Polymyxin-Induced Lipid A Deacylation in Pseudomonas aeruginosa

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Polymyxin-induced Lipid A Deacylation in Pseudomonas aeruginosa Perturbs Polymyxin Penetration and Confers High-level Resistance Mei-Ling Han, Tony Velkov, Yan Zhu, Kade D. Roberts, Anton P. Le Brun, Seong Hoong Chow, Alina D. Gutu, Samuel M. Moskowitz, Hsin-Hui Shen, and Jian Li ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00836 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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Polymyxin-induced Lipid A Deacylation in Pseudomonas aeruginosa Perturbs

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Polymyxin Penetration and Confers High-level Resistance

3 4

Mei-Ling Han1,2, Tony Velkov1,3, Yan Zhu2, Kade D. Roberts1, Anton P. Le Brun4, Seong Hoong

5

Chow2, Alina D. Gutu5, Samuel M. Moskowitz6, Hsin-Hui Shen2,7, Jian Li2

6 7

1Monash

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Parkville 3052, Victoria, Australia

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2Monash

Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade,

Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia

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3Department

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Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010,

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Australia.

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4Australian

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Organisation, Locked Bag 2001, Kirrawee DC, New South Wales 2232, Australia

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5Department

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USA

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6Vertex

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7Department

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University, Clayton, Victoria 3800, Australia

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*Corresponding authors: [email protected] (H.H.S); [email protected] (J.L.).

of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of

Centre for Neutron Scattering, Australian Nuclear Science and Technology

of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts,

Pharmaceuticals, Boston, MA, USA of Materials Science and Engineering, Faculty of Engineering, Monash

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1 ACS Paragon Plus Environment

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Abstract

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Polymyxins are last-line antibiotics against life-threatening multidrug-resistant Gram-negative

25

bacteria. Unfortunately, polymyxin resistance is increasingly reported, leaving a total lack of

26

therapies. Using lipidomics and transcriptomics, we discovered that polymyxin B induced lipid

27

A deacylation via pagL in both polymyxin-resistant and -susceptible P. aeruginosa. Our results

28

demonstrated that the deacylation of lipid A is an ‘innate immunity’ response to polymyxins

29

and a key compensatory mechanism to the aminoarabinose modification to confer high-level

30

polymyxin resistance in P. aeruginosa. Furthermore, cutting-edge neutron reflectometry

31

studies revealed that assembled outer membrane (OM) with the less hydrophobic penta-

32

acylated lipid A decreased polymyxin B penetration, compared to the hexa-acylated form.

33

Polymyxin analogues with enhanced hydrophobicity displayed superior penetration into the

34

tail regions of the penta-acylated lipid A OM. Our findings reveal a previously undiscovered

35

mechanism of polymyxin resistance, wherein polymyxin-induced lipid A remodeling affects the

36

OM packing and hydrophobicity, perturbs polymyxin penetration and thereby confers high-

37

level resistance.

38 39 40



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Introduction

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The treatment of life-threatening infections caused by multidrug-resistant (MDR) Gram-

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negative bacteria is very problematic due to the lack of new antibiotics. 1, 2 In particular, MDR

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Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae present a

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major global challenge, 3-5 and the World Health Organization (WHO) has placed them at the

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top of the 2017 Antibiotic-resistant Priority Pathogens List. 6 Polymyxins (i.e. polymyxin B and

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colistin) show significant activity against these ‘superbugs’. They are cyclic lipopeptides and

48

polycations at pH 7.4 owing to the five positively charged L-,-diaminobutyric acids (Dab)

49

(Fig S1).

50

of lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet and

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presents as an effective permeability barrier. 5, 9 Present understanding of the polymyxin mode

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of action is largely focused on the interaction with lipid A component of LPS through an initial

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polar interaction (via Dab) with the negatively charged lipid A phosphates (Fig S2).

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hydrophobic interaction between the fatty acyl tail of polymyxins and bacterial OM is essential

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for the bacterial killing, as polymyxin B nonapeptide (which has the fatty acyl-Dab1 removed)

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is devoid of antibacterial activity.

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resistance is low, the recent emergence of mcr plasmids indicates that polymyxin resistance

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may readily spread. 15, 16 In essence, resistance to polymyxins implies a total lack of antibiotics

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against MDR Gram-negative infections. The discovery of novel polymyxins to combat the

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growing threat of a ‘post-antibiotic era’ is paramount. 14, 17, 18

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The most common mechanism of polymyxin resistance is the modification of lipid A phosphate

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groups with positively charged moieties (e.g. 4-amino-4-deoxy-L-arabinose [L-Ara4N],

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phosphoethanolamine [pEtN] and galactosamine) which decreases the negative charge of

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lipid A and therefore reduces its binding to polymyxins.

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lipid A through the deacylation of R-3-hydroxydecanoate at position 3 was observed in several

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Gram-negative pathogens, including Salmonella enterica and P. aeruginosa.

