Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

A Role for Taok2 in Listeria monocytogenes Vacuolar Escape.

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

DHA-containing phospholipids control membrane fusion and transcellular tunnel dynamics.

Metabolic studies and animal knockout models point to the critical role of polyunsaturated docosahexaenoic acid (22:6, DHA)-containing phospholipids (DHA-PLs) in physiology. Here, we investigated the impact of DHA-PLs on the dynamics of transendothelial cell macroapertures (TEMs) triggered by RhoA inhibition-associated cell spreading. Lipidomic analyses showed that human umbilical vein endothelial cells (HUVECs) subjected to a DHA diet undergo a 6-fold enrichment in DHA-PLs at the plasma membrane (PM) at the expense of monounsaturated oleic acid-containing PLs (OA-PLs). Consequently, DHA-PL enrichment at the PM induces a reduction in cell thickness and shifts cellular membranes towards a permissive mode of membrane fusion for transcellular tunnel initiation. We provide evidence that a global homeostatic control of membrane tension and cell cortex rigidity minimizes overall changes of TEM area through a decrease of TEM size and lifetime. Conversely, low DHA-PL levels at the PM lead to the opening of unstable and wider TEMs. Together, this provides evidence that variations of DHA-PL levels in membranes affect cell biomechanical properties.

Dewetting: From Physics to the Biology of Intoxicated Cells.

Pathogenic bacteria colonize or disseminate into cells and tissues by inducing large-scale remodeling of host membranes. The physical phenomena underpinning these massive membrane extension and deformation are poorly understood. Invasive strategies of pathogens have been recently enriched by the description of a spectacular mode of opening of large transendothelial cell macroaperture (TEM) tunnels correlated to the dissemination of EDIN-producing strains of Staphylococcus aureus via a hematogenous route or to the induction of gelatinous edema triggered by the edema toxin from Bacillus anthracis. Remarkably, these highly dynamic tunnels close rapidly after they reach a maximal size. Opening and closure of TEMs in cells lasts for hours without inducing endothelial cell death. Multidisciplinary studies have started to provide a broader perspective of both the molecular determinants controlling cytoskeleton organization at newly curved membranes generated by the opening of TEMs and the physical processes controlling the dynamics of these tunnels. Here we discuss the analogy between the opening of TEM tunnels and the physical principles of dewetting, stemming from a parallel between membrane tension and surface tension. This analogy provides a broad framework to investigate biophysical constraints in cell membrane dynamics and their diversion by certain invasive microbial agents.

High prevalence and moderate diversity of Pseudomonas aeruginosa in the U-bends of high-risk units in hospital.

The presence of P. aeruginosa in water supply is clearly identified as a risk factor for P. aeruginosa infection in critical care units, even if routes of transmission are often unclear and remain a matter of debate. We determined here the frequency of U-bends contaminated with P. aeruginosa in high-risk units and described the population structure of this opportunistic pathogen in a non-outbreak situation. Eighty-seven U-bends from sinks of rooms in five wards were sampled 3 times and P. aeruginosa was detected in 121 of the 261 (46.4%) U-bend samples. We genotyped 123 P. aeruginosa isolates with pulsed-field gel electrophoresis and multilocus sequence typing and found 41 pulsotypes distributed in 21 Sequence Types (STs). Seven major ST (ST111, CC235, CC253, ST520, ST539, ST1216, and ST1725) were overrepresented in the collection, including the high-risk clones ST111, CC253, and CC235. The distribution of the 21 STs in the cladogram of the species was uneven with most major STs clustering into 2 clades. The major STs were found in different units and buildings and could be represented by a high diversity of pulsotypes. Altogether, this suggests a long term presence of P. aeruginosa in the hospital water network, possibly contaminated by the distribution water or by plumbing fittings before putting into service. Analysis of resistance rates showed that the deficiency of porin OprD was very frequent in U-bends isolates that may benefit from this resistance mechanism in hospital water fittings. In conclusion, our study demonstrates that U-bends of high-risk units are very frequently contaminated with P. aeruginosa with a moderate genomic diversity and with an over-representation of adapted clones.