Membrane invagination induced by Shiga toxin B-subunit: from molecular structure to tube formation.

W Pezeshkian,L Johannes,P B S Kumar,J C Shillcock,A G Hansen,H Khandelia,J H Ipsen

doi:10.1039/c6sm00464d

Abstract

The bacterial Shiga toxin is composed of an enzymatically active A-subunit, and a receptor-binding homopentameric B-subunit (STxB) that mediates intracellular toxin trafficking. Upon STxB-mediated binding to the glycolipid globotriaosylceramide (Gb3) at the plasma membrane of target cells, Shiga toxin is internalized by clathrin-dependent and independent endocytosis. The formation of tubular membrane invaginations is an essential step in the clathrin-independent STxB uptake process. However, the mechanism by which STxB induces these invaginations has remained unclear. Using a combination of all-atom molecular dynamics and Monte Carlo simulations we show that the molecular architecture of STxB enables the following sequence of events: the Gb3 binding sites on STxB are arranged such that tight avidity-based binding results in a small increment of local curvature. Membrane-mediated clustering of several toxin molecules then creates a tubular membrane invagination that drives toxin entry into the cell. This mechanism requires: (1) a precise molecular architecture of the STxB binding sites; (2) a fluid bilayer in order for the tubular invagination to form. Although, STxB binding to the membrane requires specific interactions with Gb3 lipids, our study points to a generic molecular design principle for clathrin-independent endocytosis of nanoparticles.

Highlights

In the absence of the A-subunit, STxB still forms a pentameric unit[10] and its internalization ability remains unaffected,[11] even if the uptake pathway might be modulated by the A-subunit.[12]
In the case of the related cholera toxin that like Shiga toxin induces membrane bending via interactions with its cellular receptor, the GSL GM1, it was shown that unsaturated GM1 acyl chain species favored cellular infection.[14]
We have shown that inclusions that induce a local curvature of C0 E 0.035 nmÀ1 and cluster via membrane-mediated interactions drive the formation of tubular membrane invaginations with narrow necks that connect them to the vesicle surface (Fig. 6)

Summary

Introduction

In the absence of the A-subunit, STxB still forms a pentameric unit[10] and its internalization ability remains unaffected,[11] even if the uptake pathway might be modulated by the A-subunit.[12]. Paper unilamellar vesicles (GUV) in the absence of any cytosolic machinery.[5] Interestingly, several pathogenic[14,15] or cellular lectins[16] share with STxB the capacity to induce membrane invaginations in interaction with defined GSL species, indicating that this could be a general mechanism Binding of STxB to Gb3 with saturated acyl chains (Gb3:C22:0; denoted hereafter by S-Gb3) does not lead to the formation of invaginations, whereas invaginations form upon STxB binding to Gb3 with unsaturated acyl chains (Gb3:C22:1; denoted by U-Gb3).[5] In the case of the related cholera toxin that like Shiga toxin induces membrane bending via interactions with its cellular receptor, the GSL GM1, it was shown that unsaturated GM1 acyl chain species favored cellular infection.[14] The mechanisms by which these lectins drive membrane bending have not yet been determined

Methods

Results

Conclusion