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Abstract

Background

M. abscessus (Mab) is among the most clinically challenging pathogens. There are significant gaps remaining in mechanism of β-lactams action in Mab. β-Lactams preferentially inactivate one or multiple different L,D-Transpeptidases (LDTs) and penicillin-binding proteins (PBPs). Founded on our LDT binding data and in vitro synergistic efficacy of imipenem (IPM), ceftaroline (CFT), and amoxicillin (AMX) in Mab, this study aimed to identify binding affinities, conformational changes and the thermal stability of five LDTs and PBPs via β-lactam binding for further structure-activity relationship.

Methods

Binding affinity was measured by kinetic studies, MS analysis, or Bocillin-FL inhibition assay. We investigated changes in the secondary structure of five LDTs (LDT1-5), d,d-carboxypeptidase, PBPB, and PBP-lipo in response to bind of IPM, CFT, and AMX using circular dichroism (CD) spectroscopy in the far-UV region. To assess the thermal stability of these LDTs, we employed a Differential Scanning Fluorimetry (DSF) assay.

Results
CD analysis revealed a significant negative shift of LDT1, LDT2, and PBPB upon binding with either single or combination of imipenem (IPM) and ceftriaxone (CFT), particularly in the 210-220 nm range. Using DSF, IPM and CFT binding to LDTs, DDC and PBPB were found to alter the thermal stability of these protein enzymes. IPM induced a decrease in the melting temperature (Tm) in the range of 2.6-6.8 ˚C for LDTs and PBPB, while caused an increase in Tm for DDC. CFT caused decrease in Tm in the range of 1.4-3 ˚C for LDTs but increase in Tm for DDC and PBPB. In contrast, amoxicillin (AMX) only targeted DDC, PBPB, and PBP-lipo and caused changes in Tm. The magnitude of the change in Tm varied between combinations and single β-lactams. Although there was no evidence of a correlation between changes in protein stability upon ligand binding and ligand binding affinity, IPM with high binding affinity (lower value of Ki,app) resulted in larger decreases in Tm compared to CFT.
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Conclusion

β-Lactam binding in single and combination leads to distinct structural changes of LDTs and PBPs, likely reflecting synergistic interactions between the two β-lactams. Further crystallography will be required to gain a better understanding of the induced structural changes.

Disclosures

Robert A. bonomo, MD, Entasis, Merck, VenatoRx, Wockhardt: Grant/Research Support

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Author notes

Session: 248. Treatment of Antimicrobial-Resistant Infections

Saturday, October 14, 2023: 12:15 PM

© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
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