1

Supplementary Figure 1

ATG CAA TTT ACC ACC ATC CTC TCC ATC GGT ATC ACC GTC TTC GGA CTT CTC AAC ACC GGA 60

M Q F T T I L S I G I T V F G L L N T G 20

[------------------------------------signal peptide----------------------------

GCC TTT GCA GCA CCC CAG CCT GTT CCC GAG GCT TAC GCT GTT TCT GAT CCC GAG GCT CAT 120

A F A A P Q P V P E A Y A V S D P E A H 40

---------] [-------------------------pro-peptide------------------------------

CCT GAC GAT TTT GCT GGT ATG GAT GCG AAC CAA CTT CAG AAA CGT GGA TTT GGA TGC AAT 180

P D D F A G M D A N Q L Q K R G F G C N 60

---------------------------------------------------------] [-------------------

GGT CCT TGG GAT GAG GAT GAT ATG CAG TGC CAC AAT CAC TGC AAG TCT ATT AAG GGT TAC 240

G P W D E D D M Q C H N H C K S I K G Y 80

-------------mature plectasin--------------------------------------------------

AAG GGA GGT TAT TGT GCT AAG GGG GGC TTT GTT TGC AAG TGT TAC 285

K G G Y C A K G G F V C K C Y 95

---------------------------------------------------------]

2

Supplementary Figure 2

NOEs by residue

0

10

20

30

40

50

60

1 5 10 15 20 25 30 35 40sequence

intra-residue sequential medium-range long-range

Supplementary Figure 3

3

Supplementary Figure 4

4

Supplementary Figure 5

5

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1

Pre-pro plectasin sequence. The cDNA was isolated and cloned from P. nigrella

as described in Methods.

Supplementary Figure 2

NOEs used for structure calculation of plectasin. Intra NOEs are NOEs between

protons belonging to the same sequential position. Sequential NOEs are NOEs

between pairs of protons from amino acids sequentially separated by 1 to 5

positions in the sequence and long range NOEs are NOEs from pairs of protons

from sequence positions separated by 5 or more sequence positions.

Supplementary Figure 3

Sequential NOEs listed as bars with thickness indicating the strength of the

NOE assigned. Full bar size refer to strong NOEs (distance < 2.7Å), medium

(2.7Å < distance < 4.0Å) and weak NOEs (distance > 4.0Å). For structure

calculation purposes the interpretation of NOE strength was a little different.

Strong NOEs were assigned to distances between 1.0 and 2.7 Å, medium

NOEs between 1.0 Å and 3.3 Å and weak NOEs from 1.0 Å to 5.5 Å. If NOEs

included one methyl group in the cross peak the upper level of distances

applied were added 0.5 Å.

Supplementary Figure 4

6

Ramachandran plot from the Procheck program showing the overall quality of

the backbone of the plectasin structure as derived from proton NMR data.

Supplementary Figure 5

Extract from Procheck, showing a schematic overview of the secondary

structural elements, and the average accessibility. Circular variance shows the

variation of a specific dihedral angle, and provides a measure of accuracy in

angular measures independent of the structural alignment procedure utilized.

7

Supplementary Table 1

Proton resonance assignments of Plectasin at 1 mM in 30 mM acetic acid, pH 3.8). Proton NMR spectra are recorded at 600 MHz using a temperature at 27° C.

Spin system HN HA Others:

