Deficient Lipid A Remodeling by the arnB Gene Promotes Biofilm

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Deficient Lipid A Remodeling by the arnB Gene Promotes Biofilm Formation in Antimicrobial Peptide Susceptible Pseudomonas Aeruginosa Li-av Segev-Zarko, Gal Kapach, Michaele Josten, Yoel Alexander Klug, Hans-Georg Sahl, and Yechiel Shai Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00149 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Biochemistry

Deficient Lipid A Remodeling by the arnB Gene Promotes Biofilm Formation in Antimicrobial Peptide Susceptible Pseudomonas Aeruginosa

Li-av Segev-Zarko1, Gal Kapach1, Michaele Josten2, Yoel Alexander Klug1, Hans-Georg Sahl2 and Yechiel Shai1*

1

Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot,

Israel 2

Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn,

Bonn, Germany

* Corresponding author E-mail: [email protected]

1

1 ACS Paragon Plus Environment

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Abstract Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides, and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal, atomic force microscopy and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a more robust biofilm compared to the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge which promote the formation of biofilms. Moreover, when grown under Mg2+ limitation, both the WT and arnB mutant exhibited a reduction in membrane bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a tradeoff between LPS remodeling and resistance mechanisms of biofilm formation.


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Biochemistry

Introduction Multidrug resistant bacteria are a growing phenomenon that concerns the scientific and medical communities worldwide. Bacteria utilize several resistance 1-3

mechanisms allowing them to cope against multiple environmental stresses

.A

strategy by which Gram-negative bacteria sense and response rapidly to the changing environment is remodeling of their lipopolysaccharides (LPS) 4. LPS constitutes the main element of the outer leaflet in Gram-negative bacteria 5. The LPS molecule consists of three building blocks, lipid A, the ‘core’ oligosaccharide and the O-antigen 6. Lipid A modifications reduces the net negative charge of the bacterial outer membrane, hence, contribute to bacterial resistance against antimicrobial peptides (AMPs)

7-11

. One such modification is the addition of the

positively charged 4-amino-4-deoxy-L-arabinose (L-Ara4N) sugar to the lipid A 1and 4-phosphate groups

10, 12

. In Pseudomonas aeruginosa, synthesis and

addition of L-Ara4N to lipid A is mediated by the arnBCADTEF operon either in response to low extracellular Mg2+ concentration, or the presence of AMPs13. Magnesium cations are crucial in preserving the integrity of bacterial membranes by binding in between negatively charged lipid moieties of LPS molecules

14

. The

addition of positively charged L-Ara4N molecules compensates for the lack of Mg2+ ions and stabilizes the membrane. In addition to LPS modifications, many bacteria colonize in highly resistance micro-colonies known as biofilms, in response to unfavorable environmental conditions

15

. The formation of biofilms takes place via four main

stages: (i) planktonic bacteria attach to surfaces reversibly; (ii) irreversible


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adhesion to surfaces; (iii) formation of sessile micro-colonies that secrete extracellular polymeric substance (EPS), consisting of proteins, DNA and lipopolysaccharides; and (iv) detachment of bacteria from the biofilms allowing the formation on new surfaces 16, 17. Since LPS modifications were identified also in biofilm embedded bacteria 18, 19

, we asked whether these modifications affect biofilm formation. To address

this question, we investigated biofilm formation of P. aeruginosa, an opportunistic Gram-negative bacterium with a high tendency to form biofilm, making it the major pathogen in lungs of patient with Cystic Fibrosis (CF)

20

. To block lipid A

modification, we used an arnB mutant strain, mutated in the first gene of the arnBCADTEF operon

11

. Combining biochemical and biophysical approaches

with advanced imaging techniques including confocal microscopy, transmission electron microscopy and atomic force microscopy, we examined the ability of the WT (wild-type) and arnB mutant to form biofilms. We show that when grown under Mg2+ limitation, the arnB mutant forms a robust biofilm compared to the WT. Mode of action studies revealed that: (i) the WT and arnB mutant present different phenotypes of adhesion and swarming motility that correlate with their biofilm formation; (ii) the outer surface of arnB is more hydrophobic, more negatively charged and with higher rigidity compared to the WT; and (iii) both the WT and arnB mutant showed reduced amounts of membrane bound polysaccharides that can potentially increase the overall difference between their outer surfaces. In summary, our results suggest that planktonic bacteria that are


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Biochemistry

deficient in the addition of L-Ara4N to lipid A are more susceptible to AMPs, but conversely possess properties that contribute to biofilm formation.

Materials and Methods Materials Fmoc rink amide Methybenzhyrilamine (MBHA) resin and Fmoc amino acids were purchased from Calibochem-Novabiochem AG (Laufelfinger, Switzerland). Polymyxin B sulfate (Fluka biochemika 81334) and Crystal Violet (C3886) were purchased from Sigma Aldrich (Rehovot, IL). Tetracycline hydrochloride (Calbiochem 58346) was purchased from Mercury (Rosh Ha’ayn, IL). Sterile 96well U-bottom polystyrene plates (BN36010096D) were purchased from Bar-Naor (Ramat Gan, IL). Bacterial Strains and Growth Conditions In this study we used the Pseudomonas aeruginosa PAO1 WT strain and PW7021 (arnB-G12: ISphoA/hah), an arnB mutant, given to us courtesy of Prof. R. EW. Hancock. The mutant was generated as part of a transposon mutant library and confers resistance to tetracycline (100 µg/mL)

21

. The mutant was

verified for holding a single mutation in the arnB gene using Sanger sequencing and Tn-seq

22

. Bacteria were grown at 37°C in BM2 minimal medium (62 mM

potassium phosphate buffer, pH 7.0, 7 mM (NH4)2SO4, 10 µM FeSO4, 0.2% (w/v) glucose, 0.5% (w/v) casamino acids) supplemented with low (20 µM) or high (2 mM) MgSO4. Lipopolysaccharide Extraction and Purification


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LPS from the WT and arnB mutant was purified using Tri-reagent, as previously described by Yi and Hackett

23

. Briefly, mid-log phase bacteria were washed

three times in DDW (double-distilled water), freeze and lyophilized. Dry bacteria were suspended in Tri-reagent for 10-15 minutes followed by the addition of chloroform to allow phase separation. The aqueous phase was collected to a new tube and DDW was added to the organic phase to allow additional separation. The combined aqueous phase was then dried using speed vacuum and the crude LPS was dissolved in 0.375 M MgCl2 in 95% ethanol and centrifuged. After three washes in DDW the purified LPS was lyophilized. Lipid A Isolation and MALDI-TOF Analysis In order to detect modifications in lipid A structure, lipid A was isolated from LPS by mild hydrolysis as previously described

23

. Briefly, 0.5-1 mg of purified LPS

were hydrolyzed at 100°C in 1% SDS in 10 mM sodium acetate buffer (pH 4.5) and incubated in an ultrasonic bath for 10 min. The samples were then dried using speed vacuum. The samples were washed with 100 µL distilled water and 500 µL of acidified ethanol (100 µL of 4 M HCl were mixed with 20 mL of 95% ethanol) followed by an additional wash with 95% ethanol. The isolated lipid A was

suspended

in DDW

and

lyophilized.

