoprotein MlaA performs the first step in this process via an unknown mechanism that does not require external energy input. Here we show, using X-ray crystallography, molecular dynamics simulations and in vitro and in vivo functional assays, that MlaA is a monomeric α-helical OM protein that functions as a phospholipid translocation channel, forming a ~20-Å-thick doughnut embedded in the inner leaflet of the OM with a central, amphipathic pore. This architecture prevents access of inner leaflet phospholipids to the pore, but allows outer leaflet phospholipids to bind to a pronounced ridge surrounding the channel, followed by diffusion towards the periplasmic space. Enterobacterial MlaA proteins form stable complexes with OmpF/C , but the porins do not appear to play an active role in phospholipid transport. MlaA represents a lipid transport protein that selectively removes outer leaflet phospholipids to help maintain the essential barrier function of the bacterial OM.

Evolution toward maximum transport capacity of the Ttg2 ABC system inPseudomonas aeruginosa

“…The soluble, periplasmic substrate binding protein Ttg2D and its orthologs in other species are thought to transport mislocalized phospholipids from the outer leaflet of the outer membrane (OM) to the inner membrane (IM) complex Ttg2ABCE across the periplasm 21 . In P. aeruginosa , it is not yet known if the VacJ component of the system, which delivers the lipids to Ttg2D, forms a complex with specific porins, as in E. coli , to extract the lipids from the membrane.…”

“…In agreement with our work, it has been previously shown that in P. aeruginosa, VacJ plays an important role in both virulence and antibiotic susceptibility to ciprofloxacin, chloramphenicol and tetracycline 74 . In E. coli , this protein forms an active complex with the outer membrane proteins OmpC and OmpF 21, 23, 75 . However, in P. aeruginosa there are no clear orthologs to either of these porins, increasing the singular characteristics of this system in this species and suggesting potential mechanistic differences with the more studied E. coli transporter (Fig.…”

“…The core components of this ATP-binding-cassette (ABC) transport system in the inner membrane (IM) comprise the permease (MlaE), the ATPase (MlaF) and the substrate-binding protein MlaD that are highly conserved among Gram-negative bacteria 20 . The MlaA component, an integral OM protein that forms a channel adjacent to trimeric porins, is thought to selectively remove phospholipids from the outer OM leaflet and transfer them to the soluble periplasmic substrate-binding protein MlaC 21, 22 . MlaC would then transport the phospholipids across the periplasm and deliver them to MlaD for active internalization through the IM 23 .…”

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

“…Whilst it has been well established that a host of PLs can move in a retrograde fashion from the OM to the IM (Jones and Osborn 1977), it remains unclear as to how PLs travel in the opposite direction. It has been suggested that retrograde PL transport is in part driven by the action of the OM MlaA-OmpF/C system (Abellon- Ruiz, Kaptan et al 2017). In fact, Thong et al (2016) proposed that the MlaFEDB driven hydrolysis of ATP may provide the necessary energy to indirectly remove PLs from the OM against their concentration gradient.…”

“…It was described as having a role in maintaining lipid asymmetry in the OM by removing and trafficking PLs found in the outer leaflet of the OM to the IM (Malinverni and Silhavy 2009). Through genetic knockouts, structural and modelling-based studies, the individual components have been identified and characterised ( Figure 1) (Chong, Woo et al 2015, Thong, Ercan et al 2016, Abellon-Ruiz, Kaptan et al 2017, Ekiert, Bhabha et al 2017, Yeow, Tan et al 2018.…”