Uncovering the role of nucleotide metabolism in the control of beta-lactam resistance in Staphylococcus aureus and the identification of novel treatment strategies for Methicillin Resistant S. aureus (MRSA) infections

Staphylococcus aureus is an opportunistic pathogen that can negatively impact human healthcare. It has a range of virulence factors that make it a formidable human pathogen with the ability to cause death. This is further complicated by the emergence of methicillin resistant S. aureus (MRSA) which is resistant to the most commonly used antibiotics; beta-lactams. This leads to poorer treatment outcomes and options for patients that contract MRSA infections. This thesis describes the identification and characterisation of mutations associated with increased beta-lactam resistance in MRSA strain JE2. These mutations included genes encoding transporters for xanthosine, guanosine and guanine. Specifically, transposon insertions in pbuG, pbuX and nupG were implicated in higher levels of resistance to the anti-staphylococcal beta-lactam oxacillin. Conversely, the addition of guanosine or xanthosine reduced oxacillin resistance of JE2, supporting the conclusion that guanosine and xanthosine uptake regulates beta-lactam resistance. Notably, exposure of MRSA cells to exogenous adenosine increased oxacillin resistance, while the purine inosine, which can be fluxed to ATP or GTP only marginally reduced resistance. The addition of guanosine in combination with oxacillin to culture media was shown to reduce c-di-AMP levels providing an insight on the mechanism of action of this antibiotic/adjuvant combination. These discoveries highlighted the importance of guanosine metabolism in altering c-di-AMP levels and beta-lactam susceptibility. Exposure to guanosine or adenosine resulted in contrasting effects on the MRSA proteomes and metabolomes. The abundances of enzymes responsible for cell wall synthesis including FemA, MurA, DapA, DapB and DapD were decreased by guanosine, whereas adenosine grown cells exhibited increased abundances in DapB and DapD. Guanosine grown cells also showed decreased intracellular levels of both glutamine and thymidine. Consistent with the reduced levels of thymidine, guanosine potentiated the activity of the anti-folates sulfamethoxazole (SMX), trimethoprim (TMP) and SMX-TMP as well as the pyrimidine analogues 5-fluorouracil (5-FU), and 5- fluorouridine (5-FUrd). Furthermore, the use of oxacillin at low concentrations provided “double synergy” with guanosine/thymidine inhibitor combinations. Compounds used for treatment of cancer that target nucleotide metabolism also modulated beta-lactam antibiotics in a similar manner to guanosine and adenosine. Notably these compounds regulated expression of the lysine biosynthesis operon possibly identifying a novel pathway controlling beta-lactam resistance. Regulation of lysine biosynthesis by nucleotide analogues may suggest their usefulness as betalactam adjuvants to improve the treatment of MRSA infections. Further work also revealed the ability of these compounds to inactivate biofilms particularly when coadministered with known anti-biofilm antibiotics such as rifampicin, linezolid or daptomycin.

Funder

Research Ireland

Publisher

University of Galway

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

cbn

Collections

University of Galway Theses (PhD Theses)