67

However, the pagL-dependent lipid A deacylation increases polymyxin resistance only in S.

5, 7, 8

The asymmetric Gram-negative bacterial outer membrane (OM) is composed

8, 13, 14

10-12

The

Although the current prevalence of polymyxin

18-22


The hydrophobic modification of

12, 18, 23, 24

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24

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enterica isolates which are unable to modify lipid A with either L-Ara4N or pEtN;

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ability to cause polymyxin resistance in P. aeruginosa has not been reported.

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To overcome polymyxin resistance, we have designed a series of polymyxin-like lipopeptides

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through hydrophobic modifications in the N-terminal fatty acyl chain or positions 6/7 (e.g.

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FADDI-019, FADDI-020) (Fig S1).

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interactions with lipid A and confer the lipopeptides activity against polymyxin-resistant

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isolates. 14, 25 However, it is largely unknown how polymyxins interact with Gram-negative OM

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which has limited our understanding of the mechanisms of polymyxin activity and resistance.

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In the present study, we firstly characterized the kinetics of lipid A remodeling in both

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polymyxin-resistant and -susceptible P. aeruginosa in response to polymyxin B treatment.

78

Subsequently, neutron reflectometry was employed to investigate the interaction of polymyxin

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B, FADDI-019, and FADDI-020 with Gram-negative OM which was assembled based upon

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our lipid A kinetics results. Our findings reveal a previously undiscovered facet of the

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mechanism of polymyxin resistance.

14

while its

These modifications accentuate the hydrophobic

82 83



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Results and Discussion

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Kinetics of lipid A remodeling and metabolic perturbations in L-Ara4N biosynthesis

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The kinetics of lipid A modifications in P. aeruginosa has not been characterized previously in

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the literature. In the present study, ESI-MS was employed to examine the time course of lipid

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A modifications in response to polymyxin treatment. In the wild-type strain P. aeruginosa PAK

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(polymyxin B minimum inhibitory concentration [MIC] 1 mg/L), extensive modifications of the

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lipid A phosphates with L-Ara4N (peaks at m/z 1497, 1577 and 1747) rapidly occurred even

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within 1 h after 4 mg/L polymyxin B treatment and persisted up to 24 h (Fig 1A, Fig S2 and

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Fig S3A). In contrast, lipid A in the paired polymyxin-resistant mutant, PAKpmrB6 (polymyxin

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B MIC 16mg/L) was constitutively modified with L-Ara4N over 24 h in the absence of polymyxin

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B (Fig 1B, Fig S2 and Fig S3B). 26, 27 The levels of L-Ara4N modified lipid A increased in PAK

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due to polymyxin B treatment at 4 mg/L, while no dramatic changes were observed in

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PAKpmrB6 (Fig S3). Furthermore, comparative metabolomics study showed that the levels of

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key intermediates related to L-Ara4N biosynthesis, Uridine diphosphate (UDP)-glucuronate

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and UDP-4-deoxy-4-formamido-L-arabinose (UDP-L-Ara4FN), were significantly enriched (>

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2-fold) only in PAK after polymyxin B treatment for 1h (Fig 2). The current literature on

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polymyxin resistance has largely focused on identifying transcriptional changes in the arn

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operon and the consequent modifications of the phosphate groups on lipid A. Our

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metabolomics study provides the first in-depth understanding of polymyxin resistance on the

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systems level by elucidating the changes in L-Ara4N biosynthesis pathway in response to

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polymyxin B.

105 106

Deacylation of lipid A and the role in polymyxin resistance

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Notably, without polymyxin treatment, the levels of total lipid A showed a decreased

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abundance in PAKpmrB6 compared to PAK (Fig 3A), indicating a partial LPS lost in the

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polymyxin-resistant P. aeruginosa strain. Surprisingly, the abundance of penta-acylated lipid

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A, relative to hexa-acylated lipid A, substantially increased in both PAK and PAKpmrB6 after


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polymyxin B treatment at 4 and 24 h (Fig 1, 3B and 3C, Fig S3). This intriguing finding was

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confirmed by our transcriptomics data which showed a significant upregulation of pagL

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encoding lipid A deacylase after polymyxin B treatment (4 mg/L) in both strains (Fig 3D). 22, 28

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The occurrence of lipid A deacylation in the polymyxin-resistant PAKpmrB6 strain

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demonstrated that lipid A deacylation and L-Ara4N modification can occur concomitantly;

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whereas in S. enterica, pagL-mediated deacylation of lipid A can only occur in isolates that

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are unable to modify lipid A with either L-Ara4N or PEtN.

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the arnBCDTEF-ugd operon induced by polymyxin B which are responsible for the addition of

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L-Ara4N to lipid A was only observed in the polymyxin-susceptible PAK strain (Fig 2 and Table

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S1).

