Nanoscale imaging of bacterial infections by sphingolipid expansion microscopy

Abstract

Expansion microscopy (ExM) enables super-resolution imaging of proteins and nucleic acids on conventional microscopes. However, imaging of details of the organization of lipid bilayers by light microscopy remains challenging. We introduce an unnatural short-chain azide- and amino-modified sphingolipid ceramide, which upon incorporation into membranes can be labeled by click chemistry and linked into hydrogels, followed by 4× to 10× expansion. Confocal and structured illumination microscopy (SIM) enable imaging of sphingolipids and their interactions with proteins in the plasma membrane and membrane of intracellular organelles with a spatial resolution of 10–20 nm. As our functionalized sphingolipids accumulate efficiently in pathogens, we use sphingolipid ExM to investigate bacterial infections of human HeLa229 cells by Neisseria gonorrhoeae, Chlamydia trachomatis and Simkania negevensis with a resolution so far only provided by electron microscopy. In particular, sphingolipid ExM allows us to visualize the inner and outer membrane of intracellular bacteria and determine their distance to 27.6 ± 7.7 nm.

The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy

“…PG thus poses a problem for ExM, since the original ExM protocols were developed for eukaryotic cells. Previously, it has been shown that PG has to be digested by lysozyme in order to enable isotropic expansion of bacteria in mixed communities ( Lim et al., 2019 ) or within host cells ( Gotz et al., 2020a ). In addition, MurNAc residues are modified by peptide chains that are cross-linked connecting individual PG layers ( Brott and Clarke, 2019 ).…”

“…HeLa229 cells were seeded on 15 mm glass slides (VWR) in multiwell plates (Corning) one day prior to infection. Staining of S. negevensis was achieved by membrane staining according to ( Gotz et al., 2020a ). For immunostaining, the samples were washed in 1× PBS and fixed with 4% PFA in PBS (Morphisto) for 15 min at RT.…”

“…Expansion microscopy (ExM) is an alternative approach bypassing the diffraction-limit and enabling super-resolution microscopy on standard fluorescence microscopes by physically linking a protein of interest in an expandable hydrogel. Introduced in 2015 ( Chen et al., 2015 ), various ExM protocols were developed leading to sample expansion factors of 4×, 10× ( Truckenbrodt et al., 2019 ) and even 20× ( Chang et al., 2017 ) allowing for high resolution imaging of proteins, nucleotides ( Chen et al., 2016 ) and modified lipids ( Gotz et al., 2020a ) in cell culture or tissues ( Wassie et al., 2019 ). In addition, expanded samples can be imaged by conventional confocal microscopes or in combination with super-resolution microscopy techniques thereby further improving spatial resolution ( Kunz et al., 2020 ).…”

“…The digestion of fungi thereby required a complex enzymatic mixture, consisting of a lysing enzyme of Trichoderma harzianum , driselase and chitinase ( Gotz et al., 2020b ). Recently, we applied ExM on several pathogens within host cells, such as Chlamydia trachomatis ( Kunz et al., 2019 ), Simkania negevensis , and Neisseria gonorrhoeae ( Gotz et al., 2020a ). Due to differences in cell wall structure, organism specific digestions strategies are required.…”

“…Due to differences in cell wall structure, organism specific digestions strategies are required. Expansion of C. trachomatis and S. negevensis has been performed with standard expansion protocols using only proteinase K for digestion, while expansion of N. gonorrhoeae additionally needed digestion with lysozyme ( Kunz et al., 2019 ; Gotz et al., 2020a ). ExM of Gram-positive bacteria was shown to be challenging due to their robust cell envelope.…”