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Abstract
Interactions of the bacterial lectin LecA with the host cells glycosphingolipid Gb3 have been shown to be crucial for the cellular uptake of the bacterium Pseudomonas aeruginosa. LecA-induced Gb3 clustering, referred to as lipid zipper mechanism, leads to full membrane engulfment of the bacterium. Here, we aim for a nanoscale force characterization of this mechanism using two complementary force probing techniques, atomic force microscopy (AFM) and optical tweezers (OT). The LecA-Gb3 interactions are reconstituted using giant unilamellar vesicles (GUVs), a well-controlled minimal system mimicking the plasma membrane and nanoscale forces between either bacteria (PAO1 wild-type and LecA-deletion mutant strains) or LecA-coated probes (as minimal, synthetic bacterial model) and vesicles are measured. LecA-Gb3 interactions strengthen the bacterial attachment to the membrane (1.5-8-fold) depending on the membrane tension and the applied technique. Moreover, significantly less energy (reduction up to 80%) is required for the full uptake of LecA-coated beads into Gb3-functionalized vesicles. This quantitative approach highlights that lectin-glycolipid interactions provide adequate forces and energies to drive bacterial attachment and uptake.
Highlights
Cell membranes have a highly complex composition of proteins and lipids, which frequently hampers the full characterization of cellular processes
We used atomic force microscopy (AFM)-based force probing in combination with fluorescence microscopy to measure the interaction forces between a single Pseudomonas aeruginosa (PA) bacterium, i.e. either the PAO1 wild-type strain (PAO1-WT) or the LecA-deletion mutant strain (PAO1-ΔLecA), with Gb3-functionalized giant unilamellar vesicles (GUVs)
After attaching an inactivated bacterium to a poly-Llysine-coated colloidal tip (Fig. 2A), the cantilever was transferred without dewetting to another chamber containing GUVs that have been adhered on streptavidin-coated coverslips by incorporation of 1 mol% of biotinylated lipids (Fig. 1A)
Summary
Cell membranes have a highly complex composition of proteins and lipids, which frequently hampers the full characterization of cellular processes. The internalization of plasma membrane components, extracellular substances or even pathogens is often mediated by the recruitment of endocytic coat proteins[8] or clustering of glycosphingolipids (GSLs),[9,10,11,12] which induces membrane curvature. The interaction of LecA, a lectin localized at the outer bacterial membrane,[19] with the host cell GSL globotriaosylceramide (Gb3) is sufficient to cause negative membrane curvature and even engulfment of PA in GUVs.[20] The molecular LecA–Gb3 interaction is crucial, since LecA deletion resulted in a significant reduction (about 45%) of PA engulfment in Gb3-functionalized GUVs and led to significantly less cellular invasion (60% reduction).[20] a direct inhibition of LecA–
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