Lipid

A

was

dissolved

in

Chlorofom:MeOH:H2O (4:4:1) and 1µl was mixed with 1µl matrix (5-Chloro-2mercaptobenzothiazole,

Sigma-Aldrich,

dissolved

in

methanol-chloroform

(1:1)). One microliter of that mixture was placed onto a ground steel MALDI-TOF target plate and allowed to dry at room temperature. Matrix assisted laser desorption ionization time of flight (MALDI-TOF) analyses were carried out in the


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Biochemistry

negative-ion mode on a Bruker Biflex III MALDI-TOF (Bruker Daltonics, Bremen, Germany) at a laser frequency of 9Hz within a mass range from 500 to 3,000 Da. 300 shots were accumulated for each mass spectrum. Instrument parameter settings were IS1 19 kV, IS2 16.5 kV, lens 9.5 kV, PIE 200 ns, Reflector 20kV, no gating. Data analysis was performed using flexAnalysis software (Bruker Daltonik GmbH). Transmission Electron Microscopy Bacterial cells were prepared using the freeze substitution method as previously described by Hunter and Beveridge

24

with some modifications. Centrifuged

bacteria were loaded on 100 µm depth aluminum discs (Engineering Office M. Wohlwend GmbH, Switzerland) and covered with a flat disc. Samples were frozen in a HPM010 high-pressure freezing machine (Bal-Tec, Liechtenstein). Bacterial cells were subsequently freeze-substituted in a AFS2 freeze substitution device (Leica Microsystems, Austria) in anhydrous acetone supplemented with 2% glutaraldehyde and 0.2% tannic acid osmium tetroxide for two days at −90°c and then warmed up over 24 hours to −20°C. Samples were washed with acetone, followed by incubation for one hour at room temperature with 2% osmium tetroxide and 0.2% uranyl acetate, washed with acetone and infiltrated for 7 days at room temperature in a series of increasing concentrations (10-100%) of Epon in acetone. Samples were then polymerized at 60°c, and 60– 80 nm sections were stained with uranyl acetate and lead citrate and examined in a CM-12 Spirit FEI electron-microscope; pictures were taken using a CCD camera Eagle 2kx2k FEI (Eindhoven Netherlands).


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Polysaccharide Quantification Using Flow Cytometry Overnight cultures of WT and arnB mutant strains grown in BM2 medium supplemented with low (20 µM) or high (2 mM) MgSO4 were centrifuged and resuspended in filtered saline (0.85% NaCl). Bacterial cells were stained with Syto9 for 10 minutes to determine live single bacteria. After incubation, bacterial cells were centrifuged and re-suspended with Calcofluor white (18909, Sigma Aldrich, IL), a fluorochrome that binds polysaccharides, for 20 minutes. After the cells were centrifuged they were re-suspended in filtered saline. Bacteria were analyzed on a BDTM LSRII (BD Biosciences) flow cytometer and analyzed by FlowJo software. The Calcofluor white mean fluorescence intensity was calculated from the Syto9 positive cells n=170,000. Peptide Synthesis and Purification Peptides were synthesized by an automated peptide synthesizer (433A from Applied Biosystems, Life Technologies, Foster City, California, USA) on rink amide 0.68 meq/mg MBHA resin using the Fmoc solid phase strategy. The resin bound peptide was washed thoroughly with DMF and then methylene chloride, dried and cleaved. Cleavage was done by addition of 95% trifluoroacetic acid, 2.5% H2O, and 2.5% triethylsilane. The crude peptides were purified (greater than

98%

homogeneity)

by

reverse

phase

high

performance

liquid

chromatography (RP-HPLC) using a Vydac 214TP™ C4 reversed-phase columns for polypeptides with particle size of 5 µm and pore size of 300 Å (Grace, Deerfield, IL). Purification of the peptides was done using a linear


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Biochemistry

gradient (10-90%) of acetonitrile (AcN) in water (both containing 0.1% TFA (v/v)) for 40 minutes. Minimal Inhibitory Concentration The minimal inhibitory concentrations (MICs) against polymyxin B, LL-37 and two de-novo designed peptides All L-K6L9 and D,L-K6L9 to the standard broth microdilution method

26

25

were assessed according

with some modifications27. Briefly,

peptide activity was examined in sterile 96-well plates. Overnight cultures (midlog phase) of WT and arnB mutant strains were washed and re-suspended in BM2 medium supplemented with low (20 µM) or high (2 mM) MgSO4. Aliquots of 50 µL suspended bacteria (1×106 colony forming units, CFU/mL) were added to 50 µL BM2 medium containing peptides in serial two-fold dilutions (final concentration range between 25 to 0.4 µM). Plates were incubated for ~18 hours at 37°C with agitation. Inhibition of growth was determined by measuring A600 using a microplate autoreader (Synergy HT, BioTek). The MIC was determined as the minimal concentration at which 90% inhibition of growth was observed after 18 hours of incubation. The values represent at least three technical repeats from three independent experiments. For some peptides and conditions, the MIC concentration changed between replicates and the values are presented as a range. Biofilm Formation Assay The biofilm formation was assessed as previously described

28

with some

modifications. Briefly, overnight cultures (mid-log phase) of WT and arnB mutant strains were washed and re-suspended in BM2 medium supplemented with low


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(20 µM) or high (2 mM) MgSO4. Aliquots of 100 µL suspended bacteria (1×106 colony forming units, CFU/mL) were transferred to sterile 96- well plates. Plates were incubated for 24 hours at 37°C without agitation, to allow biofilm formation. Plates were then washed from unattached bacteria and stained with 0.1% crystal violet (CV) followed by measuring A595 using a microplate autoreader (Synergy HT, BioTek). Swarming Motility Assay . Sterile 6-well Costar plates (CC-3596 Corning inc., Getter, IL) were filled with 6 mL of swarming medium (semi solid 0.5% agar) by dissolving 5% sterilized Difco bacto agar and rapidly adding it to pre- heated BM2 medium supplemented with low (20µM) MgSO4. The plates were left to solidify at room temperature for one hour, and then 1 µL of an overnight culture was placed in the middle of a plate. Plates were incubated for ~18 hours at 37°C on a solid surface. Plates were scanned using ImageScanner III (Danyel Biotech). Bacterial Adhesion Assay Bacterial adhesion assay was performed as described for the biofilm formation assay with some alterations. The plate was incubated at 37°C for one hour without agitation to allow bacterial adhesion. After washing wells to remove unattached bacteria, initial adhesion was evaluated by CV staining and measuring A595 using a microplate autoreader (Synergy HT, BioTek). Confocal Fluorescence Microscopy We visualized the WT and arnB mutant biofilm using an Olympus FV1000 confocal microscope, objective lens 40X (oil). Both strains (1×106 CFU/mL) were


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Biochemistry

incubated in an eight well chambered cover-glass (Nunc, Thermo Scientific, Waltham, Massachusetts, USA) at 45° angle, for 24 hours to allow biofilm formation. Chambers were washed three times with fresh BM2 medium and bacterial cells were stained by the provided protocol using Filmtracer live/dead biofilm viability kit (Invitrogen, Life Technologies, Carlsbad, California, USA). Syto9 (488 nm) stained live bacteria and propidium iodide (559 nm) stained dead bacteria. Data analysis was done using Olympus Fluoview (Ver. 4.1) and ImageJ, n≥46 fields. Results are reported as the fluorescence intensity of each channel and the fold change between live and dead bacteria. Tri-Calcium Phosphate Adhesion Assay The charge of the outer surface of the bacteria was examined using a modification of the hydroxyapatite adherence assay described by Lachica and Zink