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increased its polymyxin susceptibility (Fig 3E), whereas no significant change was observed

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in the pagL mutant of polymyxin-susceptible PAK (Fig 3F). Failure of lipid A deacylation to

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cause polymyxin resistance in the wild-type strain indicates that lipid A modification with

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L-Ara4N

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in polymyxin-resistant P. aeruginosa demonstrates that resistant bacterial cells need to

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activate secondary mechanisms to survive polymyxin prolonged treatment. Furthermore, the

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pagL-mediated lipid A deacylation can cause reduced toll-like receptor 4 (TLR-4) stimulatory

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inflammation in P. aeruginosa.

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weaken TLR4 signalling and change the downstream cytokine profile, compared to hexa-

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acylated lipid A. 31, 32 Therefore, our finding is crucial in understanding the pathogenesis of P.

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aeruginosa with different modifications of lipid A, and in terms of the treatments caused by this

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pathogen in the clinic.

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Our collective findings show, for the first time, that polymyxin-resistant P. aeruginosa responds

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to polymyxin treatment by deacylation, not the phosphate modification, of lipid A. This

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observation demonstrates that polymyxin resistance due to different lipid A modifications is

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multi-faceted and the deacylation of lipid A appears to confer high-level polymyxin resistance

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in resistant P. aeruginosa strains. We therefore hypothesized that lipid A deacylation through

27

24

The up-regulation of pmrAB and

Moreover, the deletion of pagL gene in polymyxin-resistant PAKpmrB6 considerably

is the first line of polymyxin resistance mechanism. A further increase in resistance

29, 30

It has been reported that penta-acylated lipid A can



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the removal of fatty acyl chain at position 3 reduces the hydrophobic interaction with

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polymyxins.

140 141

Interactions of polymyxin B with outer membrane models constructed by wild-type and

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deacylated lipid A

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Cutting-edge neutron reflectometry has been demonstrated for its unique ability to precisely

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characterize the asymmetric Gram-negative OM models and the interaction with peptides. 33, 34

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In the present study, our NR investigation of the interactions of polymyxins with OM models

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assembled by wild-type and deacylated lipid A provided unprecedented molecular level in

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understanding the mechanism of polymyxin resistance.

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The OM model was assembled by sequential Langmuir-Blodgett and Langmuir-Schaefer

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deposition methods (Fig S4). 33, 34 The outer leaflet of OM was constructed using commercially

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available penta- or hexa-acylated lipid A (Fig S5) and the inner leaflet was composed of tail-

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deuterated DPPC [d-DPPC, 1, 2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine]. While the

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lipid A of P. aeruginosa contained both mono- and di-phosphate groups (Fig 1), the OM model

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using the commercial lipid A products with the only difference at position 3 (i.e. with or without

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deacylation) was sufficient to examine the effect of lipid A deacylation on the interaction with

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polymyxins. The NR profiles obtained from the hexa-acylated lipid A : d-DPPC bilayer were

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fitted to a five-layer model to describe the interfacial structure, i.e. sequentially from silicon to

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solution, a hydrated silicon oxide layer, the d-DPPC head group, the d-DPPC fatty acyl tails,

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the lipid A fatty acyl tails, and the lipid A glucosamine (GlcN) head group (Fig S6A).

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Interestingly, the penta-acylated lipid A : d-DPPC bilayer detected in NR could only be fitted

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into a six-layer model which includes an extra lipid A head group due to the structural

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difference between penta- and hexa-acylated lipid A (Fig S6B). The thicknesses and bilayer

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coverages recorded for the assembled membranes were consistent with the literature.

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Briefly, the NR analysis revealed that asymmetric lipid compositions of both hexa- and penta-

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acylated lipid A bilayer had been deposited on the silicon-water interface. In the hexa-acylated


33

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lipid A : d-DPPC bilayer model, the inner layer was composed of 93.1% d-DPPC and 4.3%

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lipid A, while 83.2% lipid A and 10.7% d-DPPC were found in the outer layer. Similarly, the

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penta-acylated lipid A : d-DPPC bilayer was detected to be composed of 75.1% d-DPPC and

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21.0% lipid A in the inner leaflet, while 75.9% lipid A and 15.7% d-DPPC in the outer leaflet.

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These results indicated an estimated 91-97% of total bilayer coverage over the neutron beam

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illuminated area. Interestingly, our results also showed that the head group of penta-acylated

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lipid A was more densely packed than hexa-acylated lipid A (86.0% and 75.4%, respectively)

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in the OM models (Fig S6; Table S2 and S3).

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Upon the addition of polymyxin B (16 mg/L) to both OM models (Fig 4), there was a 10 Å

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increase in the thickness on the top of both penta- and hexa-acylated lipid A head groups,

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indicating the binding of polymyxins to the membrane outer leaflet. However, the thickness of

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each layer in both the hexa- and penta-acylated lipid A OM did not significantly change after

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the injection of polymyxin B onto the surface. Thus, it is unlikely that the insertion of polymyxin

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B induces significant changes in membrane curvature. 35 The well accepted structure-activity

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relationship (SAR) model of polymyxins is purported to involve an initial binding of the

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polymyxin molecule to the membrane surface via an electrostatic interaction through the

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positively charged Dab residues of the polymyxin and the negatively charged phosphate

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groups of lipid A.

