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Protective role of Cav-1 in pneumolysin-induced endothelial barrier dysfunction
Robert K Batori1, Feng Chen2, Zsuzsanna Bordan1
1Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA, United States.
Insights
Caveolin-1 protects the endothelial barrier from pneumolysin (PLY) by promoting endocytosis. Loss of Caveolin-1 enhances PLY-induced barrier disruption, highlighting its protective role against Streptococcus pneumoniae.
Area of Science:
- Cell Biology
- Microbiology
- Pathophysiology
Background:
- Pneumolysin (PLY) from *Streptococcus pneumoniae* is a key virulence factor causing pneumonia by disrupting endothelial cell (EC) barrier integrity.
- PLY targets cholesterol-rich membrane domains, leading to calcium influx and compromised EC function.
- Caveolae, cholesterol-rich microdomains in ECs, are implicated in cellular responses to toxins.
Purpose of the Study:
- To investigate the role of membrane cholesterol and caveolin-1 (Cav-1) in endothelial cell susceptibility to PLY.
- To elucidate the protective mechanisms of Cav-1 against PLY-induced endothelial barrier dysfunction.
Main Methods:
- Experiments involved cholesterol depletion, CRISPR-Cas9 knockout, and siRNA silencing of Cav-1 in human lung microvascular ECs.
- Functional assays assessed PLY-induced intracellular calcium (IC Ca2+) levels and endothelial barrier integrity.
- Dynamin-2 (DNM2) inhibition and knockdown were used to study the role of endocytosis.
Main Results:
- Contrary to hypothesis, cholesterol depletion increased PLY-induced IC Ca2+.
- Cav-1 and its scaffolding domain peptide protected ECs from PLY-induced barrier disruption.
- Loss of Cav-1 significantly exacerbated PLY's effects, while Cav-1 re-expression restored protection.
- Inhibition of endocytosis via DNM2 amplified PLY-induced EC barrier dysfunction.
Conclusions:
- Caveolin-1 protects the endothelial barrier against pneumolysin by facilitating the endocytosis of damaged membrane sections.
- This Cav-1-mediated endocytosis reduces calcium influx and downstream PLY-dependent signaling, preserving barrier integrity.
- Cav-1 plays a critical role in defending endothelial cells against bacterial toxin-induced damage.
Abstract:
Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.
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