Biology subjects
Publications and source records attributed to Tran Van Nhieu, G..
Withdrawal statementThe authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2025/668902. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: 10.1101/2025.08.06.668902
Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a "closed" and "open" D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell matrix adhesion.
Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domain that binds to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.
Streptococcus pyogenes (Group A streptococcus; GAS) is an important human pathogen responsible for mild to severe, life-threatening infections. GAS expresses a wide range of virulence factors, including the M family proteins. The M proteins allow the bacteria to evade parts of the human immune defenses by triggering the formation of a dense coat of plasma proteins surrounding the bacteria, including IgGs. However, the molecular level details of the M1-IgG interaction have remained unclear. Here, we characterized the structure and dynamics of this interaction interface in human plasma on the surface of live bacteria using integrative structural biology, combining cross-linking mass spectrometry and molecular dynamics (MD) simulations. We show that the primary interaction is formed between the S-domain of M1 and the conserved IgG Fc-domain. In addition, we show evidence for a so far uncharacterized interaction between the A-domain and the IgG Fc-domain. Both these interactions mimic the protein G-IgG interface of group C and G streptococcus. These findings underline a conserved scavenging mechanism used by GAS surface proteins that block the IgG-receptor (Fc{gamma}R) to inhibit phagocytic killing. We additionally show that we can capture Fab-bound IgGs in a complex background and identify the specific M1 epitopes targeted on live bacteria. Our results elucidate the M1-IgG interaction network involved in inhibition of phagocytosis and reveal important M1 peptides that can be further investigated as future vaccine targets.
Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems - focal adhesions and mechanosensitive ion channels, can convert mechanical features of the microenvironment into biochemical signals. We report here surprisingly that the mechanosensitive Ca2+-channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting adhesion maturation and cell spreading in normal but not in tumor cells. In the absence of Piezo1, matrix adhesions are smaller in normal cells mimicking transformed cells where adhesions do not change with or without Piezo1. A novel adhesion-targeted calcium sensor shows robust Piezo1-dependent, calcium influx at adhesions in normal cells; but not in transformed cells. A linker domain in Piezo1 is needed for binding to adhesions and overexpression of the domain blocks Piezo1 binding to adhesions decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a novel component of focal adhesions in non-transformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.
Introductory paragraphStreptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people. The mechanism underlying PN crossing of the blood brain barrier (BBB) remains poorly understood. Here, we show that the gap junctional component connexin 43 expressed in astrocytes (aCx43) plays a major role in PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. We show a role for aCx43 in the PN-induced fragmentation of astrocytic GFAP filaments associated with bacterial translocation across endothelial vessels and replication in the brain cortex. aCx43 triggers the PN- and Ply-dependent GFAP fragmentation and nuclear shrinkage in in vitro cultured astrocytes. We showed that purified pneumolysin (Ply) co-opted Cx43 to promote the permeabilization and cytosolic calcium (Ca2+) increase of host cells, a process sensitive to extracellular ATP depletion. These results point to aCx43 as a major player during bacterial meningitis and extend cytolytic mechanisms implicating other host cell plasma membrane channels proposed for small pore-forming toxins, to Ply, a cholesterol-dependent cytolysin, at concentrations relevant to bacterial infection.
The Shigella effector IpaA co-opts the focal adhesion protein vinculin to promote bacterial invasion. Here, we show that IpaA triggers an unreported mode of vinculin activation through the cooperative binding of its three vinculin-binding sites (VBSs) leading to vinculin oligomerization via its D1 and D2 head subdomains and highly stable adhesions resisting actin relaxing drugs. Using cross-linking mass spectrometry, we found that while IpaA VBSs1-2 bound to D1, IpaA VBS3 interacted with D2, a subdomain masked to other known VBSs. Structural modeling indicated that as opposed to canonical activation linked to interaction with D1, these combined VBSs interactions triggered major allosteric changes leading to D1D2 oligomerization. A cysteine-clamp preventing these changes and D1D2 oligomerization impaired growth of vinculin microclusters and cell adhesion. We propose that D1D2-mediated vinculin oligomerization occurs during the maturation of adhesion structures to enable the scaffolding of high-order vinculin complexes, and is triggered by Shigella IpaA to promote bacterial invasion in the absence of mechanotransduction. SummaryThe Shigella IpaA effector binds to cryptic vinculin sites leading to oligomerization via its head domain. This vinculin oligomerization mode appears required for the maturation and strengthening of cell adhesion but is co-opted by invasive bacteria independent of actomyosin contractility.
Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this pathovar, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections.
During Shigella cell invasion, the IpaA effector targets the focal adhesion protein vinculin through three vinculin-binding sites (VBSs). Here, we report that IpaA VBS3 also binds to talin. The 2.5 [A] resolution crystal structure indicates that IpaA VBS3 forms a tightly folded -helical bundle with talin H1-H4, contrasting with bundle unraveling upon vinculin interaction. High-affinity binding of Ipa VBS3 to talin H1-H4 requires a core of hydrophobic residues conserved in vinculin binding and a pair of electrostatic interactions accounting for talin binding specificity. IpaA VBS3 does not bind to talin H1-H5 suggesting the targeting of partially activated stretched talin but not inactive talin. Consistently, IpaA VBS3 labeled filopodial and nascent adhesions and regulated their formation through talin binding. In addition, talin-binding by IpaA VBS3 was required for bacterial capture by filopodia during Shigella invasion and for the stabilization of focal adhesions in infected cells. These findings point to the functional diversity of VBSs and a critical role for talin-binding by Shigella IpaA VBS3 and possibly talin VBSs in the regulation of adhesion structures.