29

. Overnight cultures of WT and arnB mutant were washed three times and

adjusted to A600=1 in filtered saline (0.85% NaCl). Acid washed glass tubes were filled with 100 mg of tri-calcium phosphat (Carl Roth 8450.1, BDL, IL) and 2 mL of suspended bacteria per tube. Bacterial samples were agitated with a VortexGenie mixer at the #2 settings for 2 minutes followed by 30 minutes incubation at room temperature to allow phase separation. A sample of ~700 µL from the aqueous phase of each tube was transferred to a cuvette and A600 was measured. The relative turbidity was calculated by comparison to a bacterial sample where tri-calciumphosphate was not added. Assay of Bacterial Adhesion to Hydrocarbons


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Bacterial hydrophobicity was examined using a modification of the adhesion assay described by Rosenberg et al.

30

. Bacterial cells were grown for two cycles

of 22 hours each in BM2 medium supplemented with low (20 µM) MgSO4. Bacteria were washed twice with 10 mM potassium chloride solution, then suspended in 10 mM potassium chloride supplemented with 2 M ammonium sulfate and adjusted to A600=0.3. Glass culture tubes were filled with one mL of ndodecane (112-40-3 Merck, Mercury, IL) and four mL of suspended bacteria. The mixtures were vigorously vortexed for two minutes at room temperature, followed by 15 minutes incubation at room temperature to allow phase separation. A sample of ~700 µL from the aqueous phase from each tube was transferred to a cuvette and A600 was measured. The relative turbidity was calculated by comparison to a tube with n-dodecane. Bacterial Self-Aggregation WT and arnB mutant strains were cultured in BM2 medium supplemented with low (20 µM) MgSO4 for 24 hours at room temperature by shaking at ~125 RPM (ELMI sky-line shaker, Ornat, IL). Turbidity was measured after the bacteria were allowed to settle for one minute. Then an aliquot of one mL was sampled out, transferred to a cuvette and A600 was measured. Maximal turbidity was measured after the tube was vigorously vortexed for two minutes at room temperature and an aliquot of one mL was transferred to a cuvette for A600 measurement. The relative turbidity was calculated by comparing the two measurements. Atomic Force Microscopy


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Biochemistry

Glass slides were soaked in H2SO4 diluted with ultra-pure (UP) water (4:1) for 20 minutes, then rinsed three times in UP water. The slides were left to dry overnight at room temperature. A drop of 0.1% poly- Lys, was left to dry in the middle of the slide, followed by washing the slide with UP water. Overnight cultures of WT and arnB mutant were washed and re- suspended in 300 mM sucrose. A drop of bacterial culture was placed on the glass slide and incubated for 30 minutes at room temperature. The slides were then washed in 300 mM sucrose and taken for AFM analysis, without allowing the sample to dry. AFM images of bacteria were acquired with JPK AFM (JPK Nanowizard III, Berlin, Germany). Images were recorded in the sucrose solution, at room temperature (22–24°C), in Quantitative Imaging (QITM) mode using a Scan Asyst Fluid (Bruker, Santa Barbara, CA, USA) probe with the radius of 20-60 nm.

The

deflection sensitivity was calibrated on the glass slide, and spring constant determined by the thermal noise method

31

. The QI mode yields simultaneous

topographic, stiffness, and adhesion images, as well as corresponding forcedistance curves at each pixel. The peak applied force for each such curve was adjusted to about 5 nano-newton and the scan rate was set to 15-30 µm/s. for total z travel of 1 µm. This resulted in depth of penetration of up to 150 nm on the bacteria, but only the initial penetration to depth of 30 nm was used in the analysis. Data analysis was performed using the JPK data processing software, a Herzian model and assumed Poisson ratio of 0.4 and nominal tip radius of 40 nm. Guided by the topographical image, 3 curves from the center of each bacterium were


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analyzed for n>15 individual bacteria for each bacterial strain. Numerical data presented is the mean value ±SE (standard error) of the Young’s modulus. Statistical Analysis Differences between group means were tested using two-tailed student t-test. Unless indicated differently, results are the mean ± SE of at least three technical repeats

from

three

independent

experiments

(*P < 0.05,

**P < 0.01,

***P < 0.001).

Results To test the contribution of the arnBCADTEF operon based LPS modification on biofilm formation we used the P. aeruginosa PAO1 WT strain and PW7021, an arnB mutant strain

21, 22

. Of note, the transposon insertion is not

predicted to have polar effects on the mutant strain 32. WT and arnB Mutant Lipid A Composition as Revealed by MALDI-TOF Mass Spectrometry There are several known lipid A modifications that have been demonstrated in the P. aeruginosa PAO1 laboratory strain in response to environmental changes 9, 33, 34

. To identify the modifications in the strains we performed MALDI-TOF

mass spectrometry to the lipid A moiety of both WT and arnB mutant, grown in low and high Mg2+ concentration (Figure 1). As expected, the spectrum of both strains grown in BM2 supplemented with high Mg2+ concentration reveals the same pattern of lipid A forms (Figure 1A and 1B). The peak at m/z 1,446 represents the penta-acylated, missing C10 acyl chain at position 3 of lipid A and


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the peak at m/z 1,616 represents the hexa-acylated, including C10 acyl chain at position 3 form of lipid A35. An additional peak in m/z 1,696 represents the addition of a third phosphate group to the lipid A9. The peak observed in m/z 1,632 represents the hydroxylation of the 1,616 form36. Growing bacteria in BM2 supplemented with low Mg2+ concentration resulted in a WT strain with two L-Ara4N moieties added to the penta-acylated lipid A (m/z 1,694)37 (Figure 1C). In the arnB mutant peaks at m/z 1684 and 1700 represents the pentaacylated, including palmitate lipid A and its hydroxylated form respectively (Figure 1D)38. ∆arnB

WT A

E

B 1446.168

1616.198

1616.344

High Mg2+

(i) m/ z= 1,616 1696.177

1696.320

1446.052 1366.177

1526.126

1366.056 1526.017 1275.965

(ii) m/ z= 1,446 1200

1400

1800

1600

C

2000

1200

1400

1600

1800

2000

(iii) m/ z= 1,684

D

1632.288

1 6 3 1 .9 0 1

Low Mg2+

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1650.919

1 6 8 4 .1 1 0

(iv) m/ z= 1,694 1696.208 1374.288

1 8 7 0 .2 6 1

1447.048 1533.084

1200

1400

1600

1800

2000

1200

1400

1600

1800

2000 (v) m/ z= 1,696

Figure 1: MALDI-TOF Negative-Ion Mode Analysis of Lipid A Structural Modifications of P. Aeruginosa WT and arnB Mutant. Both the penta-acylated missing C10 acyl chain at position 3, and the hydroxylated form, are presented at m/z 1,446 and m/z 1,462, respectively. The hexaacylated, including C10 acyl chain at position 3 and its hydroxylated form are presented at m/z 1,616 and m/z 1,632, respectively. MALDI-TOF mass spectrum of lipid A isolated from WT (A) or


