Exploring the therapeutic potential of cell state specific binders to the β-barrel assembly machine

Abstract

Gram-negative bacterial infections are particularly difficult to treat as the presence of the outer membrane (OM) makes them intrinsically resistant to many antibiotics used in the clinic. Understanding how the impermeable OM barrier is established might reveal novel antibiotic targets. One strategy that has been considered is to find targets that are exposed on the cell surface to circumvent the requirement to cross the OM. In fact, several of the protein machines required to assemble the OM have surface accessible regions. This thesis explores the potential to recognize the regions of one such machine, the β-Barrel Assembly Machine (Bam).

We leveraged our new, detailed understanding of Bam-mediated folding to isolate a nanobody binder to a late-stage folding intermediate state of Bam. Chapter 2 describes the development of an approach to isolate nanobodies capable of binding to Bam. This involved preparing several defined states of the Bam complex: a closed state incapable of folding substrates, a resting state capable of binding to and recognizing unfolded substrates, and an intermediate state of Bam engaged in the folding process. A nanobody yeast surface display library was used to isolate a nanobody capable of discriminating between these states.

In Chapter 3, we assessed whether this state-specific nanobody could recognize the complex in the context of a cell surface. To do this, we overexpressed two distinct states of Bam in the cell and showed that the nanobody bound to the cell surface only when we have stalled Bam. To probe what the nanobody is recognizing on the cell surface we developed a TR-FRET assay by labeling the stalled Bam substrate and the nanobody. These experiments establish that high affinity binding specifically occurs between the nanobody and the stalled complex.

Chapter 4 describes experiments to probe where slow folding substrates accumulate during folding on Bam. We probed where folding intermediates localize on the Bam complex using in vivo photocrosslinking. Significant regions of the slow folding substrate were shown to contact the interior wall of BamA and displayed a periodic pattern of crosslinking characteristic of nascent β-sheet structure. Since these stalled substrates make the OM permeable they provide proof of concept that stalling substrates while folding can cause OM defects that could be exploited in a therapeutic.

Chapter 5 integrates these proof of concept studies with an analysis of how we could use our state selective nanobody binders as therapeutics. It is proposed that binders that could stall WT substrates during folding on Bam might cause the OM to become permeable like our artificially stalled substrates.