Directed Evolution of Cefepime and Imipenem Resistance in P99 Cephalosporinase by In Vitro Mutagenesis Bukhari, S.M. and Lefurgy, S.T. Department of Chemistry, Hofstra University, Hempstead, NY 11549, USA
Abstract
Methods/Results
• Directed evolution of cefepime and imipenem resistance in bacteria will increase understanding of how class C cephalosporinases evolve antibiotic resistance. • epPCR paired with megaprimer whole plasmid PCR facilitates rapid generation of point mutants and recombined genes. • Selection of resistant variants and susceptibility testing revealed the structural determinants of cefepime and imipenem resistance.
Background
Scheme 1. Structure of cefepime, a 4th-generation cephalosporin.
Scheme 2. Structure of imipenem, a carbapenem.
Conclusions/Future Directions 1. 2. 3. 4.
Protein purification & kinetics Individual/combination of mutations(s) in P99 Test mutants in wildtype background Selection on other carbapenems (ex: doripenem, ertapenem, meropenem, etc.)
IMI
A292T/ A292P
Figure 1. Error-prone PCR (epPCR) is used to amplify a gene fragment. DNA polymerase introduces mutations into the gene. The mutated gene fragment is purified. (5)
V278 L V350 N346IF
L293P
K6Q
Table 2. Mutations and their corresponding positions in four library ep5 clones that had a higher MIC for IMI than the parent plasmid. Ep5 refers to the fifth library created. All clones have an ins204Ser in the backbone. Mutations present in the literature are highlighted. (8)
Clone & Mutation
IMI
1
2
3
4
Q120K ins204Se r
M215I
T126A Q141L
Figure 3. X-ray crystal structure of wildtype AmpC bound to IMI (PDB: 1LL5). Mutations generated via mutagenesis for library ep2 that cause an amino acid change and lead to increased FEP resistance are highlighted. Ep2 refers to the second library created.
Clone & Mutation K6Q V16I
1
2
3
4
Figure 4. X-ray crystal structures of wildtype AmpC bound to IMI (PDB: 1LL5). Mutations generated via mutagenesis that cause an amino acid change and lead to increased IMI resistance are highlighted. A potential hydrogen bond between the Q120K mutation and IMI substrate is depicted. Q141 L
IMI
Q120 K
D123E
ins204S er
S130T M215I Figure 2. Megaprimer whole-plasmid PCR. epPCR product is extended using a parent plasmid as template. DpnI removes the methylated parent plasmid, leaving only plasmids with randomly mutagenized AmpC genes. (6)
⇑
Cefepime (FEP) and imipenem (IMI) are last-line β-lactam antibiotics that kill bacteria by inhibiting cell wall synthesis (1, 2). FEP resistance is still rare, but it is anticipated that β-lactamases will evolve through natural evolution to be able to hydrolyze FEP, leading to drug-resistant bacteria. Directed evolution mimics natural evolution in an iterative process of diversity generation and selection (3). DNA acquires mutations principally by copying errors (4). Single-base changes can lead to single-substitutions of amino acids; these rarely lead to changes in substrate specificity on their own, but accumulated mutations can increase turnover of antibiotics. This process is mimicked in the test tube using error-prone polymerase chain reaction (epPCR) to generate a library of P99 genes with random mutations. These genes are installed in an expression plasmid by using the epPCR product as megaprimer for whole-plasmid PCR (MEGAWHOP). To demonstrate the effectiveness of directed evolution, the evolution of FEP resistance was modeled in the laboratory. Mutant genes were generated in vitro and selected for resistance phenotypes in noninfectious bacteria (E. coli). To reach this goal, the P99 cephalosporinase from Enterobacter cloacae P99 was used to develop a standard in vitro directed evolution protocol. Natural evolution has not yet generated mutant class C cephalosporinases that have carbapenemase activity. Directed evolution can be used to determine if class C cephalosporinases can mutate and become resistant to carbapenems. After establishing a standard in vitro directed evolution protocol, P99 was mutated to select variants that increase resistance to IMI. Elucidating the structural changes that cause IMI resistance will help inform the design of new antibiotics that exploit these mutations.
FEP
Table 1. Mutations and their corresponding positions in four library ep4 clones that had a higher MIC for FEP than the parent plasmid. Ep4 refers to the fourth library created. All clones have an ins204Ser in the backbone. Mutations reported in the literature are highlighted. (7)
A246T
T126 A
V278L S287T V291E A292T L293P
Q120 K
IMI
V299A A305V N346I
References
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