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2+

arnB mutant (B) strains grown with a high (2 mM) Mg concentration, show a peak at m/z 1,696. This corresponds to a hexa-acylated lipid A which includes a third phosphate group. MALDI-TOF mass spectrum of lipid A isolated from WT (C) or arnB mutant (D) strain grown with a low (20 µM) 2+ Mg concentration, show a peak at m/z 1,694. This corresponds to a penta-acylated lipid A including two L-Ara4N moieties. Other peaks at m/z 1,684 and m/z 1,700, correspond to a pentaacylated including lipid A palmitate and its hydroxylated form. (E) Diagram of P. aeruginosa Lipid A structure illustrating that the backbone consists of two glucosamine and four directly attached decanoate (black) and one phosphate groups on each carbohydrate (green circle). Covalent modifications include (i) addition of laurate (C12:0) (blue); (ii) removal of deaconate; (iii) addition of palmitate (C16:0) (green); (iv) addition of L-Ara4N (red ellipse) and (v) addition of a third phosphate group (green circle). Given m/z values are the average mass of two independent experiments rounded to the nearest whole number.

Polysaccharide Phenotypes of WT and arnB Mutant Strains Changes in the lipid A amount can potentially affect the morphology of the outer membrane of the bacteria. Transmission electron microscopy was used to visualize the plasma membrane of the bacteria (PM), outer membrane (OM), and LPS components. No visible changes in the PM or OM were observed between the strains and growth conditions (Figure 2). Interestingly, the polysaccharide morphology appeared different under different growth conditions, as the uranyl acetate staining of the polysaccharide is stronger for bacteria grown in high Mg2+ concentration (Figure 2A and 2B) than in low Mg2+ concentration (Figure 2C and 2D). To verify this observation, the amount of membrane bound polysaccharides was measured by staining live bacteria with Calcofluor white (a fluorochrome that binds polysaccharides) and measuring its mean fluorescence intensity

39

. Flow

cytometry analysis was used to plot the Calcofluor white fluorescence and measure the mean fluorescence intensity of both strains in either growth conditions, as shown in Figure 2 E-H. Our results reveal that the mean Calcofluor white fluorescence intensity of the WT and arnB mutant grown in limiting Mg2+ concentration is twofold lower than the WT grown in high Mg2+ concentration


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Biochemistry

(Figure 2I). The diagram in Figure 2J emphasizes the shift in the mean fluorescence intensity, hence the amount of membrane bound polysaccharides between bacteria grown in low Mg2+ concentration versus high Mg2+ concentration. Taken together, both TEM and flow cytometry results suggest that strains grown in high Mg2+ have more polysaccharides on their outer membrane. Figure 2: The P. aeruginosa WT and arnB mutant present a different LPS phenotype when grown in a limiting Mg2+ concentration. TEM micrograph thin sections of P. aeruginosa WT (A) and arnB mutant (B) cells grown either under 2+ a high (2 mM) Mg concentration or a 2+ limiting (20 µM) Mg concentration (C and D respectively). Cells were prepared by freeze-substitution. The black arrows indicate the bacterial polysaccharide (PS) and the white errors indicate the plasma membrane (PM) and the outer membrane (OM). Bars are 100 nm. Representative flow cytometry density plot showing the fluorescence intensity of the WT and arnB 2+ mutant grown in high (2 mM) Mg concentration (5,534 (E) and 5,455 (F), 2+ respectively) or limiting (20 µM) Mg concentration (2,503 (G) and 3,157 (H), respectively). Calcofluor white fluorescence was detected at 350nm. (I) Graphical summary of the mean Calcofluor white fluorescence intensity as a fold change from the WT strain grown 2+ under a high Mg concentration. Results are the mean ± SE of three independent experiments (*, p≤0.05; ***, p≤0.001). (J) Diagram presentation of the mean Calcofluor white fluorescence intensity shown in panels E-H, showing a shift in the relative amount of membrane bound polysaccharides between bacteria grown 2+ in low (20 µM) versus high (2 mM) Mg concentration.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 35

Resistance to Antimicrobial Peptides and Biofilm Biomass of WT and arnB Mutant It has been previously shown that addition of L-Ara4N to lipid A results in more resistant bacteria

10

. To find out a correlation between resistance to AMPs due to

lipid A modification and biofilm formation, we tested the minimal inhibitory concentration (MIC) of selected native and de-novo designed peptides against the wild type and arnB mutant (Table 1). As expected, the MIC values of the two native peptides polymyxin B and LL-37 were higher when tested against WT strain grown in limiting Mg2+ concentration compared to the same strain grown in high Mg2+ concentrations (12.5 µM vs. 1.56 µM and 12.5 µM vs. 6.25 µM, respectively). Furthermore, whereas in the high Mg2+ concentration there were no differences between the MICs of the arnB mutant and the WT, in the low Mg2+ concentration the arnB mutant remained susceptible to both peptides (0.78 µM for polymyxin B and 1.56 µM for LL-37). The MIC of the all L-K6L9 peptide

25, 40

was the same (3.13 µM) for both strains at both Mg2+ concentrations. The MIC of the D,L-K6L9 does not dependent on the growth conditions of both bacterial strains, but the arnB mutant is more susceptible to this peptide (1.56 µM compared to 3.125 µM of the WT). It is possible that the addition of L-Ara4N to lipid A is not sufficient to protect bacteria from these peptides and other


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Biochemistry

mechanisms can also be used. Therefore, we tested if the addition of L-Ara4N to lipid A plays a role in biofilm formation. Table 1: Minimal inhibitory concentration of various native and de-novo designed AMPs against planktonic forms of P. aeruginosa WT and arnB mutant a

MIC (µM) Polymyxin B

LL-37

All L- K6L9

D, L- K6L9

2+

Mg

Low concentration WT

6.2-12.5 0.8-1.6

∆arnB a

High

Low

High

Low

High

Low

High

12.5

6.2

3.1

3.1

3.1

3.1-6.2

1.6

3.1

3.1

3.1

1.6

1. 6

b

0.8

0.8-1.6

The MIC (µM) was measured by a serial dilution method performed in a 96-well

polystyrene plate. Plates were incubated for 12 h at 37◦C followed by A600 measurements for bacterial growth. MICs90 is the concentration that inhibited 90 % of bacterial growth. b

2+

Mg

concentrations: low= 20 µM, high= 2 mM

Biofilm biomass of both the WT and arnB mutant strains were evaluated using crystal violet staining. BM2 medium supplemented with low (20 µM) Mg2+ concentration was used since differences in lipid A composition and AMPs susceptibility were observed only under this condition. The arnB mutant biofilm


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

biomass was five times higher than the WT (A590 1.44 and 0.29, respectively) (Figure 3A). Biofilm biomass is composed of bacteria and EPS, both detected by crystal violet staining. Confocal microscopy images with Syto-9 and propidiumiodide live/dead kit staining showed differences in the amounts of biofilm embedded bacteria and the relation between the live and dead cell populations (Figure 3B-E). These results reveal that the arnB mutant biofilm contains six times more live bacteria than the WT (Figure 3C), whereas both show the same levels of dead bacteria staining (Figure 3D). Overall, the ratio of live versus dead bacteria is 4.2 for the arnB mutant and 0.7 for the WT (Figure 3E). Next, we looked for phenotypical changes between the WT and the arnB mutant strains that might explain the differences in biofilm formation.