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were similar (44-50% on the surface of the head group and ~20% in the galactosamine [GlcN]

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head group region; Table 1). Notably, a slight decrease in the volume fractions of penta-

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acylated lipid A (from 67.7% to 52.0% in the outer head groups and from 86.0% to 65.6% in

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the inner head groups) and hexa-acylated lipid A (from 75.4% to 58.4% in the head groups)

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was observed after polymyxin B penetration, indicating that 15-17% of both lipid A

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compositions in the OM models were disrupted by polymyxin B (Table 1 and Table S3).

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Moreover, a volume fraction of polymyxin B at 10% was estimated to be in the fatty acyl

190

regions of hexa-acylated lipid A, indicating a further hydrophobic interaction between

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polymyxin B and lipid A in addition to the primary electronic contact. In the contrast, the

8

The volume fractions of polymyxin B on the surface of both head groups



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unchanged scattering length densities (SLDs) of the penta-acylated lipid A fatty acyl tails and

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the inner leaflet in the penta-acylated lipid A : d-DPPC bilayer indicated that polymyxin B did

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not penetrate into the tail regions of penta-acylated lipid A layer (Table 1).

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Collectively, compared to the hexa-acylated form, the denser packing of penta-acylated lipid

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A head groups prevented deep penetration of polymyxin B into the bilayer; and the reduced

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hydrophobicity of the fatty acyl chains decreased further hydrophobic interactions with

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polymyxin B. The combination of both factors likely increased the resistance to the

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disorganizing activity of polymyxin B. Taken together with the systems biology data,

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deacylation of lipid A appears to affect the packing of bacterial OM and directly prevented the

201

penetration of antibiotics into bacteria.

202 203

Interactions of polymyxin-like lipopeptides with penta-acylated lipid A assembled

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membranes

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The interactions of synthetic lipopeptides FADDI-019 and FADDI-020 (both at 16 mg/L) with

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penta-acylated lipid A assembled OM were investigated. The OM models were re-constructed,

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in which similar lipid compositions of both bilayer models composed of 77.9% and 88.2%

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d-DPPC in the inner leaflet, while 86.1% and 88.2% lipid A in the outer leaflet were detected

209

through NR, respectively; this model was consistent with the bilayer parameters established

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for the polymyxin B studies (Table S4). Our NR analysis showed that 39.7 and 35.8% of

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FADDI-019 and FADDI-020 were found on the membrane surface, respectively; while 15-18%

212

of which penetrated into the GlcN head group regions of the penta-acylated lipid A (Fig 5;

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Table 2). These results indicated that the penetration of FADDI-019 and FADDI-020 with the

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OM involves similar polar interactions as the polymyxin B–OM interaction. Interestingly, unlike

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polymyxin B, FADDI-019 and FADDI-020 penetrated deeply into the fatty acyl regions of

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penta-acylated lipid A in the assembled membrane with a volume fraction of 16.6% and 10.5%,

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respectively. Our previous study showed that both lipopeptides with significant activity against

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polymyxin-resistant P. aeruginosa isolates have lower nephrotoxicity and better efficacy in


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animal models, compared to polymyxin B and colistin.

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the molecular level discovery that the modification of polymyxin core scaffold at position 6 (D-

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OctGly) and in the fatty acyl chain (octanoyl for FADDI-019, and biphenyacyl for FADDI-020)

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(Fig S1) significantly increased their hydrophobic interaction with Gram-negative OM, and

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therefore overcome polymyxin resistance caused by the deacylation of lipid A.

Our NR finding is the first to reveal

224 225

Conclusion

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Currently, it is believed that modification of lipid A phosphate groups with aminoarabinose

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primarily causes polymyxin resistance in P. aeruginosa. Our lipid A kinetics study revealed,

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for the first time, that rapid deacylation of lipid A was promoted by polymyxin B in both

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polymyxin-resistant and -susceptible P. aeruginosa. The transcriptomics and metabolomics

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results demonstrated that pagL was upregulated by polymyxin B in both strains; while the

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increase of the lipid A modification with L-Ara4N was only observed in polymyxin-susceptible

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PAK. Moreover, a mutant of PAKpmrB6 with pagL deletion displayed dramatically increased

233

susceptibility to polymyxin B. Here, we demonstrated that lipid A deacylation plays a key role

234

in high-level polymyxin resistance in P. aeruginosa, which is not solely due to the L-Ara4N

235

modification. Excitingly, the NR results confirmed that the deacylation of lipid A dramatically

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decreased polymyxin interaction with the Gram-negative OM, and new-generation polymyxins

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incorporating hydrophobic moieties into their scaffold overcome resistance to polymyxin B by

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increasing the hydrophobic interactions with penta-acylated lipid A. Taken together, our

239

findings reveal that the mechanism of polymyxin resistance due to lipid A modifications is

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multi-faceted, wherein polymyxin-induced lipid A deacylation plus phosphate modifications

241

confers high-level resistance by affecting the OM packing and hydrophobicity, and perturbing

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polymyxin penetration. This study highlights the dynamic and complex nature of membrane

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remodeling in Gram-negative bacteria and their impact on the interaction with the last-line

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polymyxins.



