Reconstitution of Machinery Involved in Cell Envelope Biogenesis to Understand its Regulation and Inhibition

Abstract

Antibiotic resistance is a growing public health concern. Many pathogenic strains are acquiring resistance to our current scope of antibiotics. To overcome this resistance, we can either find new antibiotic targets or modify the activity of the current classes of antibiotics. By developing a suite of biochemical reconstitutions, we herein report the progress we have made on both axes towards the machinery involved in cell envelope biogenesis. In the first set of projects, we examine the regulation of lipopolysaccharide (LPS) transport by its essential seven protein transenvelope bridge. We start by characterizing a recently reported mutation that allows a laboratory strain to dispense with LptC, one of the seven broadly conserved proteins in the bridge. We show this mutation is unable to form stable bridges in vitro using a novel flow cytometry based proteoliposome assay. We then develop a method to measure the initial rate of LPS transport through the seven-protein bridge in vitro by leveraging the binding affinity for LPS of polymyxin B nonapeptide. This reconstitution demonstrated that the amino acid identity of LptE affects the rate of transport in vitro. We explore this observation further by examining the biology of cells with defective LptE. We find that the identified LPS transport defective mutants result in reduced flux of LPS in vivo as well. Further we find that this defect is aggravated by the expression of a truncated LptC, suggesting the coordination of LPS extraction (controlled by LptC) and translocation (controlled by LptE). Briefly, we begin to characterize the drug thanatin using our novel reconstitutions. In the second project we detail advancements in understanding the mechanism of inhibition of cell wall glycopeptides. These drugs, which bind to peptidoglycan, have long been thought to share a similar mechanism. However, we show that teicoplanin has a novel mechanism of inhibition against S. aureus PBP2 and does not share the canonical mechanism with vancomycin. We find that this inhibition results in the termination of glycan strand polymerization after four couplings, turning Lipid II into Lipid VIII. This is followed by the first measurement of binding affinity between these drugs and full-length Lipid II targets. Further, we find that the relationship between structural features and mechanism of inhibition is more nuanced than previously reported. Notably, glycopeptide lipidation is not necessary for teicoplanin-like inhibition. Finally, we report that the drug-target interaction has emergent interactions with the enzyme catalyst that are important for inhibition; notably, when we treat a different class of glycosyltransferase proteins (SEDS-bPBP) we find that vancomycin and teicoplanin have swapped mechanisms.