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Biochemistry

Figure 3: Biofilm formation by P. aeruginosa WT and arnB mutant. (A) P. aeruginosa WT and arnB mutant strains were incubated statically at 37°C for 24 hours in BM2 medium 2+ supplemented with a low (20 µM) Mg concentration. Surface associated biofilm was examined using 0.1% crystal violet staining followed by A595 measurements. Results are the mean ± SE of four independent experiments (*, p≤0.05). (B) Representative images of 24-hour P. aeruginosa WT and arnB mutant biofilms stained with Syto 9 (live bacteria) and propidium iodide (dead bacteria) (Filmtracer live/dead biofilm viability kit, Invitrogen). Images were taken using an Olympus FV1000 confocal microscope, objective lens 40X (oil), white bars size= 40 µm. Mean fluorescence intensity of live (C) and dead (D) bacteria, and the live to dead bacteria ratio (E) of the two strains. Data analysis was done using Olympus Fluoview (Ver. 4.1) and ImageJ, n≥46 fields. Results are the mean ± SE of three independent experiments (***, p≤0.001).

Biofilm Formation by WT and arnB Mutant Biofilm starts from planktonic motile bacteria that can adhere to a surface, 15

proliferate and create biofilm

. One type of motility is swarming, characterized

by a fast and coordinated bacterial movement over a semi-solid surface. There is evidence supporting a relation between swarming, AMPs resistance and biofilm formation. Swarming cells were shown to be more tolerant to AMPs and create unstructured biofilms

41, 42

. Here, swarming of the WT on semi-solid agar reveals

a well distinct fractal-like pattern. Such a pattern is typical for P. aeruginosa

43

. In

comparison, the arnB mutant shows a small portion of tendrils (Figure 4A) indicative of the capacity for more prominent biofilm formation 43. The motility of planktonic bacteria can influence its initial adhesion to surfaces, which is the first stage of biofilm formation. The initial amount of bacteria that adhere to the surface can influence the mature biofilm biomass. LPS was previously shown to play a role in the initial attachment and biofilm formation of P. aeruginosa

44

. We used an initial adhesion assay to evaluate the

bacterial attachment to polystyrene after one-hour incubation. The initial attachment of arnB mutant is 1.7 times higher than the WT (A590 0.63 and 0.38, respectively) (Figure 4B). The initial adhesion of bacteria to the polystyrene can 21 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

be also affected by the properties of the bacterial outer surface. Therefore, we looked for unique biochemical and biophysical features that can shed light on the mechanism of biofilm formation, and might explain the differences between the strains phenotypes. Figure 4: Swarming motility and adhesion to polystyrene of P. aeruginosa WT and arnB mutant. (A) P. aeruginosa WT and arnB mutant swarming motility on semi-solid agar. Bacterial motility was examined after ~15 hours incubation in 37°C on 0.5% agar BM2 medium plates 2+ supplemented with low (20 µM) Mg concentration. (B) P. aeruginosa WT and arnB mutant adhesion to polystyrene after one-hour incubation (statically) at 37°C in BM2 medium 2+ supplemented with a low (20 µM) Mg concentration. Adhered bacteria were stained with 0.1% crystal violet staining followed by A595 measurements. Results are the mean ± SE of three independent experiments (*, p≤0.05).

Hydrophobicity and Charge of the Outer Surface of WT and arnB Mutant Electrostatic forces and hydrophobic interactions were previously shown to support biofilm formation

45

. Since lipid A modifications, as detected for the WT

and arnB mutant, can potentially affect the charge of the bacterial outer surface, hydrophobicity and self-aggregation, we evaluated the physical properties of the strains. The net negative charge of the WT and arnB mutant strains was evaluated by allowing bacterial precipitation with the positively charged tri-


Page 22 of 35

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Biochemistry

calcium phosphate and measuring the percentage of the remaining cells in the supernatant. As expected, the net negative charge of the WT strain was higher (25.5%) than that of the arnB mutant (14.8%) (Figure 5A). Next, we measured bacterial precipitation with n-dodecane to evaluate the relative hydrophobicity of the outer surface. The data reveal that the hydrophobicity of the outer surface of the WT is significantly higher than that of the arnB mutant (66.5% and 56.9%, respectively) (Figure 5B). We also tested the self-aggregation of the bacteria and found that the WT strain tends to aggregate more than the arnB mutant (42.4% and 35.2%, respectively) (Figure 5C). It has been suggested that the rigidity of the outer surface of bacteria is associated with resistance to AMPs

46

. We have recently reported that LTA

alteration in Gram-positive bacteria increase cell wall density and rigidity

27

. We

therefore suggest that strains of Gram-negative bacteria lacking the ability to alter their LPS, will also confer differences in their mechanical properties. This can further explain their susceptibility to AMPs and their ability to create more biofilms. In order to test the mechanical properties of the bacteria, we used atomic force microscopy (AFM) which allow us to measure nanoscale changes in the outer membrane of the bacteria, with a minimal intervention with cell stability. To restrict our measurements to the LPS layer of the bacteria, we calculated the young’s modulus referring to ~30nm from the penetration point. The rigidity of the arnB mutant was 1.6 fold higher than that of the WT (147.6±12.9kPa and 89.1±6.4kPa, respectively) (Figure 5D).


Biochemistry

In summary, our data present differences in the physical properties of the bacteria that correlate with the chemical changes found in the lipid A of the two strains. These properties explain the promoted biofilm formation of the arnB mutant strain.

*

B 80

30 25 20 15 10 5 0 WT WT

∆arnB ∆arnB

WT

**

C 80 70 60 50 40 30 20 10 0

70 60 50 40 30 20 10 0 ∆arnB

***

D 180 160 140 120 100 80 60 40 20 0

Rigidity (Young’s modulus)

**

A 35

Relative turbidity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 35

WT

∆arnB ∆arnB

WT

∆arnB ∆arnB

Figure 5: Physical characteristics of the outer surface of P. aeruginosa WT and arnB mutant. Relative charge (A) and hydrophobicity (B) as calculated by the relative turbidity (A600) of the upper aqueous phase after bacterial precipitation with tri-calcium phosphate or n-dodecane respectively. (C) Self aggregation as calculated by the relative turbidity (A600) of each strain after bacterial culturing for 24 hours in room temperature and allowing sedimentation of clumps. (D) Mean rigidity of bacterial surface as calculated by the Young’s modulus (n≥15 cells). Results are the mean ± SE of three or four independent experiments (*, p≤0.05; **, p≤0.01; ***, p≤0.001).

Discussion In this study, we aimed to shed light on the relation between biofilm formation and LPS modifications. We show that the later affect the biochemical and mechanical properties of the outer cell membrane, as well as the swarming ability of the WT and the mutant bacteria. Specifically, we focused on one major path of LPS remodeling namely the addition of a positively charged L-Ara4N to lipid A, mediated by the arnBCADTEF operon under limiting Mg2+ conditions

13, 37

Magnesium limitation promotes biofilm formation by Pseudomonas aeruginosa

.

