Abstract
Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce ‘click-AT-CLEM’, a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity.
Highlights
Sphingolipids, including ceramides, form a diverse group of structurally related lipids and are composed of a backbone of sphingoid bases coupled to a fatty acid side chain
We established a protocol based on a combination of correlative light and electron microscopy (CLEM), i.e. super-resolution array tomography[17,18] and click chemistry[19], which we call in short ‘click-AT-CLEM’
As this is very challenging with established immunolabeling approaches for electron microscopy, we developed a novel pre-embedding correlative light and electron microscopy (CLEM) protocol to achieve a more precise localization of the uptake of the two azido-modified sphingolipids, ω-N3-sphingosine and ω-N3-C6-ceramide, by N. meningitidis
Summary
Sphingolipids, including ceramides, form a diverse group of structurally related lipids and are composed of a backbone of sphingoid bases coupled to a fatty acid side chain. We established a protocol based on a combination of correlative light and electron microscopy (CLEM), i.e. super-resolution array tomography (srAT)[17,18] and click chemistry[19], which we call in short ‘click-AT-CLEM’. This combination of fluorescence and electron imaging applied here to the Gram-negative bacterium N. meningitidis allowed to determine morphological and ultrastructural changes after treatment with azido-modified sphingolipid analogs as well as to visualize their subcellular localization. Our data demonstrate that srAT technology in combination with click chemistry–based labeling reaction is ideally suited to visualize, track and quantify azido-modified antimicrobial sphingolipids in bacteria
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