IpaA reveals distinct modes of vinculin activation during Shigella invasion and cell-matrix adhesion.

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.

A Retro-orbital Sinus Injection Mouse Model to Study Early Events and Reorganization of the Astrocytic Network during Pneumococcal Meningitis.

Pneumococcal (PN) meningitis is a life-threatening disease with high mortality rates that leads to permanent neurological sequelae. Studies of the process of bacterial crossing of the blood brain barrier (BBB) are hampered by the lack of relevant in vitro and in vivo models of meningitis that recapitulate the human disease. PN meningitis involves bacterial access to the bloodstream preceding translocation across the BBB. A large number of PN meningitis models have been developed in mice, with intravenous administration via the lateral tail vein representing the main way to study BBB crossing by PN. While in humans, meningitis is not always associated with bacteremia, PN meningitis after intravenous injection in mice usually develops following sustained and very high bacteremic titers. High grade bacteremia, however, is known to favor inflammation and BBB permeabilization, thereby increasing PN translocation across the BBB and associated damages. Therefore, specific processes associated with early events of PN translocation may be blurred by overall changes in the inflammatory environment and potentially systemic dysfunction in the case of severe sepsis. Here, we report a mouse meningitis model induced by PN injection in the retro-orbital (RO) sinus. We show that, in this model, mice appear to control bacteremic levels during the first 13 h post-infection, while PN crossing of the BBB can be clearly detected by fluorescence confocal microscopy analysis of brain slices as early as 6 h post-infection. Because of the low frequency of events, however, PN translocation across brain parenchymal vessels at early time points requires a rigorous and systematic examination of the brain volume.

Role of astroglial Connexin 43 in pneumolysin cytotoxicity and during pneumococcal meningitis.

Streptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people worldwide. The mechanism underlying PN crossing of the blood brain barrier (BBB) and specifically, the role of non-endothelial cells of the neurovascular unit that control the BBB function, remains poorly understood. Here, we show that the astroglial connexin 43 (aCx43), a major gap junctional component expressed in astrocytes, plays a predominant role during PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. Immunofluorescence analysis of brain slices indicated that PN induces the aCx43-dependent destruction of the network of glial fibrillary acid protein (GFAP), an intermediate filament protein specifically expressed in astrocytes and up-regulated in response to brain injury. PN also induced nuclear shrinkage in astrocytes associated with the loss of BBB integrity, bacterial translocation across endothelial vessels and replication in the brain cortex. We found that aCx4-dependent astrocyte damages could be recapitulated using in vitro cultured cells upon challenge with wild-type PN but not with a ply mutant deficient for the pore-forming toxin pneumolysin (Ply). Consistently, we showed that purified Ply requires Cx43 to promote host cell plasma membrane permeabilization in a process involving the Cx43-dependent release of extracellular ATP and prolonged increase of cytosolic Ca2+ in host cells. These results point to a critical role for astrocytes during PN meningitis and suggest that the cytolytic activity of the major virulence factor Ply at concentrations relevant to bacterial infection requires co-opting of connexin plasma membrane channels.