Dataset Information

Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606.

ABSTRACT: The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl ?-d-1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3-O-[(R)-3-OH-C14]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A. baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth.

SUBMITTER: Richie DL

PROVIDER: S-EPMC4985137 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606.

Richie Daryl L DL Takeoka Kenneth T KT Bojkovic Jade J Metzger Louis E LE Rath Christopher M CM Sawyer William S WS Wei Jun-Rong JR Dean Charles R CR

PloS one 20160815 8

The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is es ...[more]

PMID: 27526195

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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.

Project description:Multidrug-resistant (MDR) Acinetobacter baumannii has emerged as a very problematic pathogen over the past decades, with a high incidence in nosocomial infections. Discovered in the late 1940s but abandoned in the 1970s, polymyxins (i.e., polymyxin B and colistin) have been revived as the last-line therapy against Gram-negative "superbugs," including MDR A. baumannii. Worryingly, resistance to polymyxins in A. baumannii has been increasingly reported, urging the development of novel antimicrobial therapies to rescue this last-line class of antibiotics. In the present study, we integrated genome-scale metabolic modeling with multiomics data to elucidate the mechanisms of cellular responses to colistin treatment in A. baumannii. A genome-scale metabolic model, iATCC19606, was constructed for strain ATCC 19606 based on the literature and genome annotation, containing 897 genes, 1,270 reactions, and 1,180 metabolites. After extensive curation, prediction of growth on 190 carbon sources using iATCC19606 achieved an overall accuracy of 84.3% compared to Biolog experimental results. Prediction of gene essentiality reached a high accuracy of 86.1% and 82.7% compared to two transposon mutant libraries of AB5075 and ATCC 17978, respectively. Further integrative modeling with our correlative transcriptomics and metabolomics data deciphered the complex regulation on metabolic responses to colistin treatment, including (i) upregulated fluxes through gluconeogenesis, the pentose phosphate pathway, and amino acid and nucleotide biosynthesis; (ii) downregulated TCA cycle and peptidoglycan and lipopolysaccharide biogenesis; and (iii) altered fluxes over respiratory chain. Our results elucidated the interplay of multiple metabolic pathways under colistin treatment in A. baumannii and provide key mechanistic insights into optimizing polymyxin combination therapy. IMPORTANCE Combating antimicrobial resistance has been highlighted as a critical global health priority. Due to the drying drug discovery pipeline, polymyxins have been employed as the last-line therapy against Gram-negative "superbugs"; however, the detailed mechanisms of antibacterial killing remain largely unclear, hampering the improvement of polymyxin therapy. Our integrative modeling using the constructed genome-scale metabolic model iATCC19606 and the correlative multiomics data provide the fundamental understanding of the complex metabolic responses to polymyxin treatment in A. baumannii at the systems level. The model iATCC19606 may have a significant potential in antimicrobial systems pharmacology research in A. baumannii.

Project description:The management of infections caused by Acinetobacter baumannii is hindered by its intrinsic tolerance to a wide variety of biocides. The aim of the study was to analyze the role of different A. baumannii efflux pumps (EPs) in tolerance to chlorhexidine (CHX) and benzalkonium (BZK) and identify non-toxic compounds, which can restore susceptibility to CHX and BZK in A. baumannii. A. baumannii ATCC 19606 strain was tolerant to both CHX and BZK with MIC and MBC value of 32 mg/L. CHX subMIC concentrations increased the expression of adeB and adeJ (RND superfamily), aceI (PACE family) and amvA (MFS superfamily) EP genes. The values of CHX MIC and MBC decreased by eightfold in ΔadeB and twofold in ΔamvA or ΔaceI mutants, respectively, while not affected in ΔadeJ mutant; EPs double and triple deletion mutants showed an additive effect on CHX MIC. CHX susceptibility was restored in double and triple deletion mutants with inactivation of adeB gene. BZK MIC was decreased by fourfold in ΔadeB mutant, and twofold in ΔamvA and ΔaceI mutants, respectively; EPs double and triple deletion mutants showed an additive effect on BZK MIC. BZK susceptibility was recovered in ΔadeB ΔaceI ΔadeJ and ΔamvA ΔadeB ΔadeJ triple mutants. The structural comparison of AdeB and AdeJ protomers showed a more negatively charged entrance binding site and F-loop in AdeB, which may favor the transport of CHX. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) EP inhibitor reduced dose-dependently CHX MIC in A. baumannii ATCC 19606 and in ΔadeJ, ΔaceI, or ΔamvA mutants, but not in ΔadeB mutant. Either piperine (PIP) or resveratrol (RV) at non-toxic concentrations inhibited CHX MIC in A. baumannii ATCC 19606 parental strain and EPs gene deletion mutants, and CHX-induced EP gene expression. Also, RV inhibited BZK MIC and EP genes expression in A. baumannii ATCC 19606 parental strain and EPs mutants. These results demonstrate that tolerance to CHX and BZK in A. baumannii is mediated by the activation of AdeB, AceI and AmvA EPs, AdeB playing a major role. Importantly, inhibition of EP genes expression by RV restores CHX and BZK susceptibility in A. baumannii.

Dataset Information

Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606.

Publications

Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606.

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