Staphylococcus aureus is a major pathogen, which has to defend against reactive
oxygen and electrophilic species encountered during
infections. Activated macrophages produce the immunometabolite
itaconate as potent electrophile and antimicrobial upon pathogen
infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of
itaconic acid in S. aureus. In the
RNA-seq transcriptome,
itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the
urease-encoding operon, revealing an
acid and oxidative stress response and impaired proteostasis. Neutralization using external
urea as
ammonium source improved the growth and decreased the expression of the
glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced
ammonium starvation upon
itaconic acid stress. In the extracellular metabolome, the amounts of
acetate and
formate were decreased, while secretion of
pyruvate and the neutral product
acetoin were strongly enhanced to avoid intracellular acidification. Exposure to
itaconic acid affected the
amino acid uptake and metabolism as revealed by the strong intracellular accumulation of
lysine,
threonine,
histidine,
aspartate,
alanine,
valine,
leucine,
isoleucine,
cysteine and
methionine. In the
proteome,
itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive
antioxidant and metabolic
enzymes,
ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the
catalase KatA, the low molecular weight
thiol bacillithiol and the
urease provided protection against
itaconic acid-induced oxidative and
acid stress in S. aureus. Altogether, our results revealed that under physiological
infection conditions, such as in the acidic phagolysome,
itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak
acid causing an
acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation.