PhD ThesisStaphylococcus aureus is a Gram positive bacterium which is predominantly commensal,
but which can also be a human and animal pathogen, able to cause serious infections. It
is becoming an increasing problem due to it becoming resistant to modern antibiotics. In
common with most bacteria, S. aureus requires small quantities of the essential metal
copper, but they also experience toxicity when exposed to high concentrations of
copper, a metal that has been used for its antimicrobial properties for centuries.
However, the mechanism of such toxicity remains elusive.
Here, the effect of copper toxicity on S. aureus has been investigated in order to
understand how excess copper ions affect its physiology. The growth of S. aureus was
found to be inhibited in media containing elevated copper, and the extent of this
inhibition was shown to be dependent on the type of growth medium used. Analysis of
soluble extracts from S. aureus cells exposed to elevated copper led to the identification
of a cytoplasmic enzyme, GapA, which binds copper. GapA is a member of the wellcharacterised
family of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
enzymes, and is not a metalloprotein. Copper inhibition of the activity of the S. aureus
GapA enzyme was demonstrated, both in S. aureus cell extracts and with recombinant
enzyme in vitro, using a specific enzyme activity assay.
Analysis of the purified recombinant GapA enzyme in vitro demonstrated a copper
binding stoichiometry of one Cu(I) ion to each GapA monomer using analytical sizeexclusion
chromatography and spectrophotometry, and evidence is presented that
suggests high affinity Cu(I) binding of biochemical relevance. Some preliminary
evidence is provided that the Cu(I) binding site on the GapA protein includes the thiol
group of cysteine 151 located within the active site, consistent with predictions based on
published crystal structures, and explaining the observed copper-dependent inhibition
of enzyme activity.
This is the first evidence of copper binding to a non-metalloprotein within the cytoplasm
of S. aureus and adds to mounting evidence that aberrant binding of copper to cytosolic
proteins contributes to bacterial copper toxicity
