Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

Hughes, Gareth W.; Hall, Stephen B.; Laxton, Claire S.; Sridhar, Pooja; Mahadi, Amirul H.; Hatton, Caitlin; Piggot, Thomas J.; Wotherspoon, Peter; Leney, Aneika C.; Ward, Douglas G.; Jamshad, Mohammed; Spana, Vaclav; Cadby, Ian T.; Harding, Christopher John; Isom, Georgia L.; Bryant, Jack A.; Parr, Rebecca J.; Yakub, Yasin; Jeeves, Mark; Huber, Damon; Henderson, Ian R.; Clifton, Luke A.; Lovering, Andrew L.; Knowles, Timothy J.

doi:10.1038/s41564-019-0481-y

Cited by 100 publications

(175 citation statements)

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“…The Mla pathway may function either to maintain outer membrane homeostasis/asymmetry (retrograde transport) 19,20,22,23 or to export nascent phospholipids to the outer membrane (anterograde transport) 21,27 . In both cases, one might predict that higher levels of Mla transport activity will be required during conditions of rapid growth or envelope stress, while transport needs may be minimal when growth is limited.…”

Section: Discussionmentioning

confidence: 99%

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Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

Kolich

Chang

Coudray

et al. 2020

Preprint

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ABC transporters facilitate the movement of a diverse array of molecules across cellular membranes, using power from ATP hydrolysis. While the overall mechanism of the transport cycle has been characterized in detail for several important members of this transporter family, it is less well understood how the activity of ABC transporters is regulated in the cell post-translationally. Here we report the X-ray crystal structure of MlaFB from E. coli, an ABC nucleotide binding domain (MlaF) in complex with its putative regulatory subunit (MlaB). MlaFB constitutes the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid transport across the bacterial envelope and is important for maintaining the integrity of the outer membrane barrier. Our data show that the regulatory subunit MlaB, a STAS domain protein, binds to the nucleotide binding domain and is required for its stability. Our structure also implicates a unique Cterminal tail of the ABC subunit, 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 the activity of an important bacterial lipid transporter may be regulated by small binding proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family, from bacteria to humans.

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Section: Discussionmentioning

confidence: 99%

“…to the OM as the cell grows 21,27 , and the direction of lipid transport mediated by Mla remains to be firmly established.…”

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confidence: 99%

Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

Kolich

Chang

Coudray

et al. 2020

Preprint

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“…In addition, the protein may carry simultaneously two PL with different head groups. Considering recent studies 29, 31 , the structural signatures of the ATPase and permease models from the ortholog Mla system in E. coli 26 , characteristic of importer and exporter ABC cassettes, we propose, in addition to the aforementioned role in retrograde phospholipid trafficking, a second potential mode of action as an anterograde trafficking system (dashed lines) that would contribute to the maintenance of phospholipid distribution asymmetry.…”

Section: Discussionmentioning

confidence: 91%

“…A countercurrent model would explain how asymmetries in lipid distribution in the two membranes might be achieved 54 . Although genetic and functional evidences have mainly suggested that the Ttg2/Mla pathway is a retrograde transport system 19, 55, 56 , recent studies in E. coli have shown that MlaD spontaneously transfers phospholipids to MlaC in vitro 31, 57 .…”

Section: Discussionmentioning

confidence: 99%

“…Deletion of the genes of this system is known to destabilize the OM, and bacterial strains lacking any of the Mla components are more susceptible to membrane stress agents 19, 24, 25, 26, 27, 28, 29, 30 . More recently, the retrograde transport hypothesis has been questioned and a new role for this system in anterograde phospholipid transport has been suggested 29, 31 .…”