Gly-1 3.68, 3.58 Phe-2 8.58 3.98 HB#a: 2.847, HB#b: 2.711, HD#: 6.818, HE#: 7.180Gly-3 8.07 4.58, 3.40 Cys-4 7.57 4.69 HB1: 2.590, HB2: 2.740Asn-5 8.07 5.12 HB1: 3.015, HB2: 2.782, HD2#a: 7.461, HD2#b: 6.872Gly-6 7.13 3.37, 1.53 Pro-7 3.96 HB#a: 1.201, HB#b: 1.858, HG#a: 1.501, HG#b: 1.759, HD#a: 2.923, HD#b: 3.083Trp-8 7.26 4.74 HB#a: 3.139, HB#b: 3.424, HD1: 7.138, HE1: 10.147, HE3: 7.658, HH2: 7.335, HZ2: 7.566, HZ3: 7.221Asp-9 7.48 4.65 HB1: 2.493, HB2: 2.406Glu-10 8.31 4.83 HB#a: 2.070, HB#b: 1.922, HG#a: 2.475, HG#b: 2.226Asp-11 8.44 4.86 HB1: 3.052, HB2: 2.588Asp-12 8.38 4.35 HB1: 2.713, HB2: 2.944Met-13 8.37 4.40 HB#a: 2.183, HG#a: 2.603, HE#: 2.013Gln-14 7.63 4.09 HB1: 2.013, HB2: 2.102, HG#a: 2.454, HG#b: 2.197, HE2#a: 7.700, HE2#b: 6.960Cys-15 7.34 4.84 HB#a: 3.720, HB#b: 2.585His-16 9.03 3.81 HB#a: 3.507, HB#b: 3.655, HD2: 7.353, HE1: 8.476Asn-17 8.60 4.30 HB1: 2.780, HB2: 2.889, HD2#a: 6.906, HD2#b: 7.544His-18 8.14 4.28 HB1: 3.593, HB2: 3.255, HD2: 6.664, HE1: 8.037Cys-19 8.59 3.68 HB#a: 2.234, HB#b: 2.126Lys-20 7.78 3.73 HB#a: 1.552, HG#a: 1.191, HD#a: 1.453, HE#: 2.963Ser-21 7.19 4.32 HB#a: 3.992, HB#b: 3.857Ile-22 7.56 3.99 HB: 2.004, HG1#a: 0.729, HG1#b: 1.327, HG2#: 0.804, HD1#: 0.483Lys-23 8.19 3.89 HB#a: 1.730, HB#b: 1.638, HG#a: 1.304, HG#b: 1.422, HD#: 1.631, HE#: 2.939Gly-24 8.56 4.06, 3.45 Tyr-25 7.69 4.90 HB#a: 3.131, HB#b: 2.794, HD#: 7.016, HE#: 6.656Lys-26 10.73 4.41 HB#a: 1.750, HB#b: 1.843, HG#a: 1.519, HG#b: 1.463, HD#a: 1.760, HE#: 3.060Gly-27 7.38 3.26, 3.52 Gly-28 8.45 4.23, 4.84 Tyr-29 8.49 4.80 HB1: 3.232, HB2: 3.068, HD#: 7.013, HE#: 6.656Cys-30 8.98 5.49 HB1: 2.567, HB2: 2.852Ala-31 9.44 4.70 HB#: 1.437Lys-32 8.86 3.97 HB#a: 1.841, HB#b: 1.950, HG#a: 1.437, HD#a: 1.699, HE#: 2.986Gly-33 8.77 4.24, 3.63 Gly-34 7.54 3.57, 3.15 Phe-35 7.48 4.29 HB#a: 3.297, HB#b: 2.706, HD#: 7.295, HE#: 7.348, 7.397Val-36 7.62 4.36 HB: 1.725, HG1#: 0.808, HG2#: 0.922Cys-37 8.78 5.09 HB1: 3.245, HB2: 2.576Lys-38 9.03 4.32 HB#a: 1.545, HG#a: 1.153, HD#a: 1.225, HE#: 2.988Cys-39 7.91 5.29 HB1: 1.962, HB2: 1.327Tyr-40 8.23 4.60 HB#a: 3.220, HB#b: 2.750, HD#: 6.959, HE#: 6.646

8

Supplementary Table 2

Structural statistics of the solution structure of plectasin

Distance constraints#: 635

Intra-residue 272

Sequential (|i-j|=1) 140

Medium-range (1<|i-j|<5) 111

Long-range (|i-j|≥5) 112

Dihedral angle restraints totally: 37

Φ angles 21

χ1 angles 16

Average number of all NOE restraint violations

0.0 Å to 0.1 Å 20.7 ± 5.3

> 0.1 Å 0

Average number of dihedral restraint violations

> 1 0

Deviations from idealized geometry

Impropers 0.09° ± 0.02° (0 violations above

5°)

Angles 0.23° ± 0.01° (0 violations above 5°)

Bonds 0.0007 Å ± 0.0002 Å (0 violations above

0.05 Å)

Energies##

Total energy 12.8 ± 2.9

Bond 0.31 ± 0.12

Angle 8.2 ± 0.8

Improper 0.42 ± 0.16

Repel 3.8 ± 1.9

9

NOE 3.0 ± 1.8

Torsion 0.017 ± 0.018

Atomic root mean square values for 10 calculated structures versus average coordinates:

atoms included: RMS [Å]

backbone (2-39) 0.78 ± 0.17

heavy atoms (2-39) 1.20 ± 0.17

PROCHECK Ramachandran plot statistics

Residues in most favoured regions 80.0%

Residues in additional allowed regions 19.7%

Residues in generously allowed regions 0.3%

Residues in disallowed regions 0.0%

# All distance restraints are based on NOESY cross peaks.

##Energies (kcal mol-1), measured with simplified repel potential used in the simulated annealing protocol,

kNOE=50 kcal mol-1 Å-2, ktor=200 kcal mol-1 rad-2, kimpr=500 kcal mol-1 rad-2, kbond=1000 kcal mol-1 Å-2, kang=500

kcal mol-1 rad-2, and krepel=4 kcal mol-1 rad-2 (vdw radii set 0.80 relative to normal values )

