The Purification and Antibiotic Inhibition of L,D-transpeptidase in Enterobacter cloacae
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Date
2023-12
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The Ohio State University
Abstract
β-lactam antibiotics such as penicillin have been particularly important in modern medicine through their ability to disrupt cross-linkages within bacterial cell walls. They work by binding to penicillin-binding proteins, or PBPs, which prevents cross-linkage between peptide strands of the cell wall from occurring, resulting in an overall weaker cell wall which subsequently kills bacterial cells. Many bacterial species, such as Enterobacter cloacae, also express several L,D-transpeptidases or Ldts, which can also cross-link peptidoglycan through an alternative pathway and are not as easily recognized by β-lactams. Based on these properties of the bacterial cell wall, it can be hypothesized that the presence of Ldts influence the effectiveness of β-lactam antibiotics. This project aims to quantify the activity of LdtE from E. cloacae in the presence of different β-lactam antibiotics. LdtE was over-expressed and purified as a recombinant protein from an E. coli expression system. Nitrocefin was used to quantify enzyme activity and inhibition in the presence of various β-lactam antibiotics. Through quantification of these interactions, it was determined that meropenem and nafcillin had the greatest inhibitory effect on LdtE. While a related carbapenem, imipenem, showed no inhibition, suggesting that the substituents on the carbapenem core play a critical role in enabling Ldt inhibition. Based on that data, we will also determine how the Ldts effect β-lactam susceptibility in E. cloacae by knocking out the genes and then performing assays to determine the minimum-inhibitory concentrations of antibiotic necessary to inhibit bacterial growth.
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Keywords
Antibiotic resistance, L,D-transpeptidase, β-lactam, Biochemistry, Enterobacter cloacae