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

Abstract

The Mla pathway is believed to be involved in maintaining the asymmetrical Gramnegative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

LetB Structure Reveals a Tunnel for Lipid Transport across the Bacterial Envelope

“…A series of studies have unraveled how the E. coli LPS transport system employs an ABC transporter in the IM to drive movement of LPS molecules across a periplasmic bridge to an OM complex that mediates their insertion into the outer leaflet (Li et al, 2019;May et al, 2015;Owens et al, 2019;Ruiz et al, 2009;Sherman et al, 2018). How phospholipids are trafficked across the cell envelope is less well established, and MCE proteins (which contain Mammalian Cell Entry domains) have emerged as candidates for phospholipid transport (Awai et al, 2006;Ekiert et al, 2017;Hughes et al, 2018;Kamischke et al, 2019;Malinverni and Silhavy, 2009;Thong et al, 2016). Members of this transporter family play an important role in the OMs of both Gramnegative bacteria (Isom et al, 2017;Kamischke et al, 2019;Malinverni and Silhavy, 2009) and Mycobacteria (Cantrell et al, 2013;Klepp et al, 2012) and are critical during host infection by various bacterial pathogens (Carpenter et al, 2014;Gioffré et al, 2005;Isom et al, 2018;Nakamura et al, 2011;Pandey and Sassetti, 2008;Senaratne et al, 2008;Zhang et al, 2012).…”

“…Of these, the Mla system is the best characterized and has been implicated in the Maintenance of OM Lipid Asymmetry in E. coli (Malinverni and Silhavy, 2009;Powers and Trent, 2018). In contrast to the periplasmic bridge used for LPS transport, the Mla system consists of a soluble periplasmic protein (MlaC) that ferries lipids between an ABC transporter complex in the IM (MlaFEDB) and a protein complex in the OM (MlaA-OmpF/ C) (Abelló n- Ruiz et al, 2017;Ekiert et al, 2017;Hughes et al, 2018;Kamischke et al, 2019;Malinverni and Silhavy, 2009;Thong et al, 2016;Chong et al, 2015).…”

Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

“…The best characterised MCE transport system, called Mla, has been implicated in the Maintenance of OM Lipid Asymmetry in E. coli by importing mislocalised phospholipids from the OM to the IM 6 , and consists of three main parts: 1) an OM complex, MlaA-OmpC, that may mediate the extraction of lipids from the OM, 2) a lipid carrier protein, MlaC, that ferries lipids across the periplasm, and 3) an IM ABC transporter complex, MlaFEDB, that may mediate the insertion of lipids into the IM. More recent work has suggested, however, that the Mla system may in fact drive lipid export, wherein MlaFEDB may use ATP hydrolysis to extract lipids from the IM and MlaA-OmpC may mediate their insertion in the OM 16,17 . Thus, the directionality of the Mla phospholipid transporter and potentially other MCE protein family members remains an open question.…”