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Methods

246

Materials

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All chemicals were purchased from Sigma-Aldrich at the highest research grade. Ultrapure

248

water was from Fluka (Castle Hill, NSW, Australia). Penta-acylated lipid A (Cat# 699852P),

249

hexa-acylated lipid A (Cat# 699800P) and 1, 2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine

250

(d-DPPC, Cat# 860355P) were obtained from Avanti Polar Lipids (>99% purity; Alabaster,

251

USA). FADDI-019 and FADDI-020 were prepared as previously described. 14 Stock solutions

252

of polymyxin B, FADDI-019, and FADDI-020 (5 mg/mL) were prepared in Milli-Q water

253

(Millipore, Australia) and filtered through 0.22-µm syringe filters (Sartorius, Australia).

254

Bacterial strains, growth conditions, and polymyxin susceptibility testing

255

P. aeruginosa PAK and PAKpmrB6 and their pagL mutants were obtained from Moskowitz

256

Laboratory (Massachusetts General Hospital, MA, USA).

257

subpopulations of PAK and PAKpmrB6 and their pagL mutants was conducted using

258

population analysis profiles (PAPs);

259

mid-logarithmic bacterial cell suspension and/or its serial saline dilutions on Mueller-Hinton

260

agar plates with and without polymyxin B (0, 0.25, 0.5, 1, 2 and 4 mg/L for PAK and its pagL

261

mutant, while 0, 4, 8, 16, 32 and 64 mg/L for PAKpmrB6 and its pagL mutant). Colonies were

262

counted after 24-h incubation at 37 C with a limit of detection of 20 CFU/mL.

263

Lipid A isolation and purification and LC-MS analysis

264

Lipid A was isolated by mild acid hydrolysis as described previously,

265

preparative

266

(chloroform/methanol/pyridine/acetic acid/water, 10:5:4:3:2, v/v). Structural and semi-

267

quantitative analysis of lipid A was performed on a Dionex U3000 high-performance liquid

268

chromatography system (HPLC) in tandem with a Q-Exactive Orbitrap mass spectrometer

269

(Thermo Fisher) in negative mode with a resolution at 70,000. The mass scanning range was

270

from 167 to 2,500 m/z. The electrospray voltage was set as 3.50 kV and nitrogen was used

thin-layer

3

26

Analysis of polymyxin-resistant

three replicates by spiral plating 50 µL of the starting

chromatography

with

multi-developing


10

and purified using solvents

system

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271

as collision gas. The Phenomenex Synergi Hydro-RP 80 Å column (50  2 mm, 4 µm) was

272

maintained at 40 C, while the samples were stored at 4 C. Lipid A samples in

273

chloroform/methanol/isopropanol/water (1:1:1:0.5, v/v) were eluted using a gradient that

274

consisted of 40% of isopropanol (IPA) and 60% of Milli-Q water with 8 mM ammonium formate

275

and 2 mM formic acid as mobile phase A, while 98% of IPA and 2% of Milli-Q water with 8 mM

276

ammonium formate and 2 mM formic acid as mobile phase B. The flow rate was 0.2 mL/min

277

within the first 15 min, and increased to 0.5 mL/min from 16 min to 22 min. The gradient started

278

with 70% mobile phase A and 30% mobile phase B, followed by a linear gradient to a final

279

composition of 100% mobile phase B which was maintained for 4 min. A 3-min re-equilibration

280

of the column with 70% mobile phase A was performed prior to the next injection.

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Preparation of cellular metabolites and LC-MS analysis

282

Cellular metabolites of PAK and PAKpmrB6 in response to 4 mg/L polymyxin B at 1, 4 and

283

24 h were prepared and analyzed through LC-MS by the previously reported method in

284

detail. 23

285

RNA extraction, library preparation and RNA sequencing (RNA-Seq)

286

RNAprotect (Qiagen) was used for the sample collection in order to preserve gene expression

287

profiles. RNA was isolated using RNeasy minikit (Qiagen) in accordance with the

288

manufacturer’s instructions. RNA-Seq was undertaken using Illumina HiSeq. The

289

transcriptome was assembled from the RNA-Seq data using Trinity RNA-Seq software and

290

RNA-Seq reads were aligned with the genome sequence of P. aeruginosa PAK and

291

PAKpmrB6 strains using Subread. 36 The RNA sequence data from biological replicates were

292

analyzed using the voom and limma linear modeling methods via Degust interactive Web-

293

based RNA-Seq visualization software (http://www.vicbioinformatics.com/degust/). 37

