Mostafavi, Mina, Wang, Lisha, Xie, Lili, Takeoka, Ken, Richie, Daryl, Casey, Fergal, Ruzin, Alexey, Sawyer, William, Rath, Christopher, Wei, Jun-Rong and Dean, Charles (2018) Interplay of Klebsiella pneumoniae fabZ and lpxC Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis. mSphere.

Abstract

Tight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging of Klebsiella pneumoniae in increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations in fabZ and lpxC occurring together (encoding either FabZR121L/LpxCV37G or FabZF51L/LpxCV37G). K. pneumoniae mutants having only LpxCV37G or LpxCV37A or various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37G accumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37G indicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37G was overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.

Item Type:	Article
Date Deposited:	20 Nov 2018 00:45
Last Modified:	20 Nov 2018 00:45
URI:	https://oak.novartis.com/id/eprint/37490