How Blocking Lipopolysaccharide Synthesis and Transport Affects Cell Survival

Abstract

Gram-negative bacteria are defined by their asymmetric second membrane. The outer leaflet, exposed to the cell surface, is composed of lipopolysaccharide (LPS), a large glycolipid. Conserved structural features of this molecule allow it to create a strong permeability barrier that blocks entry to many compounds, including antibiotics, making Gram-negative infections particularly difficult to treat in the clinic. LPS synthesis and transport to the cell surface is essential in most Gram-negative species, making it an attractive target for antimicrobial development, though no known clinically used antibiotics currently target either of these pathways. There are currently a small number of Gram-negative species, such as Acinetobacter baumannii, that can survive in the absence of LPS; however, what allows them, and not others, to do so is not well understood. This work establishes that polymyxin B (PMB), a critical antibiotic of last resort, targets LPS transport to the cell surface as its mechanism of killing. PMB is known to bind LPS at the cell surface, which permeablizes the cell, but the understanding of its mechanism of action has been vague and nonspecific. Through a biochemical reconstitution of LPS transport, in vivo crosslinking experiments, and imaging studies, we provide evidence that PMB binds LPS at the inner membrane and prevents entry into LptFG, which are components of the Lpt machine that extract LPS from the inner membrane and transport and insert it into the outer membrane. This causes a specific build-up of LPS in the inner membrane which leads to cell death. This dissertation also characterizes the phenotypes associated with LPS loss in A. baumannii and defines conditions in which growth and morphological defects are minimal. Using detailed growth analysis in a variety of conditions we develop a hypothesis about why LPS loss is tolerated in A. baumannii, but not other species such as Escherichia coli. We propose a model for how an outer membrane must be built without LPS and explain why we believe this to be rate limiting for growth. If our hypothesis is correct, we believe we can find conditions that will enable us to engineer an E. coli strain that can survive without LPS.