Abstract
Gram positive (G+) infections make up ∼50% of all acute lung injury cases which are characterized by extensive permeability edema secondary to disruption of endothelial cell (EC) barrier integrity. A primary cause of increased permeability are cholesterol-dependent cytolysins (CDCs) of G+-bacteria, such as pneumolysin (PLY) and listeriolysin-O (LLO) which create plasma membrane pores, promoting Ca2+-influx and activation of PKCα. In human lung microvascular endothelial cells (HLMVEC), pretreatment with the nitric oxide synthase (NOS) inhibitor, ETU reduced the ability of LLO to increase microvascular cell permeability suggesting an endothelial nitric oxide synthase (eNOS)-dependent mechanism. LLO stimulated superoxide production from HLMVEC and this was prevented by silencing PKCα or NOS inhibition suggesting a link between these pathways. Both LLO and PLY stimulated eNOS T495 phosphorylation in a PKC-dependent manner. Expression of a phosphomimetic T495D eNOS (human isoform) resulted in increased superoxide and diminished nitric oxide (NO) production. Transduction of HLMVEC with an active form of PKCα resulted in the robust phosphorylation of T495 and increased peroxynitrite production, indicative of eNOS uncoupling. To determine the mechanisms underlying eNOS uncoupling, HLMVEC were stimulated with LLO and the amount of hsp90 and caveolin-1 bound to eNOS determined. LLO stimulated the dissociation of hsp90, and in particular, caveolin-1 from eNOS. Both hsp90 and caveolin-1 have been shown to influence eNOS uncoupling and a peptide mimicking the scaffolding domain of caveolin-1 blocked the ability of PKCα to stimulate eNOS-derived superoxide. Collectively, these results suggest that the G+ pore-forming toxins promote increased EC permeability via activation of PKCα, phosphorylation of eNOS-T495, loss of hsp90 and caveolin-1 binding which collectively promote eNOS uncoupling and the production of barrier disruptive superoxide.
Highlights
Gram positive (G+) infections make up,50% of acute respiratory distress syndrome (ARDS) cases and Streptococcus pneumoniae infections account for 45% of all community-acquired pneumonia (CAP) cases
In cells pretreated with ETU or Go 6976 there was a significant reduction in the ability of LLO to disrupt the endothelial barrier (Figure1A-B) suggesting functional roles for PKC and endothelial nitric oxide synthase in the ability of G+ toxins to disrupt the endothelial barrier
In human lung microvascular endothelial cells (HLMVEC) we found that an inhibitor of nitric oxide synthases prevented the ability of G+-toxins to decrease transendothelial resistance, suggesting that endothelial nitric oxide synthase (eNOS) was a crucial mediator of barrier dysfunction
Summary
Gram positive (G+) infections make up ,50% of acute respiratory distress syndrome (ARDS) cases and Streptococcus pneumoniae infections account for 45% of all community-acquired pneumonia (CAP) cases. There are currently no effective therapeutics for ARDS- and CAP- related pulmonary barrier dysfunction. These facts provide a strong rationale for more intensive research into the molecular mechanisms of endothelial barrier regulation. Bactericidal antibiotics can promote significant release of G+toxins and cause extensive and enduring injury even in a sterile lung These thiol-sensitive gram positive (G+) virulence factors oligomerize in the presence of cholesterol to form plasma membrane pores that stimulate calcium entry in various cell types and stimulate phospholipase C and protein kinase C alpha (PKCa) as we and others have recently shown [5,6,7]. Despite the greater appreciation of the importance of G+ toxins, the mechanisms by which PLY and LLO induce endothelial barrier disruption are poorly understood
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