Menaquinone biosynthesis potentiates haem toxicity in Staphylococcus aureus

Abstract

Summary Staphylococcus aureus is a pathogen that infects multiple anatomical sites leading to a diverse array of diseases. Although vertebrates can restrict the growth of invading pathogens by sequestering iron within haem, S. aureus surmounts this challenge by employing high-affinity haem uptake systems. However, the presence of excess haem is highly toxic, necessitating tight regulation of haem levels. To overcome haem stress, S. aureus expresses the detoxification system HrtAB. In this work, a transposon screen was performed in the background of a haem-susceptible, HrtAB-deficient S. aureus strain to identify the substrate transported by this putative pump and the source of haem toxicity. While a recent report indicates that HrtAB exports haem itself, the haem-resistant mutants uncovered by the transposon selection enabled us to elucidate the cellular factors contributing to haem toxicity. All mutants identified in this screen inactivated the menaquinone (MK) biosynthesis pathway. Deletion of the final steps of this pathway revealed that quinone molecules localizing to the cell membrane potentiate haem-associated superoxide production and subsequent oxidative damage. These data suggest a model in which membrane-associated haem and quinone molecules form a redox cycle that continuously generates semiquinones and reduced haem, both of which react with atmospheric oxygen to produce superoxide.

Menaquinone Analogs Inhibit Growth of Bacterial Pathogens

“…P value for survival difference was determined by Fisher's exact test. (29)(30)(31). Menadione is a precursor for menaquinone, so it was interesting that this molecule was antimicrobial for these two bacteria.…”

Differential Activation of Staphylococcus aureus Heme Detoxification Machinery by Heme Analogues

“…MP accumulation was measured similarly to previously described methods (10). Briefly, cells were cultured overnight in untreated TSB and diluted to an OD 600 of 0.2 in the morning.…”

“…All experiments in this study were performed in S. aureus clinical isolate Newman (20). Generation of the isogenic mutants lacking hrtB, hssRS, hemA, or menB and the strain expressing the phrtB::xylE reporter fusion was previously described (6,10,12,13,21). S. aureus cultures were grown on tryptic soy broth (TSB) solidified with 1.5% agar at 37°C or in TSB at 37°C with shaking at 180 rpm.…”

“…Oxidative protein damage was monitored by immunoblotting as previously described (10). Briefly, cells were cultured overnight in untreated TSB and diluted to an optical density at 600 nm (OD 600 ) of 0.2 in the morning.…”

“…Upon exposure to atmospheric oxygen, membrane-associated heme molecules can autooxidize to generate damaging superoxide radicals. This reaction is potentiated by the presence of menaquinone, which reduces heme and allows multiple rounds of auto-oxidation to occur (10).…”