Section: Introductionmentioning

confidence: 99%

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

Yero

Costenaro

Conchillo-Solé

et al. 2019

Preprint

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22In Pseudomonas aeruginosa, Ttg2D is the soluble periplasmic phospholipid-binding 23 component of an ABC transport system thought to be involved in maintaining the 24 asymmetry of the outer membrane. The crystallographic structure of Ttg2D at 2.5Å 25 resolution reveals that this protein can bind two diacyl phospholipids. Native and 26 denaturing mass spectrometry experiments confirm that Ttg2D binds two 27 phospholipid molecules, which may have different head groups. Analysis of the 28 available structures of Ttg2D orthologs allowed us to classify this protein family as a 29 novel substrate-binding protein fold and to venture the evolutionary events that 30 differentiated the orthologs binding one or two phospholipids. In addition, gene 31 knockout experiments in P. aeruginosa PAO1 and multidrug-resistant strains show 32 that disruption of this system leads to outer membrane permeabilization. This 33 demonstrates the role of this system in low-level intrinsic resistance against certain 34 antibiotics that use a lipid-mediated pathway to permeate through membranes. 35 36 Introduction 37Pseudomonas aeruginosa are amongst the most important multidrug-resistant (MDR) 38 human pathogens 1 , showing inherent resistance to an important number of the 39 presently available antibiotics 2 . P. aeruginosa are responsible for chronic lung 40 infections in individuals with chronic obstructive pulmonary disease or cystic fibrosis 41 (CF) 3 and account for over a tenth of all nosocomial infections 4 . A number of effective 42 drugs and formulations can treat P. aeruginosa infections, even in CF patients 5 . 43These include frontline antibiotics such as piperazillin-tazobactam, ceftazidime, 44 aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, tobramycin, amikacin, 45 and colistin 6 . Yet, resistance to most of these antimicrobials is being increasingly 46 reported 7 . The basis for the inherently high resistance of these microorganisms is 47primarily their low outer-membrane (OM) permeability 8, 9 , complemented by the 48 production of antibiotic-inactivating enzymes (e.g. β-lactamases), the constitutive 49 expression of efflux pumps 10, 11 and the capacity to form biofilms 1, 12 , among other 50 mechanisms. The susceptibility of P. aeruginosa to antimicrobials can be additionally 51 reduced by the acquisition of inheritable traits, including horizontal gene transfers 52 and mutations that decrease uptake and efflux pump overexpression 13, 14, 15 . 53Although a number of genes and mechanisms of resistance to antibiotics are already 54 known in P. aeruginosa, the complex mechanisms controlling the basal, low-level 55 resistance to these compounds are still poorly understood 16, 17 . 56 57The OM of P. aeruginosa is known to be central to its antibiotic-resistance 58 phenotype. Its intrinsically low permeability is partly determined by inefficient OM 59 porin proteins that provide innate resistance to several antimicrobial compounds, 60 mainly hydrophilic 1, 8, 10 . On the other hand, the loss of specific efflux pump ...

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Metabolic engineering of Escherichia coli to efficiently produce monophosphoryl lipid A

Wang

Zhao

Wang

et al. 2023

Biotech and App Biochem

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Monophosphoryl lipid A (MPL), mainly isolated from Salmonella minnesota R595, has been used as adjuvant in several vaccines. In this study, an Escherichia coli strain that can efficiently produce the MPL has been constructed. The gene clusters related to the biosynthesis of O‐antigen, core oligosaccharide, enterobacterial common antigen, and colanic acid were sequentially removed to save the carbon source and to increase the activity of PagP in E. coli MG1655. Then, the genes pldA, mlaA, and mlaC related to the phospholipid transport system were further deleted, resulting in the strain MW012. Finally, the genes lpxE from Francisella novicida and pagP and pagL from Salmonella were overexpressed in MW012 to modify the structure of lipid A, resulting in the strain MW012/pWEPL. Lipid A species were isolated from MW012/pWEPL and analyzed by thin‐layer chromatography and liquid chromatography–mass spectrometry. The results showed that mainly two MPL species were produced in E. coli MW012/pWEPL, one is hexa‐acylated, and the other is penta‐acylated. More importantly, the proportion of the hexa‐acylated MPL, which is the most effective component of lipid A vaccine adjuvant, reached 75%. E. coli MW012/pWEPL constructed in this study provided a good alternative for the production of lipid A vaccine adjuvant MPL.

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Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system

Cited by 100 publications

References 90 publications

Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

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

Metabolic engineering of Escherichia coli to efficiently produce monophosphoryl lipid A

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