Probing the mechanism of the bacterial Lipid II flippase, MurJ

Abstract

Antimicrobial resistance (AMR) describes the ability of microorganisms such as bacteria to become insensitive to the growth-curbing effects of antibiotics. Resistant strains are able to grow and divide in the presence of these drugs, posing a significant threat to otherwise healthy patients. As AMR spreads across nations and continents, it has quickly become one of the most pressing global health issues of the 21st century. AMR is a process which is expected to occur naturally over time in bacterial populations, but has been accelerated by the misuse and overuse of antibiotics in the clinic. Up until recently, newly discovered classes of antibiotics allowed us to keep pace with growing resistance, however the past two decades have seen such attempts reach a standstill. This is mainly attributable to the fact that current antibiotics all target the same protein classes with known mechanisms of resistance. It is commonly understood that in order to curb rising AMR, new protein targets need to be discovered and studied in order to design new classes of small molecules. One of these promising new targets is a protein called MurJ, which plays a critical role in the construction of the bacterial cell wall. MurJ transports the precursor to the cell wall, Lipid II, from its site of synthesis (the cytoplasm) to its eventual site of polymerization (the periplasm). In addition to being essential, MurJ is highly conserved across all bacteria that make a cell wall. MurJ was discovered in 2008—relatively recently compared to the other proteins in its biosynthetic pathway. Since then, a number of studies have elucidated its three-dimensional structure and identified some functionally important residues. However, very little is known about the mechanistic details of how it carries out its catalytic activity. In this thesis, I describe the development of a novel cross-linking methodology to capture intermediates of Lipid II flipping on MurJ in living cells. Being able to capture such intermediates, and comparing their relative amounts across different mutants, allowed me to identify a key energetic bottleneck in the flipping process, and to implicate a set of essential residues in this key step. These residues had previously been hypothesized to participate in the extraction of the substrate from the membrane, a step which I show to be independent of the presence of said residues. In collaboration with Dr. Kumar from the lab of Prof. Natacha Ruiz (Ohio State), I also investigated the energy source powering Lipid II transport across the membrane. We found that the homolog from Thermosipho africanus requires chloride ions to function, but that the same dependency is absent in the homolog from Escherichia coli. Importantly, it appears as if chloride plays the same role as Lipid II (albeit in reverse), by facilitating the conformational transition of MurJ through an otherwise high-energy state. Last but not least, I describe the discovery of the first known interaction partner of MurJ, a divisome protein called DamX. I describe my efforts to purify and obtain a structure of the complex, and to test the hypothesis that DamX recruits MurJ to sites of active cell wall synthesis. In summary, my thesis work covers diverse aspects of MurJ physiology, ranging from atomic-level studies of substrate engagement and energetics to broader questions of cellular-level localization. I hope that the newly developed tools and paradigms outlined herein can eventually be used to exploit MurJ as a novel target for the development of a new class of broad-spectrum antibiotics.