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.Author summaryThe role of non-endothelial cells constituting the neurovascular unit during infectious meningitis is poorly appreciated despite their key regulatory functions on the blood-brain barrier integrity. Here, we show that Streptococcus pneumoniae or pneumococcus, a major causative agent of bacterial meningitis, targets astroglial cells to translocate across brain endothelial vessels. We found that astroglial connexin 43, a gap junctional component, played a major role during PN meningitis in mice. PN translocation and replication in the brain cortex were associated with connexin-dependent fragmentation of astrocytic the GFAP network, a process associated with brain injury. These findings were recapitulated and extended in vitro using cultured primary astrocytes and the major PN virulence determinant Pneumolysin. Ply-mediated cytotoxicity was linked to Ca2+ increase and required aCx43, arguing against a direct toxin activity. The results reveal a key role for astroglial signaling during PN crossing of the BBB and shed light on the mechanism of Ply-mediated cytotoxicity during meningitis.