294

Deposition of the Gram-negative bacterial outer membrane (OM) and neutron

295

reflectometry measurements



296

The Gram-negative OM model was deposited onto the silicon oxide surface using LB

297

deposition for the deuterated DPPC (d-DPPC) inner layer and followed by deposition of the

298

penta-/hexa-acylated lipid A outer layer via Langmuir Schaefer (LS) method as described

299

before (Fig S4). 34, 38 NR measurements of the OM models were undertaken on the Platypus

300

time-of-flight neutron reflectometer and a cold neutron spectrum (2.8 Å   18 Å) at the OPAL

301

20 MW research reactor (Sydney, Australia). 39 Neutron pulses of 24 Hz were generated using

302

a disc chopper system (EADS Astrium GmbH, Germany) set to a Δλ/λ ≈ 8% and were recorded

303

on a 2D 3He detector (Denex GmbH, Germany). The reflected intensity was measured using

304

a footprint of 65 mm at two glancing angles of 0.45 for 15 min and 1.6 for 60 min as a function

305

of the momentum transfer, Qz (Qz=[4π sin θ]/λ, where λ is the wavelength and θ is the incident

306

angle). Data were reduced using the SLIM reduction software, 40 which stitches the two angles

307

together at the appropriate overlap region, re-bins the data to instrument resolution, and

308

corrects for background and detector efficiency. The silicon wafers were placed on a variable-

309

angle sample stage in the NR instrument and the inlet to the liquid cell was connected to a

310

liquid chromatography pump (Knauer BlueShadow 40P HPLC pump); which allowed for easy

311

exchange of the solution isotopic contrast (5 mL volume) within the solid-liquid sample cell.

312

The bilayer structure was analysed in three solution isotopic contrasts: (i) 100% D2O, (ii) 38%

313

D2O and 62% H2O [which has the same neutron scattering length density (nSLD) as the silicon

314

wafer, and thus called silicon-matched water, SMW] and (iii) 100% H2O. For each change of

315

the isotopic contrast solution, a total of 5 mL of 10 mM pH 7.4 HEPES buffer combined with 5

316

mM CaCl2 and 150 mM NaCl solution was pumped through the cell at a speed of 1 mL/min.

317

For the interaction of polymyxins with the model OM bilayers, polymyxin B, FADDI-019 and

318

FADDI-020 were respectively dissolved in buffer solutions at pH 7.4 containing 20 mM

319

HEPES, 5mM CaCl2 and 150 mM NaCl in D2O at 16 mg/L, and then injected into the sample

320

cells.

321

Neutron reflectometry data analysis

322

Neutron reflectometry data were analyzed using MOTOFIT which employs the Abeles


Page 14 of 28

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323

Formalism to fit multiple contrast NR data. 40, 41 A least square fitting routine selects the best

324

fit by minimizing 2 values through varying the thickness, interfacial roughness and nSLD of

325

each layer. The interface was described as a series of slabs, each of which was characterized

326

by its nSLD, thickness and roughness. The reflectometry for the model starting point was then

327

calculated and compared with the experimental data. In all cases, the model which contained

328

the least number of layers was selected to adequately describe the data. In this study, the

329

systems for NR data were asymmetrically deposited bilayers (i.e. d-DPPC [inner layer] : lipid

330

A [outer layer]). Each bilayer was examined under three isotopic contrasts (D 2O, SMW, and

331

H2O) to yield three reflectometry profiles for each bilayer. A single profile of layer thickness

332

and roughness was fitted for the three reflectometry profiles in the silicon deposited bilayer,

333

while the nSLD of each individual layer was allowed to vary in order to account for the volume

334

fraction. The parameter fit values and the nSLD profiles described above were used to

335

determine the bilayer structure across and the surface coverage. The volume fractions and

336

bilayer asymmetry were calculated according to the method described previously,

337

nSLDs of the deuterated tail regions of d-DPPC and the hydrogenated fatty acyl chains of lipid

338

A were employed to determine lipid asymmetry. The volume fractions of lipid A and d-DPPC

339

in the head group layers were determined according to the minimal isotopic contrast between

340

the head groups of lipid A glucosamine (GlcN) and d-DPPC. The total amount of lipid A and

341

d-DPPC head groups was determined by measuring the volume fraction of water in the inner

342

and outer head group regions and comparing the fitted nSLDs of the differing solution

343

contrasts to the known nSLD of H2O and D2O. The nSLDs of polymyxin B, FADDI-019 and

344

FADDI-020 were calculated using nSLD calculator and changes in the nSLD due to labile

345

hydrogen exchange with D2O, SMW, and H2O. The coverage of polymyxins in their binding

346

layers was determined by comparing the fitted nSLD values of these layers to the calculated

347

nSLD values of lipopeptides, lipids, and water (Table S2).


38

with the


348

ASSOCIATED CONTENT

349

Supporting Information

350

The Supporting Information is available free of change on the ACS Chemical Biology website

351

at DOI: XXXX

352

Supporting Tables S1S4, and Figures S1S6.

