Investigation of the cellular location of Multicopper Oxidase and its contribution to copper tolerance and immune resistance in Staphylococcus aureus

Abstract

In high amounts copper exerts toxic effects on bacteria, and therefore its cellular concentrations must be tightly controlled. Staphylococcus aureus is a WHO priority pathogen responsible of an enormous burden on healthcare systems globally. S. aureus has acquired copper tolerance systems to evade killing by toxic levels of copper.

The role of the relatively uncharacterised multicopper oxidase Mco of S. aureus was examined in this thesis. An isogenic deletion mutant of mco was generated. This mutant MRSA252Δmco, had a reduced growth in copper supplemented growth conditions, but did not have a reduced survival in human blood or neutrophils when compared to wild type indicating that mco contributes to copper tolerance in MRSA252 but not to survival in human blood or neutrophils. This copper sensitive phenotype was successfully complemented by introducing the mco gene back onto the chromosome under the transcriptional control of its native promoter.

The cellular location of Mco was studied by linking DNA encoding a 3X-FLAG tag to the 3' end of mco before generating subcellular fractions of the bacteria. 3X-FLAG tagged Mco was detected in the membrane fraction only, indicating that it is a membrane protein. The first 26 amino-acids of Mco contained a cleavable secretory signal peptide that delivered a reporter protein to the culture supernatant. This signal peptide was not recognized by the twin-arginine translocation system of S. aureus. This is the first reported bacterial multicopper oxidase that is not exported by the twin-arginine translocation system.

A link was found between the S. aureus cadmium resistance genes cadA and cadC and copper tolerance by analysis of growth of isogenic cadAC deletion mutants in copper supplemented media. The effect of copper on biofilm formation of S. aureus strains isolated from biofilm related infections in Irish hospitals was also examined, uncovering that copper can significantly reduce biofilm formation of many S. aureus isolates, but can increase or stimulate biofilm production by some isolates. Future studies may identify a use for copper as an effective anti-biofilm therapy.