From the membrane to central metabolism: New insights into the diverse mechanisms controlling β-lactam resistance in MRSA

Staphylococcus aureus is an important human pathogen, with the ability to colonise and cause disease, ranging in severity from superficial skin infections to life threatening invasive disease. The emergence of healthcare and community-acquired strains of methicillin resistant Staphylococcus aureus (MRSA), and their resistance to antibiotics makes it difficult to eradicate infections that would otherwise be treatable. This leads to poorer outcomes and increased morbidity and mortality for patients. We have identified novel genetic factors which affect β-lactam resistance in community acquired-MRSA which will contribute to the understanding and development of therapeutic targets. Disruption of the cycA gene, encoding an alanine permease, led to an increased susceptibility of MRSA to β-lactam antibiotics. In chemically defined media supplemented with glucose, the lack of CycA permease activity led to a reduction in alanine uptake by the cell, affecting peptidoglycan structure and cross-linking, rendering the cycA mutant susceptible to β-lactams. Exposure of the cycA mutant to the drug D-cycloserine, which inhibits alanine racemase and D-alanine ligase enzymes, exacerbated the impact of impaired alanine uptake and metabolism. This demonstrates the potential for D-cycloserine/oxacillin combinations as a new therapeutic option for the treatment of MRSA infections. The phenotypes of impaired alanine uptake and oxacillin susceptibility were not evident in a cycA mutant in CDM, indicating a role for another transporter in these growth conditions. Mutation of the TCA cycle gene sucC in MRSA resulted in increased β-lactam susceptibility. Analysis of this mutant and the parent strain JE2 revealed that mutation of sucC was associated with altered concentrations of TCA cycle metabolites including an accumulation of succinyl-CoA, and perturbation of the succinylome. The sucC mutant showed differences in the succinylation pattern of the major autolysin Atl, and sucC had reduced autolytic activity. This work reveals 10 succinyl-CoA and protein succinylation as a potential therapeutic target, in the effort to sensitise MRSA to β-lactams. Future work will provide insight as to whether the autolysis and resistance phenotypes are interlinked, potentially identifying additional targets. LspA is a signal peptidase of the lipoprotein processing pathway. We discovered that mutation of the lspA gene or inhibition of LspA activity by the drug globomycin led to increased resistance in MRSA. This effect does not appear to be linked to cell wall changes, and we hypothesize that accumulation of diacylglycerol or mislocalisation of lipoproteins at the cell membrane may be causing this phenotype. This work also demonstrates that the combination of an LspA inhibitor such as globomycin should not be used as treatment in combination with β-lactams, due to the risk that it would increase β-lactam resistance in an MRSA strain