353 354

AUTHOR INFORMATION

355

Corresponding authors:

356

Email: [email protected].

357

Email: [email protected].

358

ORCID:

359

Mei-Ling Han: 0000-0002-0861-2437

360

Tony Velkov: 0000-0002-0017-7952

361

Hsin-Hui Shen: 0000-0002-8541-4370

362

Jian Li: 0000-0001-7953-8230

363

Author Contributions

364

J.L. conceived the project and all authors involved in the design of the experiments. M.L.H.

365

performed the experiments, A.D.G. performed mutant construction, and M.L.H., H.H.S., T.V.,

366

and Y.Z. analyzed the results. All authors reviewed the manuscript.

367 368

ACKNOWLEDGEMENTS

369

This research was supported by a research grant from the National Institute of Allergy and

370

Infectious Diseases of the National Institutes of Health (R01 AI111965, J.L. and T.V.). The

371

content is solely the responsibility of the authors and does not necessarily represent the official

372

views of the National Institute of Allergy and Infectious Diseases or the National Institutes of

373

Health. J.L. is an Australian National Health and Medical Research Council (NHMRC) Senior

374

Research Fellow. H.H.S. and T.V. are Australian NHMRC Career Development Research


Page 16 of 28

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375

Fellows. The authors would like to thank AINSE and ANSTO for the financial assistance

376

(awards of neutron beam time, P4787 and P5344; AINSE-PGRA).

377 378

Competing Financial Interests: The authors declare no competing financial interests.

379



380

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381 382

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Lee, H., Hsu, F. F., Turk, J., and Groisman, E. A. (2004) The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica, J. Bacteriol. 186, 4124-4133.

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Miller, A. K., Brannon, M. K., Stevens, L., Johansen, H. K., Selgrade, S. E., Miller, S. I., Hoiby, N., and Moskowitz, S. M. (2011) PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients, Antimicrob. Agents Chemother. 55, 5761-5769.

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507


Tables Table 1. Thicknesses and volume fractions of hexa- and penta-acylated lipid A : d-DPPC bilayer in the presence of polymyxin B (16 mg/L).

[a]

Layer

Thickness (Å)

[b]

% d-DPPC

[b]

% Lipid A

[b]

% Water

% Polymyxin B

[b]

Hexa-acylated lipid A : d-DPPC bilayer d-DPPC head

13.5±0.5

d-DPPC tail

15.9±1.2

80.2±3.1

Lipid A tail

15.3±1.2

9.6±1.4

Lipid A head

13.3±1.1

Polymyxin B

10.2±0.3

82.3±2.3

[c]

58.4±10.0 N/A

11.8±1.9

5.9±0.5

4.8±2.0

2.2±1.5

12.8±4.5

76.3±3.1

3.7±2.2

10.4±6.0

18.7±6.1

22.9±4.0

49.6±7.9

50.4±7.9

[c]

[d]

N/A

[d]

Penta-acylated lipid A : d-DPPC bilayer d-DPPC head

14.9±4.1

86.0±3.5

d-DPPC tail

19.3±1.1

75.1±2.6

Lipid A tail

15.1±0.9

15.7±2.3

Lipid A inner head

6.5±2.1

Lipid A outer head

9.7±2.4

N/A

Polymyxin B

10.2±0.3

N/A

65.6±7.6 [d]

[c]

NF

21.0±3.4

3.9±0.9

NF

75.9±0.4

8.4±2.7

NF

[c]

52.0±5.4

[d]

[e]

14.0±3.5

N/A

[d]

[e]

[e]

10.7±5.4

23.8±2.2

24.8±2.0

23.2±3.5

55.8±3.8

44.2±3.8

Roughness of each layer was fitted at 4.0  1.0 Å. [b]Volume fraction of each constituent within the fitted layer. [c]Total volume fraction of lipid A and d-DPPC head groups within the fitted layer, which was determined according to the minimal isotopic contrast between the head groups of lipid A and d-DPPC, while bilayer asymmetry was calculated according to the neutron scattering length densities (nSLDs) of the tail regions of lipid A and d-DPPC in three isotopic contrast (D2O, SMW, and H2O). [d]N/A: not available. [e]NF: not found. [a]



Page 22 of 28

Table 2. Thicknesses and volume fractions for penta-acylated lipid A: d-DPPC bilayer in the presence of new-generation polymyxins at 16 mg/L.

