Massive antibody discovery used to probe structure–function relationships of the essential outer membrane protein LptD

The overuse and misuse of antibiotics has led to the rise of multi-drug resistant bacteria which threaten global public health. Antibiotics interfere with essential processes in bacteria so they are unable to divide or survive, but over time, the microbes have found ways to become immune to the drugs. New antibiotics are now desperately needed. Gram-negative bacteria are wrapped in an outer membrane made of large molecules called lipopolysaccharides. This structure is an extra barrier to molecules (such as drugs) that try to enter the cell, but it could also hold new targets for antibiotics to exploit. A protein called LptD is embedded in the outer membrane, where it inserts new lipopolysaccharides. It is critical for bacteria to grow and survive, and is a relatively new potential target for antibiotic development. The protein has a number of ‘extracellular loops’ that extend into the environment, but their roles in the structure and the activity of LptD are still largely unknown. This is partly due to a lack of tools to investigate these elements. In response, Storek et al. built a library of over 3,000 custom antibodies, which are small Y-shaped proteins that can each recognise a specific portion in one of the extracellular loops and potentially incapacitate LptD. The antibodies were used to target LptD in its native environment, when it is embedded in the bacteria. In parallel, mutant bacteria were created in which the loops were genetically removed one by one to assess their importance for LptD activity. The experiments revealed that although the antibodies could target most extracellular loops, they could not target the few loops that were essential for LptD to work properly. This suggests that antibody-accessible loops are expendable and that these structures could serve to shield other regions of LptD which are critical for survival. The findings will help to prioritise research that develops other approaches to inhibit LptD. Finally, the antibody workflow designed by Storek et al. can serve as a road map to study other membrane proteins in their native cellular environment.