Abstract
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.
Highlights
Acinetobacter baumannii American Type Culture Collection (ATCC) 19606 can grow without lipid A, the major component of lipooligosaccharide
In Escherichia coli, where this metabolic pathway is best understood, lipid A biosynthesis is initiated by enzymes LpxA, LpxC, and LpxD, which together catalyze the addition of two -hydroxyacyl chains to UDP-GlcNAc to form UDP-2,3-diacyl-GlcN
A. baumannii ATCC 19606 is capable of growth under standard laboratory conditions in the absence of LOS, and the LpxA, LpxC, and LpxD enzymes that catalyze early steps of lipid A biosynthesis were shown to be dispensable for growth [11, 28]
Summary
Acinetobacter baumannii ATCC 19606 can grow without lipid A, the major component of lipooligosaccharide. 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. Such LPS-deficient organisms are hypersusceptible to multiple antibiotics; inhibitors of lipid A biosynthesis may be expected to potentiate the activity of many antibiotics, opening the possibility of combination therapies Enzymes such as LpxA and LpxC are not essential in A. baumannii ATCC 19606, at least under standard laboratory conditions, other enzymes in the lipid A biosynthetic pathway may be essential for growth since several pathway intermediates, such as lipid X and UDP-2,3-diacyl-GlcN (which have critical micelle concentrations below 25 M), are detergent-like [26] and blocking certain pathways steps might cause the toxic accumulation of these intermediates. The growth defect due to LpxH depletion was ameliorated by chemical inhibition of LpxC, upstream of LpxH, presumably by blocking the synthesis of detergent-like intermediates and thereby preventing their accumulation [27]
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