Oxidation of hemoproteins byStreptococcus pneumoniaecollapses the cell cytoskeleton and disrupts mitochondrial respiration leading to cytotoxicity of human lung cells

Abstract

Streptococcus pneumoniae (Spn) causes pneumonia that kills millions through acute toxicity and invasion of the lung parenchyma. During aerobic respiration, Spn releases hydrogen peroxide (Spn-H2O2), as a by-product of enzymes SpxB and LctO, and causes cell death with signs of both apoptosis and pyroptosis by oxidizing unknown cell targets. Hemoproteins are molecules essential for life and prone to oxidation by H2O2. We recently demonstrated that during infection-mimicking conditions, Spn-H2O2 oxidizes the hemoprotein hemoglobin (Hb), releasing toxic heme. In this study, we investigated details of the molecular mechanism(s) by which the oxidation of hemoproteins by Spn-H2O2 causes human lung cell death. Spn strains, but not H2O2-deficient SpnΔspxBΔlctO strains caused time-dependent cell cytotoxicity characterized by the rearrangement of the actin, the loss of the microtubule cytoskeleton and nuclear contraction. Disruption of the cell cytoskeleton correlated with the presence of invasive pneumococci and an increase of intracellular reactive oxygen species. In cell culture, the oxidation of Hb or cytochrome c (Cytc) caused DNA degradation and mitochondrial dysfunction from inhibition of complex I-driven respiration, which was cytotoxic to human alveolar cells. Oxidation of hemoproteins resulted in the creation of a radical, which was identified as a protein derived side chain tyrosyl radical by using electron paramagnetic resonance (EPR). Thus, we demonstrate that Spn invades lung cells, releasing H2O2 that oxidizes hemoproteins, including Cytc, catalyzing the formation of a tyrosyl side chain radical on Hb and causing mitochondrial disruption, that ultimately leads to the collapse of the cell cytoskeleton.

Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria

“…39,54 Spn-H O oxidizes hemoproteins such as hemoglobin and cytochrome C (Cyt c ) and this oxidation creates two other toxic products, labile heme and a tyrosyl radical ( • Hb+Fe 4+ ). 17,19,20 Here, we discovered that oxidative reactions driven by Spn-H O contributes to lung toxicity by oxidizing hemoglobin contributing to the degradation of cytosolic proteins, and facilitating the differentiation of pneumococci into encapsulated bacteria. Accordingly, the differentiation predominantly takes place within the alveoli and endothelial cells of pulmonary arteries.…”

“…We made a fascinating discovery related to the mechanism of lung cell death that correlated with the loss of the lung’s cellular structure. Proteins of the cell cytoskeleton involved in structural stability and protein trafficking, such as β-tubulin and β-actin, and cytosolic GAPDH from the glycolytic pathway were degraded during infection of human alveolar cells, or bronchial cells 20 , with H 2 O 2 -producing pneumococci. Degradation of cytoskeletal proteins correlated with microtubule collapse (catastrophe) and cell swelling due to loss of actin stress fibers (not shown and 20 ).…”

“…Proteins of the cell cytoskeleton involved in structural stability and protein trafficking, such as β-tubulin and β-actin, and cytosolic GAPDH from the glycolytic pathway were degraded during infection of human alveolar cells, or bronchial cells 20 , with H 2 O 2 -producing pneumococci. Degradation of cytoskeletal proteins correlated with microtubule collapse (catastrophe) and cell swelling due to loss of actin stress fibers (not shown and 20 ). Structural damage of microtubules has been observed in ventricular cardiomyocytes exposed to H O (100 µm).…”

“…Spn-H 2 O 2 oxidation of hemoglobin causes the release of heme, its degradation, and the creation of highly reactive tyrosyl radical. 20,30,31,80,81 As such, heme destruction is extremely toxic. For example, intravascular hemolysis with the accumulation of labile heme during hemolytic diseases such as malaria and SCD 23 has been linked to proinflammatory and prothrombotic complications, 82 as well as cell death through PANoptosis.…”

“…17,18 The oxidation of hemoglobin by Spn produced potent oxidants, including labile heme 19 and a hemoglobin-derived side chain tyrosyl radical. 20 These toxic products are important in the pathophysiology of human diseases that are accompanied by intravascular hemolysis, such as sickle cell disease (SCD), and in inflammatory conditions, such as sepsis. 21–23 In children with SCD, hemoglobin (Hb)S is oxidized to met-hemoglobin resulting in an acute hemolytic vascular inflammatory process inducing the polymerization of hemoglobin, and consequently acute lung injury.…”