Characterizing a feedback signal balancing lipopolysaccharide and glycerophospholipid synthesis in Gram negative bacteria

ABSTRACT Due to increasing antibiotic resistance, infectious bacteria once again pose a direct threat to health. Gram- negative pathogens are particularly dangerous as these organisms are intrinsically resistant to many antibiotics. This resistance arises from the asymmetric outer membrane of the Gram-negative cell envelope, with glycerophospholipids (GPL) in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. The impermeability of the outer membrane is however extremely vulnerable to imbalances between synthesis and transport of GPL and LPS. Still, there remains a major gap in our understanding regarding how the cell enforces the appropriate GPL/LPS balance. Recent findings in Escherichia coli point to two proteins, YejM and YciM, that use LPS transiting through the periplasm (periplasmic LPS) as a homeostatic signal to regulate the proteolysis of LpxC, the key enzyme in LPS synthesis. We hypothesize that many signals proposed previously must indirectly modulate LpxC proteolysis by altering periplasmic LPS levels, and thus are detected through the YejM-YciM system. In Aim 1, we will continue our development of a novel analytical tool for quantifying LPS transport intermediates, including periplasmic LPS. Parallel quantification of LpxC and LPS metabolic precursors by mass spectrometry will elucidate the kinetics of the YejM-YciM system and identify the influence of upstream metabolism on LPS synthesis and LpxC degradation. Aim 2 will investigate whether previously reported stimuli (e.g. exogenous fatty acids, envelope stress) that alter LPS synthesis also work via the YejM-YciM signaling system. Our findings will potentially clarify our understanding of GPL/LPS balancing by indicating that periplasmic LPS acts as the main regulator of LpxC proteolysis. Furthermore, the data-drive quantitative models developed here can be used to predict effects of antibiotics on cell envelope integrity. The YejM-YciM regulatory system is found in numerous pathogens, therefore our findings will be broadly applicable. Furthermore, the tool can be used to determine whether periplasmic (or cytoplasmic) LPS plays a role in Gram-negative pathogens that lack the YejM-YciM system.