Abstract
Phagocyte-derived production of a complex mixture of different oxidants is a major mechanism of the host defense against microbial intruders. On the protein level, a major target of these oxidants is the thiol group of the amino acid cysteine in proteins. Oxidation of thiol groups is a widespread regulatory post-translational protein modification. It is used by bacteria to respond to and to overcome oxidative stress. Numerous redox proteomic studies have shown that protein thiols in bacteria, such as Escherichia coli react towards a number of oxidants in specific ways. However, our knowledge about protein thiols in bacteria exposed to the complex mixture of oxidants encountered in the phagolysosome is still limited. In this study, we used a quantitative redox proteomic method (OxICAT) to assess the in vivo thiol oxidation status of phagocytized E. coli. The majority (65.5%) of identified proteins harbored thiols that were significantly oxidized (> 30%) upon phagocytosis. A substantial number of these proteins are from major metabolic pathways or are involved in cell detoxification and stress response, suggesting a systemic breakdown of the bacterial cysteine proteome in phagocytized bacteria. 16 of the oxidized proteins provide E. coli with a significant growth advantage in the presence of H2O2, when compared to deletion mutants lacking these proteins, and 11 were shown to be essential under these conditions.
Highlights
Neutrophils are key players of the innate immune response
We showed that the genetically encoded fluorescent redox probe roGFP2-Orp1 is promptly oxidized in bacteria that are phagocytized by neutrophils [14]
We studied the effects of neutrophil phagocytosis on the thiol proteome of bacteria
Summary
Neutrophils are key players of the innate immune response. In response to invading microorganisms, they are recruited to sites of infection, where they internalize pathogens into compartments called phagosomes. Oxidants that are produced in the phagosome can react with and damage major cellular components of pathogens, including DNA, lipids and proteins. Bacteria have their own mechanisms to protect themselves from oxidative stress. The oxidized proteins were part of protein, nucleotide and carbohydrate metabolic pathways but were involved in cell detoxification and stress response, which indicate a systemic oxidation of protein thiols. This suggests a total break-down of E. colis thiol proteome after encountering neutrophil phagocytosis. As revealed by subsequent growth rate assays, 16 mutants, which lack proteins identified in our redox proteomic experiments, showed increased sensitivity towards oxidative stress. 11 of the genes encoding those proteins were essential for the growth of E. coli under otherwise sublethal oxidative stress conditions
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