Characterizing outer membrane protein biogenesis in Escherichia coli using a small-molecule inhibitor of BamA and its resistant mutation

Abstract

Gram-negative bacteria are especially resistant to antibiotics due to the presence of an additional layer to the cell envelope called the outer membrane. Outer membrane proteins (OMPs) are transmembrane proteins found in this outer membrane, and their synthesis and assembly are of great research interest, as disrupted OMP assembly causes cell death and could be a target for new antibiotics. The β-barrel assembly machinery (Bam) complex, which is composed of five subunits (BamA, B, C, D, and E), takes unfolded OMPs and assembles them for insertion into the outer membrane. The exact mechanism of this assembly is still not completely understood. The primary goal of this study was to identify compounds that target the Bam complex, as their interaction with the Bam complex could help better characterize the Bam mechanism and lead to novel antibiotic development. We conducted screens with small-molecule compounds that showed potential in disrupting OMP biogenesis. The compound TJS-300 killed Gram-negative bacteria that possessed functional efflux pumps and demonstrated increased potency against strains with disrupted OMP assembly, suggesting that it acts by interacting with and inhibiting BamA on the outer membrane surface. A resistant mutation to TJS-300, bamAE470K, does not alter BamA levels or cause activation of the σE stress response, suggesting that it causes only a conformational change in BamA and does not confer resistance via any change in function. Thus, TJS-300 most likely directly inhibits BamA on the outer membrane surface. This study contributes to our understanding of the mechanism of OMP biogenesis and to development of potential antibiotics that target this pathway.