[a]

Layer

Thickness (Å)

[b]

% d-DPPC

[b]

% Lipid A

[b]

% Water

% FADDI-019/-020

[b]

FADDI-019 90.9±2.0[c]

9.1±2.0

NF[e]

19.1±0.1

3.0±0.6

NF[e]

71.6±6.4

1.0±0.5

16.6±7.4

71.4±6.5[c]

9.9±3.2

18.7±4.3

N/A[d]

72.5±2.1

12.1±5.5

60.3±3.7

39.7±3.7

11.9±5.7

NF[e]

d-DPPC head

16.6±2.7

d-DPPC tail

18.9±1.7

77.9±0.5

Lipid A tail

15.5±2.6

10.8±1.0

Lipid A inner head

9.6±2.3

Lipid A outer head

8.1±3.1

FADDI-019

10.3±0.2

N/A[d]

N/A[d]

FADDI-020 88.1±5.7[c]

d-DPPC head

15.0±2.5

d-DPPC tail

19.8±5.6

88.2±0.4

6.9±0.9

4.9±1.3

NF[e]

Lipid A tail

13.5±5.7

7.1±1.9

79.2±4.3

3.2±0.1

10.5±5.7

Lipid A inner head

11.1±0.4

12.5±3.8

17.0±4.2

Lipid A outer head

10.6±2.1

N/A[d]

59.3±8.7

22.8±11.2

18.0±3.4

FADDI-020

10.4±0.3

N/A[d]

N/A[d]

64.2±1.7

35.8±1.7

70.8±8.3[c]

Roughness of each layer was fitted at 4.0  1.0 Å. [b]Volume fraction of each constituent within the fitted layer. [c]Total volume fraction of lipid A and d-DPPC head groups within the fitted layer, which was determined according to the minimal isotopic contrast between the head groups of lipid A and d-DPPC, while bilayer asymmetry was calculated according to the neutron scattering length densities (nSLDs) of the tail regions of lipid A and d-DPPC in three isotopic contrast (D2O, SMW, and H2O). [d]N/A: not available. [e]NF: not found. [a]

508 509


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510


Figures

511 512

Figure 1. (A) MS of lipid A modifications with L-Ara4N addition and deacylation (i.e. removal of

513

R-3-hydroxydcanoate from the 3 position of lipid A by pagL) in the wild-type PAK; and (B) only

514

deacylation of lipid A in PAKpmrB6 in response to polymyxin B (PMB) treatment at 4 mg/L for

515

4 h. The lipid A samples were analyzed by LC-MS/MS in negative ion mode; therefore, each

516

m/z value in the MS spectra corresponds to a specific lipid A with the loss of one hydrogen

517

([MH]). Data was collected based on three replicates of each condition, and each strain with

518

or without polymyxin B treatment was incubated from the inoculum of ~108 CFU/mL and

519

normalized according to OD600nm at 0.50.02. Red and blue numbers indicate wild-type and



520

L-Ara4N

521

hexa-acylated lipid A, respectively.

modified lipid A, respectively. Green and purple dashed boxes indicate penta- and

522


Page 24 of 28

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523 524

Figure 2. Perturbations of the intermediate metabolites and genes related to

525

biosynthesis in PAK and PAKpmrB6 in response to 4 mg/L polymyxin B at 1 h. Metabolomics

526

data were collected based on five biological replicates, while transcriptomics data were based

527

on three biological replicates. Each strain with or without polymyxin B treatment was incubated

528

from the inoculum of ~108 CFU/mL, and normalized according to OD600nm at 0.500.02. The

529

red box indicates metabolites that were significantly increased by more than 2-fold after

530

polymyxin B treatment for 1 h in PAK; while metabolites in black boxes were not detected

531

using metabolomics method. p < 0.05, p < 0.01; p < 0.001 (student t-test).

532 533


L-Ara4N


534 535

Figure 3. (A) Levels of total lipid A in PAK and PAKpmrB6 analyzed by LC-MS/MS, in which

536

the relative abundance was estimated from the sum of the peak area of each lipid A molecule;

537

Fold changes of penta- to hexa-acylated lipid A in (B) PAK and (C) PAKpmrB6 in response to

538

4 mg/L polymyxin B; (D) Upregulation of pagL in PAK and PAKpmrB6 due to 4 mg/L polymyxin

539

B treatment; Effect of pagL deletion on polymyxin resistance in (E) PAKpmrB6 and (F) PAK.

540

The results were based on three replicates. p< 0.05, p< 0.01, p< 0.001 (student t-test).

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Page 26 of 28

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Figure 4. NR profiles (upper), fitted SLDs and thicknesses (middle) in different solution

545

isotopic (D2O, SMW and H2O) contrasts, and schematic representations (lower) of the

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interfacial structures of (A) hexa- and (B) penta-acylated lipid A : d-DPPC bilayer after the

547

addition of 16 mg/L polymyxin B (PMB). SMW: silicon matched water, which contains 38%

548

D2O and 62% H2O.

549 550



551 552

Figure 5. NR profiles (upper) and fitted SLDs and thicknesses (lower) of the interfacial

553

structures for new-generation polymyxins (A) FADDI-019 (F019) and (B) FADDI-020 (F020)

554

interactions with penta-acylated lipid A : d-DPPC bilayer in three different isotopic (D2O, SMW,

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and H2O) contrasts; (C) Schematic representation of the structures was based on the fitted

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parameters and the calculated volume fractions.

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Page 28 of 28

Polymyxin-Induced Lipid A Deacylation in Pseudomonas aeruginosa

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