SUMMARY How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles.

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 pivoting βsheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the inner membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provides evidence of an apparatus for lipid export away from the inner membrane and suggests that the Mla pathway may have a role in anterograde phospholipid transport.

Highlights d Cryo-EM structures of LetB reveal a tunnel that spans the bacterial cell envelope d Lipids bind inside the tunnel, supporting a role for LetB in lipid transport d Different conformations reveal tunnel dynamics, with implications for transport

In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.

Bacterial proteins with MCE domains were first described as being important for Mammalian Cell Entry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli, the singledomain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli, PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/159053 doi: bioRxiv preprint first posted online Jul. 3, 2017; 2 defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and fig. S1). Other multi-MCE domain-containing proteins were detected in Proteobacteria, but at much lower frequencies than type III and IV proteins (Supplementary table S1, Supplementary fig. S1).Some MCE domains were detected in proteins from eukaryotic genomes (Supplementary fig. S2). Type I proteins were found in plant phyla Chlorophyta and Streptophyta. In Arabidopsis they are involved in the trafficking of phosphatidic acid from the outer to the inner membrane of chloroplasts 18 . A small number of MCE proteins were identified in animal genomes. Manual inspection of the DNA sequences encoding these proteins revealed that all but one could be attributed to contamination with bacterial DNA, the exception being in Trichoplax adhaerens, an animal known to have an unusually large mitochondrial genome 22,23 .Protein clustering and evolution of multi-domain proteins. To understand the evolutionary relationships between MCE proteins, protein-protein similarity networks were constructed and coloured by architecture type and phylum (Fig. 2). MCE proteins generally cluster within phyla, suggesting that little or no horizontal transmission of these genes has occurred and variants have arisen through speciation.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The (Fig. 3B). In many of these peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/159053 doi: bioRxiv preprint first posted online Jul. 3, 2017The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/159053 doi: bioRxiv preprint first posted online Jul. 3, 2017; 7 neighbourhoods, for example in Neisseria meningitidis 27 , the outer membrane component MlaA (VacJ) is found in ...

ABC transporters facilitate the movement of diverse molecules across cellular membranes, but how their activity is regulated post-translationally is not well understood. Here we report the crystal structure of MlaFB from E. coli, the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid trafficking across the bacterial envelope to maintain outer membrane integrity. MlaB, a STAS domain protein, binds the ABC nucleotide binding domain, MlaF, and is required for its stability. Our structure also implicates a unique C-terminal tail of MlaF in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how an important bacterial lipid transporter may be regulated by small proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family.

Mutations in σ-regulated lipoproteins have previously been shown to impact bacterial viability under conditions of stress and during infection. YraP is conserved across a number of Gram-negative pathogens, including, where the homolog is a component of the Bexsero meningococcal group B vaccine. Investigations using laboratory-adapted K-12 have shown that mutants have elevated sensitivity to a range of compounds, including detergents and normally ineffective antibiotics. In this study, we investigate the role of the outer membrane lipoprotein YraP in the pathogenesis of serovar Typhimurium. We show that mutations in Typhimurium result in a defective outer membrane barrier with elevated sensitivity to a range of compounds. This defect is associated with attenuated virulence in an oral infection model and during the early stages of systemic infection. We show that this attenuation is not a result of defects in lipopolysaccharide and O-antigen synthesis, changes in outer membrane protein levels, or the ability to adhere to and invade eukaryotic cell lines.