Cargando…

LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates

Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. However, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway) prevented growth of this strain due to toxic accumulation of lipid A pathway interme...

Descripción completa

Detalles Bibliográficos
Autores principales: Wei, Jun-Rong, Richie, Daryl L., Mostafavi, Mina, Metzger, Louis E., Rath, Christopher M., Sawyer, William S., Takeoka, Kenneth T., Dean, Charles R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555675/
https://www.ncbi.nlm.nih.gov/pubmed/28815210
http://dx.doi.org/10.1128/mSphere.00199-17
_version_ 1783256956015738880
author Wei, Jun-Rong
Richie, Daryl L.
Mostafavi, Mina
Metzger, Louis E.
Rath, Christopher M.
Sawyer, William S.
Takeoka, Kenneth T.
Dean, Charles R.
author_facet Wei, Jun-Rong
Richie, Daryl L.
Mostafavi, Mina
Metzger, Louis E.
Rath, Christopher M.
Sawyer, William S.
Takeoka, Kenneth T.
Dean, Charles R.
author_sort Wei, Jun-Rong
collection PubMed
description Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. However, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway) prevented growth of this strain due to toxic accumulation of lipid A pathway intermediates. Here, we explored whether a similar phenomenon occurred with depletion of LpxK, a kinase that phosphorylates disaccharide 1-monophosphate (DSMP) at the 4′ position to yield lipid IV(A). An A. baumannii ATCC 19606 derivative with LpxK expression under the control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-regulated expression system failed to grow without induction, indicating that LpxK is essential for growth. Light and electron microscopy of LpxK-depleted cells revealed morphological changes relating to the cell envelope, consistent with toxic accumulation of lipid A pathway intermediates disrupting cell membranes. Using liquid chromatography-mass spectrometry (LCMS), cellular accumulation of the detergent-like pathway intermediates DSMP and lipid X was shown. Toxic accumulation was further supported by restoration of growth upon chemical inhibition of LpxC (upstream of LpxK and the first committed step of lipid A biosynthesis) using CHIR-090. Inhibitors of fatty acid synthesis also abrogated the requirement for LpxK expression. Growth rescue with these inhibitors was possible on Mueller-Hinton agar but not on MacConkey agar. The latter contains outer membrane-impermeable bile salts, suggesting that despite growth restoration, the cell membrane permeability barrier was not restored. Therefore, LpxK is essential for growth of A. baumannii, since loss of LpxK causes accumulation of detergent-like pathway intermediates that inhibit cell growth. IMPORTANCE Acinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A. baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials.
format Online
Article
Text
id pubmed-5555675
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher American Society for Microbiology
record_format MEDLINE/PubMed
spelling pubmed-55556752017-08-16 LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates Wei, Jun-Rong Richie, Daryl L. Mostafavi, Mina Metzger, Louis E. Rath, Christopher M. Sawyer, William S. Takeoka, Kenneth T. Dean, Charles R. mSphere Research Article Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. However, we previously reported that depletion of LpxH (the fourth enzyme in the lipid A biosynthetic pathway) prevented growth of this strain due to toxic accumulation of lipid A pathway intermediates. Here, we explored whether a similar phenomenon occurred with depletion of LpxK, a kinase that phosphorylates disaccharide 1-monophosphate (DSMP) at the 4′ position to yield lipid IV(A). An A. baumannii ATCC 19606 derivative with LpxK expression under the control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-regulated expression system failed to grow without induction, indicating that LpxK is essential for growth. Light and electron microscopy of LpxK-depleted cells revealed morphological changes relating to the cell envelope, consistent with toxic accumulation of lipid A pathway intermediates disrupting cell membranes. Using liquid chromatography-mass spectrometry (LCMS), cellular accumulation of the detergent-like pathway intermediates DSMP and lipid X was shown. Toxic accumulation was further supported by restoration of growth upon chemical inhibition of LpxC (upstream of LpxK and the first committed step of lipid A biosynthesis) using CHIR-090. Inhibitors of fatty acid synthesis also abrogated the requirement for LpxK expression. Growth rescue with these inhibitors was possible on Mueller-Hinton agar but not on MacConkey agar. The latter contains outer membrane-impermeable bile salts, suggesting that despite growth restoration, the cell membrane permeability barrier was not restored. Therefore, LpxK is essential for growth of A. baumannii, since loss of LpxK causes accumulation of detergent-like pathway intermediates that inhibit cell growth. IMPORTANCE Acinetobacter baumannii is a Gram-negative pathogen for which new therapies are needed. The lipid A biosynthetic pathway has several potential enzyme targets for the development of anti-Gram-negative agents (e.g., LpxC). However, A. baumannii ATCC 19606 can grow in the absence of LpxC and, correspondingly, of lipid A. In contrast, we show that cellular depletion of LpxK, a kinase occurring later in the pathway, inhibits growth. Growth inhibition results from toxic accumulation of lipid A pathway intermediates, since chemical inhibition of LpxC or fatty acid biosynthesis rescues cell growth upon loss of LpxK. Overall, this suggests that targets such as LpxK can be essential for growth even in those Gram-negative bacteria that do not require lipid A biosynthesis per se. This strain provides an elegant tool to derive a better understanding of the steps in a pathway that is the focus of intense interest for the development of novel antibacterials. American Society for Microbiology 2017-07-26 /pmc/articles/PMC5555675/ /pubmed/28815210 http://dx.doi.org/10.1128/mSphere.00199-17 Text en Copyright © 2017 Wei et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Wei, Jun-Rong
Richie, Daryl L.
Mostafavi, Mina
Metzger, Louis E.
Rath, Christopher M.
Sawyer, William S.
Takeoka, Kenneth T.
Dean, Charles R.
LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates
title LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates
title_full LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates
title_fullStr LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates
title_full_unstemmed LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates
title_short LpxK Is Essential for Growth of Acinetobacter baumannii ATCC 19606: Relationship to Toxic Accumulation of Lipid A Pathway Intermediates
title_sort lpxk is essential for growth of acinetobacter baumannii atcc 19606: relationship to toxic accumulation of lipid a pathway intermediates
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555675/
https://www.ncbi.nlm.nih.gov/pubmed/28815210
http://dx.doi.org/10.1128/mSphere.00199-17
work_keys_str_mv AT weijunrong lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT richiedaryll lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT mostafavimina lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT metzgerlouise lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT rathchristopherm lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT sawyerwilliams lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT takeokakennetht lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates
AT deancharlesr lpxkisessentialforgrowthofacinetobacterbaumanniiatcc19606relationshiptotoxicaccumulationoflipidapathwayintermediates