Dataset Information

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport.

ABSTRACT: The asymmetric outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier to the environment. Perturbations to OM lipid asymmetry sensitize the cell to antibiotics. As such, mechanisms involved in lipid asymmetry are fundamental to our understanding of OM lipid homeostasis. One such mechanism, the Maintenance of lipid asymmetry (Mla) pathway has been proposed to extract mislocalized glycerophospholipids from the outer leaflet of the OM and return them to the inner membrane (IM). Work on this pathway in Acinetobacter baumannii support conflicting models for the directionality of the Mla system being retrograde (OM to IM) or anterograde (IM to OM). Here, we show conclusively that A. baumannii mla mutants exhibit no defects in anterograde transport. Furthermore, we identify an allele of the GTPase obgE that is synthetically sick in the absence of Mla; providing another link between cell envelope homeostasis and stringent response.

SUBMITTER: Powers MJ

PROVIDER: S-EPMC7500953 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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The Mla pathway in <i>Acinetobacter baumannii</i> has no demonstrable role in anterograde lipid transport.

Powers Matthew J MJ Simpson Brent W BW Trent M Stephen MS

eLife 20200903

The asymmetric outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier to the environment. Perturbations to OM lipid asymmetry sensitize the cell to antibiotics. As such, mechanisms involved in lipid asymmetry are fundamental to our understanding of OM lipid homeostasis. One such mechanism, the Maintenance of lipid asymmetry (Mla) pathway has been proposed to extract mislocalized glycerophospholipids from the outer leaflet of the OM and return them to the inne ...[more]

PMID: 32880370

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Project 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 IVA. 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. IMPORTANCEAcinetobacter 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.

Dataset Information

The Mla pathway in Acinetobacter baumannii has no demonstrable role in anterograde lipid transport.

Publications

The Mla pathway in <i>Acinetobacter baumannii</i> has no demonstrable role in anterograde lipid transport.

Similar Datasets

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