47

via the activation of the PhoPQ two-components system (TCS), which directly repress RetS, a biofilm repressor

48

. In addition, PhoPQ TCS upregulates the 24

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Biochemistry

transcription of arnBCADTEF operon and pagP, genes responsible for several lipid A modifications

13

. To elucidate the potential role of lipid A modifications on

the biochemical and biophysical properties of the outer surface of the bacteria that promote biofilm formation. We investigated two P. aeruginosa strains, WT PAO1 and PAO1 with a loss of function mutation in the arnB gene. The data revealed that the arnB mutant is susceptible to AMPs when in a planktonic form and possesses physical surface features such as a higher net negative charge, elevated hydrophobicity and impaired swarming motility. Differences in lipid A composition between the strains are expected only when grown in a low Mg2+ concentration, that triggers LPS remodeling13. Indeed, when grown in a high Mg2+ concentration, both strains show the same three main species of lipid A; penta-acylated, hexa-acylated and hexa-acylated with the addition of a phosphate group, common to these growth conditions

9, 35, 36, 49

.

Growing the strains in low Mg2+ concentration resulted in the absence of a lipid A moiety containing a third phosphate group. In the WT strain, a penta-acylated lipid A with two positively charged L-Ara4N moieties masks the phosphate charge

37

. This is in line with its reduced net negative charge, which makes it

more resistant to AMPs. The lipid A of arnB mutant contains penta-acylated lipid A with addition of palmitate

38

. Palmitoylation indicates activation of the PhoPQ

TCS, as expected in low Mg2+ conditions. Palmitate can potentially lower the membrane fluidity and make it more rigid

50

, which indeed correlates with our

AFM analysis. It was shown that the cell surface of planktonic bacteria is less rigid than

the cell surface of biofilm embedded bacteria


51

. We show that

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 35

modifications of the rigidity of lipid A alter the bacterial outer surface. Furthermore, we show positive correlation between the rigidity of the outer surface and biofilm formation. Though more rigid, the arnB mutant did not show elevated resistance, in line with a previous study suggesting that addition of palmitate does not qualify for bacterial resistance against AMPs 52. With respect to biofilm formation, it appears that the addition of L-Ara4N results in reduced biofilm formation. Biofilm growth can be promoted by; (i) bacterial motility; (ii) adhesion to the surface; (iii) recruitment and proliferation inside the biofilm. Swarming motility can affect the formation of biofilms

53

. We show that

the swarming phenotype is different for cells that go through specific lipid A modifications. The impaired swarming phenotype of the arnB mutant shows a biofilm like centered shape pattern. This suggests that biofilm formation and swarming are opposing behaviors of P. aeruginosa. This is in line with the Salmonella

pmrHFIJKLM

operon

(parallel

to

arnBCADTEF

operon

Pseudomonas) that also adds L-Ara4N, is upregulated during swarming

in 41

,

suggesting a link between swarming motility and the addition L-Ara4N to lipid A. LPS is a major wetting agent in P. aeruginosa, which is essential for swarming motility. A mutation in the LPS O-antigen was reported to eliminate swarming motility

54

, presumably by affecting surface "wettability". We suggest that lipid A

modifications are additional factors that can affect bacterial motility, however the exact mechanism should be further investigated. Non-motile cells can potentially bind faster to the surface, and also detach less from the mature biofilm

53

. Taken

together, with the physical properties of the arnB mutant (discussed below), this


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Biochemistry

finding can explain its improved initial reversible adhesion and bacterial permanent recruitment to the constructed biofilm, as supported by our confocal images (Figure 3). When both strains were exposed to a low concentration of Mg2+, we observed a reduced amount of membrane bound polysaccharides on the bacterial outer membrane (see TEM and FACS results). A similar phenotype was previously reported when chelating agents such as EDTA and Tris were used

55

. These data suggest that the loss of the polysaccharide shield exposes

membranes with different features between the strains, hence affecting their mechanism of biofilm formation.

Hydrophobic forces encourage bacterial

adhesion to abiotic surfaces 56. Together with electrostatic interactions, these two 45

forces were shown to support biofilm formation

. We show that lipid A

modifications affects the bacterial outer surface hydrophobicity and negative charge. Compared to the WT, the palmitoylated arnB mutant is more hydrophobic and possesses a higher net negatively charge, allowing better adhesion and biofilm formation. Hydrophobic properties have been shown to affect bacterial self-aggregation

57

, which directly correlate to biofilm formation

58

.

In contrast, the tendency of the arnB mutant to aggregate is lower than the WT despite its elevated hydrophobicity. These findings suggest that self-aggregation does not necessarily correlate to biofilm formation. It is possible that a higher net negative

charge

causes

bacterial repulsion,

which

results in

reduced

aggregation. This can make the arnB mutant more accessible for surface interactions. During the process of biofilm formation, bacteria secrete EPS. P. aeruginosa EPS mainly contains three central polysaccharides (alginate, pel and


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

psl) along with bacterial DNA and proteins

Page 28 of 35

59

. The contribution of EPS secretion

to biofilm formation was reported in the past

60

. Lipid A modifications can

potentially alter the content and form of secreted polysaccharides, which affect biofilm formation. In conclusion, lipid A modifications were shown to promote bacterial resistance in subpopulations found in biofilms

18, 19

, and observed in WT and

remodeling impaired mutant isolates taken from the lungs of CF patients

37, 61

.

However, it is unknown whether such modifications contribute to biofilm formation. In this study, we showed to our knowledge for the first time that the arnBCADTEF operon that adds L-Ara4N to lipid A is directly involved in biofilm formation. Phenotypes that were previously suggested to either contribute to biofilm formation or observed in biofilm embedded bacteria can now be explained by modifications in the lipid A. We suggest that this LPS modification and biofilm formation are opposing behaviors of bacteria. When planktonic stage bacteria encounter stress conditions, the WT bacteria remodel its lipid A by adding LAra4N, hence improving its resistance to AMPs. On the other hand, the arnB mutant, which forms more biofilm, adds palmitate to the lipid A, a phenotype that is well characterized in biofilm embedded bacteria, especially lung isolates from CF patients

8, 61, 62

. Together with impaired swarming motility and distinct

biochemical characteristics, we suggest that enhanced biofilm formation can compensate for the loss of L-Ara4N. These insights into the factors driving the bacteria to adopt one resistance mechanism over another may help in the future to design drugs and treatment regimens against multidrug-resistant bacteria.


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Biochemistry

Acknowledgments: The authors thank Batya Zarmi for her invaluable help with peptide purification, Vladimir Kiss for technical assistance with the confocal imaging, Dr Reinat Nevo for advising on the confocal analysis, Dr. Eyal Shimoni for technical assistance with the transmission electron microscope and Dr. Sidney Cohen for technical assistance with the atomic force microscope. P. aeruginosa PAO1 and arnB mutant strains were provided courtesy of Prof. R. EW. Hancock (Department of Microbiology and Immunology, University of British Columbia, Canada). Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions: LSZ prepared the materials, performed and designed most of the experiments, analyzed the data and wrote the paper. GK and MJ designed and performed experiments. YK advised on data analysis and writing of the paper. HGS advised on experimental design and writing of the paper. YS directed the project, designed the experiments and advised on writing the paper.

Funding: This work was partially supported by the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement [278998] and the German Israeli Foundation.


