Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors

Abstract

The GTPase RhoA is a major regulator of the assembly of actin stress fibers and the contractility of the actomyosin cytoskeleton. The epidermal cell differentiation inhibitor (EDIN) and EDIN-like ADP-ribosyltransferases of Staphylococcus aureus catalyze the inactivation of RhoA, producing actin cable disruption. We report that purified recombinant EDIN and EDIN-producing S. aureus provoke large transcellular tunnels in endothelial cells that we have named macroapertures (MAs). These structures open transiently, followed by the appearance of actin-containing membrane waves extending over the aperture. Disruption of actin cables, either directly or indirectly, through rhoA RNAi knockdown also triggers the formation of MAs. Intoxication of endothelial monolayers by EDIN produces a loss of barrier function and provides direct access of the endothelium basement membrane to S. aureus.

Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I‐BAR proteins

“…Tunnels open up to a maximum diameter of about 10-20 μm within 1-2 min, depending on the type of cell intoxication ( Figure 1). Next, membrane waves rich in actin filaments (F-actin) extend around the edge of the TEM driving aperture closure in about 3-5 min ( Figure 1) (Boyer et al, 2006;Maddugoda et al, 2011; Gonzalez-Rodriguez et al, 2012). As discussed in the following section from liquid dewetting to cellular dewetting, recent physical modelling of TEM dynamics suggests a new framework to help decipher the relationship between actomyosin contractility and membrane tension, and how this interplay affects cell shape organisation (Gonzalez-Rodriguez et al, 2012).…”

“…Excessive contraction or disruption of the actin cytoskeleton cortex leads to the local detachment of the membrane associated with induction of membrane blebs (Sheetz et al, 2006;Ridley, 2011;Spangler et al, 2011;Salbreux et al, 2012). In contrast, a reduction of actomyosin cable contractility produces large tunnels across thin endothelial cells, referred to as transendothelial cell macroaperture tunnels (TEMs) (Boyer et al, 2006;Maddugoda et al, 2011;Gonzalez-Rodriguez et al, 2012). Internal cellular regulation usually inhibits the spontaneous formation of such TEMs, but some bacterial toxins or leucocytes have the capability to induce TEMs to cross the endothelial barrier .…”

“…Recently, studies conducted on a group of bacterial toxins, now referred to as tunnel-forming toxins (TFTs), have shed light on the molecular and biophysical aspects of the dynamic of TEM formation (Maddugoda et al, 2011;Gonzalez-Rodriguez et al, 2012). These toxins reduce actomyosin contractility and allow for the spontaneous opening of large apertures (macroapertures) across endothelial cells (Boyer et al, 2006;Maddugoda et al, 2011). Tunnels open up to a maximum diameter of about 10-20 μm within 1-2 min, depending on the type of cell intoxication ( Figure 1).…”

ADP-ribosylating toxins modifying the actin cytoskeleton

“…An original effect triggered by EDIN and related C3 enzymes involves perturbation of endothelial permeability by forming transcellular channels. Thereby, EDIN-mediated inactivation of RhoA in endothelial cells causes a reorganization of the actin cytoskeleton that forms transient macroapertures termed large transendothelial cell macroaperture tunnels (TEMs) [5,52,53]. Increased endothelial permeability facilitates the binding of S. aureus to extracellular matrix proteins and subsequent dissemination from the bloodstream to underlying tissues.…”

Activation of p61Hck Triggers WASp- and Arp2/3-dependent Actin-comet Tail Biogenesis and Accelerates Lysosomes

“…To functionally inhibit Rho, cytosolic extracts were treated with C3 exoenzyme (1 g of enzyme per 1 mg of total proteins) for 10 min at 37°C before being used in the actin-comet tail assay described below. To assess the level of Rho inhibition in the cell extracts treated with active or inactive C3 exoenzyme, the extracts were further incubated with an excess of active C3 exoenzyme (100 g of enzyme per 1 mg of total proteins) for in vitro ADP-ribosylation, which was performed for 30 min at 37°C in the presence of 50 Ci of adenine adenylate-32 P dinucleotide per 1 mg of total proteins (34). Under these conditions, 83% of endogenous Rho were inhibited.…”