Adding insult to injury: mechanistic basis for how AmpC mutations allow Pseudomonas aeruginosa to accelerate cephalosporin hydrolysis and evade avibactam
Slater, Cole Lee
Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide and notorious for its broad-spectrum resistance to antibiotics. A key mechanism that confers extensive resistance to β-lactam antibiotics is the inducible expression of AmpC, a highly efficient Ambler class C β-lactamase enzyme. Unfortunately, several P. aeruginosa clinical isolates expressing mutated forms of AmpC have been found to be clinically resistant to the novel antipseudomonal β-lactam/β-lactamase inhibitor (BLI) combinations ceftolozane/tazobactam and ceftazidime/avibactam. The objective of this thesis was to investigate the enzymatic activity of four of these reported AmpC mutants, E247K, G183D, T96I, and ∆G229–E247 (alongside wild-type (WT) AmpC from P. aeruginosa PAO1), to gain detailed insights into how these mutations circumvent these clinically vital antibiotic/inhibitor combinations. The effect of these mutations on the catalytic cycle of AmpC was found to be two-fold. First, they reduced the stability of the enzyme, which presumably increased its flexibility. This appeared to accelerate deacylation of the enzyme-bound β-lactam, which resulted in greater catalytic efficiencies towards ceftolozane and ceftazidime. Second, these mutations reduced the affinity of avibactam for AmpC by increasing the apparent activation energy barrier of the enzyme acylation step. The catalytic turnover of ceftolozane and ceftazidime was not influenced by this significantly, as deacylation was found to be the rate-limiting step for the breakdown of these antibiotics. It is remarkable that these mutations enhance the catalytic efficiency of AmpC towards ceftolozane and ceftazidime while simultaneously reducing susceptibility to inhibition by avibactam. It is our hope that the knowledge gained from the molecular analysis of these and other AmpC resistance mutants will aid the design of β-lactams and BLIs with reduced susceptibility to mutational resistance.
Microbiology, Enzymology, Antibiotic resistance