A High-Affinity Nanobody Selectively Recognizing KPC-2/KPC-3: Biochemical and Structural Insights.

Area of Science:

• Microbiology
• Biotechnology
• Immunology

Background:

• Carbapenemase-producing Enterobacteriaceae (CPE) are a major global health concern, driven by resistance to last-resort antibiotics.
• The KPC-3 variant is a prevalent carbapenemase, necessitating rapid detection methods for effective clinical management.
• Conventional antibodies face limitations in stability and epitope access, highlighting the need for alternative detection tools.

Purpose of the Study:

• To generate and characterize a nanobody (VHH) with high specificity for the KPC-3 carbapenemase.
• To evaluate the nanobody's potential as a diagnostic tool for detecting KPC-producing bacteria.
• To explore the nanobody's binding characteristics and potential for future therapeutic applications.

Main Methods:

• Construction and screening of an immune VHH phage display library against KPC-3.
• Validation of nanobody binding using ELISA and Western blot assays.
• Analysis of nanobody recognition of KPC variants in bacterial periplasmic extracts and structural modeling.

Main Results:

• A nanobody targeting KPC-3 was successfully generated and validated.
• The nanobody demonstrated high binding affinity for both KPC-2 and KPC-3 variants.
• Structural modeling indicated favorable interaction surfaces for nanobody binding.

Conclusions:

• This study presents the first nanobody targeting KPC-3, capable of recognizing a conserved epitope shared by KPC-2 and KPC-3.
• The developed nanobody shows significant promise as a molecular tool for detecting KPC variants in clinical settings.
• Further affinity maturation of this nanobody could lead to novel therapeutic strategies against carbapenemase-producing bacteria.