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Replacing plasma membrane outer leaflet lipids with exogenous lipid without damaging membrane integrity.

ABSTRACT: We recently introduced a M?CD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025-14030). Here, we show if the lipid-to- M?CD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step by incubation in complete growth medium. After exchange and transfer to complete growth medium cell growth was similar to that of untreated cells. In some cases, cell damage was also prevented by carrying out exchange at close to room temperature (rather than at 37°C). Exchange with lipids that do (sphingomyelin) or do not (unsaturated phosphatidylcholine) support a high level of membrane order in lipid vesicles had the analogous effect on plasma membrane order, confirming exogenous lipid localization in the plasma membrane. Importantly, changes in lipid composition and plasma membrane properties after exchange and recovery persisted for several hours. Thus, it should be possible to use lipid exchange to investigate the effect of plasma membrane lipid composition upon several aspects of membrane structure and function.

SUBMITTER: Li G

PROVIDER: S-EPMC6779269 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Replacing plasma membrane outer leaflet lipids with exogenous lipid without damaging membrane integrity.

Li Guangtao G Kakuda Shinako S Suresh Pavana P Canals Daniel D Salamone Silvia S London Erwin E

PloS one 20191007 10

We recently introduced a MαCD-based method to efficiently replace virtually the entire population of plasma membrane outer leaflet phospholipids and sphingolipids of cultured mammalian cells with exogenous lipids (Li et al, (2016) Proc. Natl. Acad. Sci USA 113:14025-14030). Here, we show if the lipid-to- MαCD ratio is too high or low, cells can round up and develop membrane leakiness. We found that this cell damage can be reversed/prevented if cells are allowed to recover from the exchange step ...[more]

PMID: 31589646

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Project description:UNLABELLED:Previous work from our laboratory showed that the Gram-negative aquatic pathogen Vibrio cholerae can take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability of V. cholerae to exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling the V. cholerae membrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrio outer membrane lysophospholipase A), required for these activities. The enzyme is well conserved in Vibrio species, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling. IMPORTANCE:Our understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogen Vibrio cholerae to utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.

Dataset Information

Replacing plasma membrane outer leaflet lipids with exogenous lipid without damaging membrane integrity.

Publications

Replacing plasma membrane outer leaflet lipids with exogenous lipid without damaging membrane integrity.

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