Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

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 part...

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Autores principales:	Mina Mostafavi, Lisha Wang, Lili Xie, Kenneth T. Takeoka, Daryl L. Richie, Fergal Casey, Alexey Ruzin, William S. Sawyer, Christopher M. Rath, Jun-Rong Wei, Charles R. Dean
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	LpxC fabZ lipid A toxic accumulation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/1e5ff2b427e345449955f5246fb5ce69
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spelling	oai:doaj.org-article:1e5ff2b427e345449955f5246fb5ce692021-11-15T15:22:26ZInterplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis10.1128/mSphere.00508-182379-5042https://doaj.org/article/1e5ff2b427e345449955f5246fb5ce692018-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00508-18https://doaj.org/toc/2379-5042ABSTRACT 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. IMPORTANCE Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.Mina MostafaviLisha WangLili XieKenneth T. TakeokaDaryl L. RichieFergal CaseyAlexey RuzinWilliam S. SawyerChristopher M. RathJun-Rong WeiCharles R. DeanAmerican Society for MicrobiologyarticleLpxCfabZlipid Atoxic accumulationMicrobiologyQR1-502ENmSphere, Vol 3, Iss 5 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	LpxC fabZ lipid A toxic accumulation Microbiology QR1-502
spellingShingle	LpxC fabZ lipid A toxic accumulation Microbiology QR1-502 Mina Mostafavi Lisha Wang Lili Xie Kenneth T. Takeoka Daryl L. Richie Fergal Casey Alexey Ruzin William S. Sawyer Christopher M. Rath Jun-Rong Wei Charles R. Dean Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis
description	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. IMPORTANCE Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.
format	article
author	Mina Mostafavi Lisha Wang Lili Xie Kenneth T. Takeoka Daryl L. Richie Fergal Casey Alexey Ruzin William S. Sawyer Christopher M. Rath Jun-Rong Wei Charles R. Dean
author_facet	Mina Mostafavi Lisha Wang Lili Xie Kenneth T. Takeoka Daryl L. Richie Fergal Casey Alexey Ruzin William S. Sawyer Christopher M. Rath Jun-Rong Wei Charles R. Dean
author_sort	Mina Mostafavi
title	Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis
title_short	Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis
title_full	Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis
title_fullStr	Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis
title_full_unstemmed	Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis
title_sort	interplay of <named-content content-type="genus-species">klebsiella pneumoniae</named-content> <italic toggle="yes">fabz</italic> and <italic toggle="yes">lpxc</italic> mutations leads to lpxc inhibitor-dependent growth resulting from loss of membrane homeostasis
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/1e5ff2b427e345449955f5246fb5ce69
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Interplay of <named-content content-type="genus-species">Klebsiella pneumoniae</named-content> <italic toggle="yes">fabZ</italic> and <italic toggle="yes">lpxC</italic> Mutations Leads to LpxC Inhibitor-Dependent Growth Resulting from Loss of Membrane Homeostasis

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