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Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter

Abstract

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and β-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml−1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100–200 nM (1.5–3.0 µg ml−1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and β-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

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All data generated or analyzed during this study are included in this article and its supplementary figures and can be provided by the corresponding author upon request.

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Acknowledgements

We are grateful to Dr. Vijay Satav of Dr. Satav’s pathological laboratories for providing us with the clinical samples of P aeruginosa as well as the other pathogens for developing and testing the specificity of the camelid antibodies. Late Mr. Anil Nahar for constant encouragement and funding to build up the infrastructure required for the execution of the project. The authors acknowledge Mrs. Manisha Sabnis, Agastya Suresh, and Rija Nada for the initial development of the phage display and protein purification protocols, Dr. Aditi Ambekar for the critical evaluation of the manuscript and Siddharth Umarje for artistic depiction of the hypothesis.

Funding

This work was partially funded by BIRAC (Govt of India) under SIBRI BT/SIBRI 1358/30/16 and Bill and Melinda Gates Foundation, Grand Challenges Grant GCE-INDIA/R4/2018/007.

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Authors

Contributions

SB formulated the concept and the therapeutic approach and oversaw the overall planning of the project. AN, PP, and MB developed the protein purification strategies and executed the animal studies. MS developed the VHH libraries and identified the hits and conducted the cell-based assays. MK conducted the in vitro microbiology assays. All authors read and approved the manuscript.

Corresponding author

Correspondence to Sanjiban K. Banerjee.

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Conflict of interest

AN declares that he has no conflict of interest or competing interests. MS declares that she has no conflict of interest. MK, PP, and MB declare no conflict of interest. SB declares that he has no conflict of interest.

Ethics approvals and consent to participate

All applicable international, national, and/or institutional guidelines were followed. Experiments in AbGenics were carried out with Institutional Biosafety Committee Approvals. All animal experiments were approved by the ethics committee of PRADO Preclinical Research and Development Organisation (India) with all relevant ethical regulations for animal testing.

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Nagraj, A.K., Shukla, M., Kulkarni, M. et al. Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter. J Antibiot (2024). https://doi.org/10.1038/s41429-024-00748-w

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