Pneumococcal hydrogen peroxide regulates host cell kinase activity

Abstract

Introduction: Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H₂O₂ on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells.

Methods: We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnΔlctOΔspxB -a deletion mutant strongly attenuated in H₂O₂ production- to assess the impact of pneumococcal hydrogen peroxide (H₂O₂) on global protein kinase activity profiles.

Results: Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H₂O₂. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnΔlctOΔspxB strain, which lacks H₂O_2. In particular, we describe for the first time H₂O₂-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H₂O₂-mediated phosphorylation.

Keywords: Akt; Lck; Streptococcus pneumoniae; hydrogen peroxide; kinome analysis.