Oxidation of bacillithiol during killing ofStaphylococcus aureusUSA300 inside neutrophil phagosomes

Abstract

Targeting immune evasion tactics of pathogenic bacteria may hold the key to treating recalcitrant bacterial infections. Staphylococcus aureus produces bacillithiol (BSH), its major low‐molecular‐weight thiol, which is thought to protect this opportunistic human pathogen against the bombardment of oxidants inside neutrophil phagosomes. Here, we show that BSH was oxidized when human neutrophils phagocytosed S. aureus, but provided limited protection to the bacteria. We used mass spectrometry to measure the oxidation of BSH upon exposure of S. aureus USA300 to either a bolus of hypochlorous acid (HOCl) or a flux generated by the neutrophil enzyme myeloperoxidase. Oxidation of BSH and loss of bacterial viability were strongly correlated (r = 0.99, p < 0.001). BSH was fully oxidized after exposure of S. aureus to lethal doses of HOCl. However, there was no relationship between the initial BSH levels and the dose of HOCl required for bacterial killing. In contrast to the HOCl systems, only 50% of total BSH was oxidized when neutrophils killed the majority of phagocytosed bacteria. Oxidation of BSH was decreased upon inhibition of myeloperoxidase, implicating HOCl in phagosomal BSH oxidation. A BSH‐deficient S. aureus USA300 mutant was slightly more susceptible to treatment with either HOCl or ammonia chloramine, or to killing within neutrophil phagosomes. Collectively, our data show that myeloperoxidase‐derived oxidants react with S. aureus inside neutrophil phagosomes, leading to partial BSH oxidation, and contribute to bacterial killing. However, BSH offers only limited protection against the neutrophil's multifaceted killing mechanisms.

Inside the phagosome: A bacterial perspective

“…94,95 Although bacterial strains lacking their major LMW thiol are more sensitive to reagent HOCl, 37,90,96,97 MSH and BSH were shown to provide no or only limited protection in neutrophil killing assays with M. smegmatis and S. aureus, respectively. 37,38 Deletion of the bacillithiol disulfide reductase in a different S. aureus strain did, however, decrease bacterial survival by about half compared with the wildtype, supporting a role for the bacillithiol redox system in coping with oxidative stress in the neutrophil phagosome. 98 Apart from attempting to modulate exposure to phagosomal oxidants, bacteria may also facilitate their survival by efficiently repairing oxidative damage.…”

Scylla, Charybdis, and navigating antimicrobial action in the neutrophil phagosome

“…Although many features of phagocytosis have been characterized since that time, fundamental aspects of the antimicrobial action of neutrophils remain unknown. The report by Ashby et al 2 . provides a refined and nuanced look at the interface between an ingested microbe, Staphylococcus aureus , and HOCl generated by the myeloperoxidase (MPO)–H 2 O 2 –chloride system in neutrophil phagosomes and represents a holistic approach to the analysis of bactericidal mechanisms that recognizes contributions from both phagocyte and its ingested prey.…”

“…With this recognition in mind, Ashby et al. examine the contribution of bacillithiol, a prominent thiol in S. aureus , to microbial defense against the MPO‐dependent generation of HOCl and other reactive chlorine species 2 . Oxidation of bacillithiol reflects HOCl production by neutrophils and parallels the loss of staphylococcal viability.…”

“…Excluding from discussion those situations where the sheer number of organisms, with or without local tissue damage, overwhelm local host defenses, we need to ask, as do Ashby et al., not only how neutrophils kill but also what mechanisms allow some bacteria ( S. aureus in Ref. 2 ) to survive and to persist in neutrophils.…”

“…examine the contribution of bacillithiol, a prominent thiol in S. aureus , to microbial defense against the MPO‐dependent generation of HOCl and other reactive chlorine species. 2 Oxidation of bacillithiol reflects HOCl production by neutrophils and parallels the loss of staphylococcal viability. In a simpler system, the bacillothiol deletion mutant, unable to combat HOCl‐induced damage, would be more susceptible to neutrophil killing.…”