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References [1] Nuri, R., Shprung, T., and Shai, Y. (2015) Defensive remodeling: How bacterial surface properties and biofilm formation promote resistance to antimicrobial peptides, Biochimica et biophysica acta 1848, 3089-3100. [2] Band, V. I., and Weiss, D. S. (2015) Mechanisms of Antimicrobial Peptide Resistance in Gram-Negative Bacteria, Antibiotics 4, 18-41. [3] Costerton, J. W., Stewart, P. S., and Greenberg, E. P. (1999) Bacterial biofilms: a common cause of persistent infections, Science 284, 13181322. [4] Li, Y., Powell, D. A., Shaffer, S. A., Rasko, D. A., Pelletier, M. R., Leszyk, J. D., Scott, A. J., Masoudi, A., Goodlett, D. R., Wang, X., Raetz, C. R., and Ernst, R. K. (2012) LPS remodeling is an evolved survival strategy for bacteria, Proceedings of the National Academy of Sciences of the United States of America 109, 8716-8721. [5] Silhavy, T. J., Kahne, D., and Walker, S. (2010) The bacterial cell envelope, Cold Spring Harbor perspectives in biology 2, a000414. [6] Raetz, C. R., and Whitfield, C. (2002) Lipopolysaccharide endotoxins, Annual review of biochemistry 71, 635-700. [7] Steimle, A., Autenrieth, I. B., and Frick, J. S. (2016) Structure and function: Lipid A modifications in commensals and pathogens, International journal of medical microbiology : IJMM 306, 290-301. [8] Ernst, R. K., Moskowitz, S. M., Emerson, J. C., Kraig, G. M., Adams, K. N., Harvey, M. D., Ramsey, B., Speert, D. P., Burns, J. L., and Miller, S. I. (2007) Unique lipid a modifications in Pseudomonas aeruginosa isolated from the airways of patients with cystic fibrosis, The Journal of infectious diseases 196, 1088-1092. [9] Nowicki, E. M., O'Brien, J. P., Brodbelt, J. S., and Trent, M. S. (2014) Characterization of Pseudomonas aeruginosa LpxT reveals dual positional lipid A kinase activity and co-ordinated control of outer membrane modification, Molecular microbiology 94, 728-741. [10] Needham, B. D., and Trent, M. S. (2013) Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis, Nature reviews. Microbiology 11, 467-481. [11] Breazeale, S. D., Ribeiro, A. A., and Raetz, C. R. (2003) Origin of lipid A species modified with 4-amino-4-deoxy-L-arabinose in polymyxin-resistant mutants of Escherichia coli. An aminotransferase (ArnB) that generates UDP-4-deoxyl-L-arabinose, The Journal of biological chemistry 278, 24731-24739. [12] Bhat, R., Marx, A., Galanos, C., and Conrad, R. S. (1990) Structural studies of lipid A from Pseudomonas aeruginosa PAO1: occurrence of 4-amino-4deoxyarabinose, Journal of bacteriology 172, 6631-6636. [13] McPhee, J. B., Bains, M., Winsor, G., Lewenza, S., Kwasnicka, A., Brazas, M. D., Brinkman, F. S., and Hancock, R. E. (2006) Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg2+-


Page 30 of 35

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Biochemistry

induced gene regulation in Pseudomonas aeruginosa, Journal of bacteriology 188, 3995-4006. [14] Clifton, L. A., Skoda, M. W., Le Brun, A. P., Ciesielski, F., Kuzmenko, I., Holt, S. A., and Lakey, J. H. (2015) Effect of divalent cation removal on the structure of gram-negative bacterial outer membrane models, Langmuir : the ACS journal of surfaces and colloids 31, 404-412. [15] Flemming, H. C., Wingender, J., Szewzyk, U., Steinberg, P., Rice, S. A., and Kjelleberg, S. (2016) Biofilms: an emergent form of bacterial life, Nature reviews. Microbiology 14, 563-575. [16] Flemming, H. C., and Wingender, J. (2010) The biofilm matrix, Nature reviews. Microbiology 8, 623-633. [17] Costerton, J. W., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., and Marrie, T. J. (1987) Bacterial biofilms in nature and disease, Annual review of microbiology 41, 435-464. [18] Haagensen, J. A., Klausen, M., Ernst, R. K., Miller, S. I., Folkesson, A., Tolker-Nielsen, T., and Molin, S. (2007) Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms, Journal of bacteriology 189, 28-37. [19] Pamp, S. J., Gjermansen, M., Johansen, H. K., and Tolker-Nielsen, T. (2008) Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes, Molecular microbiology 68, 223-240. [20] Hoiby, N., Krogh Johansen, H., Moser, C., Song, Z., Ciofu, O., and Kharazmi, A. (2001) Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of growth, Microbes Infect 3, 23-35. [21] Jacobs, M. A., Alwood, A., Thaipisuttikul, I., Spencer, D., Haugen, E., Ernst, S., Will, O., Kaul, R., Raymond, C., Levy, R., Chun-Rong, L., Guenthner, D., Bovee, D., Olson, M. V., and Manoil, C. (2003) Comprehensive transposon mutant library of Pseudomonas aeruginosa, Proceedings of the National Academy of Sciences of the United States of America 100, 14339-14344. [22] Held, K., Ramage, E., Jacobs, M., Gallagher, L., and Manoil, C. (2012) Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1, Journal of bacteriology 194, 6387-6389. [23] Yi, E. C., and Hackett, M. (2000) Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria, The Analyst 125, 651-656. [24] Hunter, R. C., and Beveridge, T. J. (2005) High-resolution visualization of Pseudomonas aeruginosa PAO1 biofilms by freeze-substitution transmission electron microscopy, Journal of bacteriology 187, 7619-7630. [25] Segev-Zarko, L., Saar-Dover, R., Brumfeld, V., Mangoni, M. L., and Shai, Y. (2015) Mechanisms of biofilm inhibition and degradation by antimicrobial peptides, The Biochemical journal 468, 259-270. [26] Wayne, P. (2012) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition., CLSI document M07-A9. 31 ACS Paragon Plus Environment

