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UBC Theses and Dissertations

Understanding Staphylococcus aureus β-lactam resistance : a structural investigation Alexander, John Andrew Nelson


Methicillin-resistant Staphylococcus aureus (MRSA) infections increase mortality and morbidity worldwide, threatening public health. MRSA is resistant to many classes of antibiotics including the most commonly prescribed β-lactam antibiotic class, making treatment of infections difficult. In MRSA β-lactam resistance is primarily mediated by PBP2a, a β-lactam resistant penicillin-binding protein and to some extent, PC1, a β-lactamase. Additionally, β- lactam resistance in S. aureus has also been recently shown to be facilitated independently of PBP2a by mutations in the gene coding for penicillin-binding protein 4 (PBP4), though the mechanisms of resistance have remained mysterious. In an effort to understand the mechanism of PBP4-mediated β-lactam resistance, two ligand-free and six acyl-enzyme intermediate X-ray crystallographic structures of mutant and wild-type PBP4 were solved. Localised within the transpeptidase active site cleft, the two substitutions appear to have different effects depending on the drug. Kinetic analysis shows the missense mutations impaired the KM value for ceftobiprole 150-fold, decreasing the proportion of inhibited PBP4. However, ceftaroline resistance appeared to be mediated by other factors, possibly including mutation of the pbp4 promoter. These findings suggest PBP4 mediated β- lactam resistance is mediated by at least two separate mechanisms. The expression of the genes coding PC1 and PBP2a are controlled by two integral membrane proteins: BlaR1 and MecR1 respectively, which consist of a zinc metalloprotease domain and an extracellular C-terminal β-lactam sensing domain which activates the proteolytic domain when acylated by a β-lactam antibiotic. Here, avibactam, a diazabicyclooctane β- lactamase inhibitor, was found to induce expression of pbp2a (which codes for PBP2a) and blaZ (which codes for PC1) in a clinical strain of MRSA. The X-ray crystallographic structures of the iv BlaR1 and MecR1 sensor domains show avibactam binds to MecR1 as has been observed for the Class-D β-lactamases. In contrast, BlaR1 has two avibactam binding poses orientated 180° to each other. As avibactam upregulates expression of blaZ and pbp2a antibiotic resistance genes, we suggest further research is needed to explore the effect of administering β-lactam-avibactam combinations to treat MRSA infections. Together, these findings improve our understanding of β-lactam resistance in MRSA and provide molecular details to facilitate improved inhibitors of MRSA.

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