A Model for the Regulation of Lipopolysaccharide Synthesis during Outer Membrane Biogenesis in Escherichia Coli

Abstract

The role of systems biology in the interpretation and analysis of important biological events is gaining rapid acceptance in a number of biological fields. Here a computational systems approach was applied to investigate the production and regulation of Escherichia coli’s (E. coli) outer membrane. The outer membrane comprises of phospholipids in the inner leaflet, and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans and its biosynthesis is in part, regulated via degradation of LpxC and WaaA enzymes by the protease FtsH. Despite a substantial amount of research conducted on LPS synthesis, there is remarkably little information on its regulation. The model of the outer membrane synthesis was completed in two phases; firstly a model of lipid A (representing the LPS pathway) was constructed followed by an integrated pathway model which incorporated fatty acids biosynthesis pathway (representing phospholipid production). The parameters used to construct the model were derived from published datasets where available, and estimated when necessary prior to model fitting. Model validation was carried out using a combination of published datasets alongside subsequent experimental data from this research. Model findings suggested that the FtsH-mediated LpxC degradation signal arises from levels of lipid A disaccharide, the substrate for LpxK. This was subsequently validated experimentally using an lpxK overexpression system. Analysis of the integrated model further refined this mechanism indicating the catalytic activity of LpxK appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the fatty acids and lipid A biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration, but could be induced in vitro by palmitic acid. The biological relevance of this acute mechanism is not obvious; however, experiments aimed at causing abrupt damage to the cell wall or membrane (by antimicrobials) suggest that under conditions which directly damage membrane structure, LPS regulation via this unidentified protease may be crucial. Computational analysis into the regulation of WaaA suggested that its proteolytic regulation does not affect the LPS synthetic rate. Subsequent experimental analysis provided evidence that WaaA regulation is aimed at controlling the quality of LPS synthesized by preventing glycosylation of undesirable lipid acceptors. Overexpression of waaA resulted in increased levels of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) sugar whereas, levels of heptose were not elevated in comparison to non-overexpressed cells. This implies that an uncontrolled production of WaaA does not increase LPS level but rather re-glycosylates lipid A precursors. This is the first time experimental data has been produced attempting to explain the regulation of WaaA. Computation of flux coefficient indicates that LpxC is the rate-limiting step when pathway regulation is ignored, but LpxK becomes the limiting step if feedback regulation is included as it is in vivo. Thus, in contrast to LpxC, LpxK may represent a more appropriate target for novel drug development. Overall, the findings of this work provide novel insights into the complex biogenesis of the E. coli outer membrane.