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[27] Saar-Dover, R., Bitler, A., Nezer, R., Shmuel-Galia, L., Firon, A., Shimoni, E., Trieu-Cuot, P., and Shai, Y. (2012) D-alanylation of lipoteichoic acids confers resistance to cationic peptides in group B streptococcus by increasing the cell wall density, PLoS pathogens 8, e1002891. [28] Segev-Zarko, L. A., and Shai, Y. (2017) Methods for Investigating Biofilm Inhibition and Degradation by Antimicrobial Peptides, Methods Mol Biol 1548, 309-322. [29] Lachica, R. V., and Zink, D. L. (1984) Plasmid-associated cell surface charge and hydrophobicity of Yersinia enterocolitica, Infection and immunity 44, 540-543. [30] Rosenberg, M., Perry, A., Bayer, E. A., Gutnick, D. L., Rosenberg, E., and Ofek, I. (1981) Adherence of Acinetobacter calcoaceticus RAG-1 to human epithelial cells and to hexadecane, Infection and immunity 33, 2933. [31] Hutter, J. L. B., J. (1993) Calibration of atomic‐force microscope tips, Review of Scientific Instruments 64, 1868-1873 [32] Fernandez, L., Gooderham, W. J., Bains, M., McPhee, J. B., Wiegand, I., and Hancock, R. E. (2010) Adaptive resistance to the "last hope" antibiotics polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel two-component regulatory system ParR-ParS, Antimicrob Agents Chemother 54, 3372-3382. [33] Bishop, R. E., Gibbons, H. S., Guina, T., Trent, M. S., Miller, S. I., and Raetz, C. R. (2000) Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria, The EMBO journal 19, 5071-5080. [34] Guo, L., Lim, K. B., Poduje, C. M., Daniel, M., Gunn, J. S., Hackett, M., and Miller, S. I. (1998) Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides, Cell 95, 189-198. [35] Gellatly, S. L., Needham, B., Madera, L., Trent, M. S., and Hancock, R. E. (2012) The Pseudomonas aeruginosa PhoP-PhoQ two-component regulatory system is induced upon interaction with epithelial cells and controls cytotoxicity and inflammation, Infection and immunity 80, 31223131. [36] King, J. D., Kocincova, D., Westman, E. L., and Lam, J. S. (2009) Review: Lipopolysaccharide biosynthesis in Pseudomonas aeruginosa, Innate immunity 15, 261-312. [37] Moskowitz, S. M., Brannon, M. K., Dasgupta, N., Pier, M., Sgambati, N., Miller, A. K., Selgrade, S. E., Miller, S. I., Denton, M., Conway, S. P., Johansen, H. K., and Hoiby, N. (2012) PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin-treated cystic fibrosis patients, Antimicrob Agents Chemother 56, 1019-1030. [38] Thaipisuttikul, I., Hittle, L. E., Chandra, R., Zangari, D., Dixon, C. L., Garrett, T. A., Rasko, D. A., Dasgupta, N., Moskowitz, S. M., Malmstrom, L., Goodlett, D. R., Miller, S. I., Bishop, R. E., and Ernst, R. K. (2014) A divergent Pseudomonas aeruginosa palmitoyltransferase essential for cystic fibrosis-specific lipid A, Molecular microbiology 91, 158-174.


Page 32 of 35

Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

[39] Puga, C. H., Orgaz, B., and SanJose, C. (2016) Listeria monocytogenes Impact on Mature or Old Pseudomonas fluorescens Biofilms During Growth at 4 and 20 degrees C, Frontiers in microbiology 7, 134. [40] Papo, N., Oren, Z., Pag, U., Sahl, H. G., and Shai, Y. (2002) The consequence of sequence alteration of an amphipathic alpha-helical antimicrobial peptide and its diastereomers, The Journal of biological chemistry 277, 33913-33921. [41] Kim, W., Killam, T., Sood, V., and Surette, M. G. (2003) Swarm-cell differentiation in Salmonella enterica serovar typhimurium results in elevated resistance to multiple antibiotics, Journal of bacteriology 185, 3111-3117. [42] Shrout, J. D., Chopp, D. L., Just, C. L., Hentzer, M., Givskov, M., and Parsek, M. R. (2006) The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional, Molecular microbiology 62, 1264-1277. [43] Tremblay, J., and Deziel, E. (2010) Gene expression in Pseudomonas aeruginosa swarming motility, BMC genomics 11, 587. [44] Ramsey, M. M., and Whiteley, M. (2004) Pseudomonas aeruginosa attachment and biofilm development in dynamic environments, Molecular microbiology 53, 1075-1087. [45] Krasowska, A., and Sigler, K. (2014) How microorganisms use hydrophobicity and what does this mean for human needs?, Frontiers in cellular and infection microbiology 4, 112. [46] Joo, H. S., Fu, C. I., and Otto, M. (2016) Bacterial strategies of resistance to antimicrobial peptides, Philosophical transactions of the Royal Society of London. Series B, Biological sciences 371(1695), 20150292. [47] Mulcahy, H., and Lewenza, S. (2011) Magnesium limitation is an environmental trigger of the Pseudomonas aeruginosa biofilm lifestyle, PLoS One 6, e23307. [48] Goodman, A. L., Kulasekara, B., Rietsch, A., Boyd, D., Smith, R. S., and Lory, S. (2004) A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa, Dev Cell 7, 745-754. [49] Bedoux, G., Vallee-Rehel, K., Kooistra, O., Zahringer, U., and Haras, D. (2004) Lipid A components from Pseudomonas aeruginosa PAO1 (serotype O5) and mutant strains investigated by electrospray ionization ion-trap mass spectrometry, J Mass Spectrom 39, 505-513. [50] Whitfield, C., and Trent, M. S. (2014) Biosynthesis and export of bacterial lipopolysaccharides, Annual review of biochemistry 83, 99-128. [51] Benamara, H., Rihouey, C., Jouenne, T., and Alexandre, S. (2011) Impact of the biofilm mode of growth on the inner membrane phospholipid composition and lipid domains in Pseudomonas aeruginosa, Biochimica et biophysica acta 1808, 98-105. [52] Vaara, M., and Nurminen, M. (1999) Outer membrane permeability barrier in Escherichia coli mutants that are defective in the late acyltransferases of lipid A biosynthesis, Antimicrob Agents Chemother 43, 1459-1462. 33 ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[53] Verstraeten, N., Braeken, K., Debkumari, B., Fauvart, M., Fransaer, J., Vermant, J., and Michiels, J. (2008) Living on a surface: swarming and biofilm formation, Trends Microbiol 16, 496-506. [54] Toguchi, A., Siano, M., Burkart, M., and Harshey, R. M. (2000) Genetics of swarming motility in Salmonella enterica serovar typhimurium: critical role for lipopolysaccharide, Journal of bacteriology 182, 6308-6321. [55] Vaara, M. (1992) Agents that increase the permeability of the outer membrane, Microbiological reviews 56, 395-411. [56] Renner, L. D., and Weibel, D. B. (2011) Physicochemical regulation of biofilm formation, MRS bulletin 36, 347-355. [57] Doyle, R. J. (2000) Contribution of the hydrophobic effect to microbial infection, Microbes Infect 2, 391-400. [58] Sorroche, F. G., Spesia, M. B., Zorreguieta, A., and Giordano, W. (2012) A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from Argentina, Applied and environmental microbiology 78, 4092-4101. [59] Wei, Q., and Ma, L. Z. (2013) Biofilm matrix and its regulation in Pseudomonas aeruginosa, Int J Mol Sci 14, 20983-21005. [60] Ghafoor, A., Hay, I. D., and Rehm, B. H. (2011) Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture, Applied and environmental microbiology 77, 5238-5246. [61] Chalabaev, S., Chauhan, A., Novikov, A., Iyer, P., Szczesny, M., Beloin, C., Caroff, M., and Ghigo, J. M. (2014) Biofilms formed by gram-negative bacteria undergo increased lipid a palmitoylation, enhancing in vivo survival, mBio 5(4), e01116-14. [62] Ciornei, C. D., Novikov, A., Beloin, C., Fitting, C., Caroff, M., Ghigo, J. M., Cavaillon, J. M., and Adib-Conquy, M. (2010) Biofilm-forming Pseudomonas aeruginosa bacteria undergo lipopolysaccharide structural modifications and induce enhanced inflammatory cytokine response in human monocytes, Innate immunity 16, 288-301.


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Biochemistry

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