10

Supplementary Methods

Transposon-assisted signal trapping (TAST)

To trap clones encoding a signal peptide, the recombinant plasmid pool was prepared

from 30,000 transformants according to the Qiagen protocol (Qiagen Inc.). After

treatment with transposon SigA2 and MuA transposase (Finnizymes, Oy)23, the plasmid

mixture was again electroporated into E. coli DH10B (Invitrogen). An ORF of the E.

coli beta-lactamase gene product, lacking a signal peptide was inserted into the EcoRI-

NotI cloning sites directly adjacent to the left transposon border. Insertion of the

transposon in a secreted gene can result in an in-frame fusion, resulting in secretion of

the beta-lactamase fusion peptide and rendering the E. coli colonies ampicillin resistant.

To estimate transposition frequency, LB agar with kanamycin (50 mg/L) and

chloramphenicol (12mg/L) were used. To recover signal trapped cDNA clones, 15 mg/L

ampicillin was included in the selective plates. Colonies were observed after 2 days at

28 oC. After three days, trappants were replica plated on LB

kanamycin/chloramphenicol plates supplemented with 50 mg/L ampicillin. Plasmids

were isolated from surviving colonies and sequenced to identify the transposed

sequence. Vector sequence removal, low quality sequence trimming and sequence

assembly of the signal trapped clones were done with PhredPhrap package

(http://www.phrap.org). The resulting contigs were searched against the EnsemblPep,

GeneSeqP, PDB, Swall, and SwissProt data bases, using BlastX, a fast global alignment

algorithm47. Additional analysis tools included SignalP and SSEARCHp48,49.

Killing kinetics

In vitro time kill curves for plectasin, penicillin (Leo Pharma A/S, Ballerup, Denmark)

and vancomycin (Sigma-Aldrich, MO, US), respectively, were determined against

11

Streptococcus pneumoniae strain D39 (serotype 2; MIC mg/L: plectasin 1, penicillin

0.032, vancomycin 0.25), strain 68034 (serotype 3; MIC mg/L: plectasin 1, penicillin

0.016, vancomycin 0.25) and strain 6A (serotype 6A; MIC mg/L: plectasin 0.5,

penicillin 0.016, vancomycin 0.5). The bactericidal effect of plectasin was tested at 1, 5

and 10 times MIC, vancomycin at 1 and 10 times MIC and penicillin at 1 and 8 times

the MIC according to the method described in the NCCLS guideline (M26-A). In brief,

a suspension prepared of fresh overnight colonies was added to a total of 20 ml Mueller

Hinton BBLII broth (Becton Dickinson, NJ, US), and incubated on a water bath at

35°C. After one hour of incubation, 1 ml broth from each flask was replaced with the

test solution. Samples for colony determinations were taken at time 0, 1, 3 and 5 hours

after addition of the test solutions. To minimize drug carry-over effect, the non-diluted

samples were streaked on an agar plate, and all further serial sample-dilutions applied as

20 µl spots on agar plates. Colony counts were determined after 20-24 hours incubation

at 35°C in 5% CO2.

Hemolysis, cytotoxicity, serum stability

To measure hemolytic activity, freshly obtained washed human red blood cells (2% v/v

final) were incubated for 1 h in phosphate buffered saline with serial two-fold dilutions

of plectasin, and hemoglobin released to the supernatant was measured at 540 nm.

Cytotoxicity was measured by the neutral red uptake procedure of Borenfreund and

Puerner50, using mouse L929 fibroblasts (ATCC CCL-1) and a 24 hr exposure to

purified plectasin (maximal concentration 512 mg/L). To assess serum stability,

plectasin (100 mg/L) was incubated at 37°C in 10% and 90% fresh normal human

serum for 0, 1, 3, 6 & 24 hours. Residual activities were measured by performing radial

diffusion assays with Staphylococcus carnosus.

12

Pharmacokinetics

The kinetics of plectasin was investigated in NMRI female mice, 25-30 gram (Harlan

Scandinavia Aps), after a single 14 mg/kg dose, administered by either intraveneous,

subcutaneous or intraperitoneal routes. Plectasin concentrations in serum samples were

determined 5, 15, 30, 60, 120 and 180 minutes after dosing, using two mice at each time

point. At each time point, urine was collected, then mice were anaesthetized with CO2

and blood collected. The blood samples were centrifuged and serum collected. The

concentration of plectasin in serum and urine were determined by LCMSMS.

Sample preparation

Serum and urine samples were diluted 1:10 or 1:20 with 0.01% TFA in polypropylene

tubes. The diluted samples were subsequently diluted additional 10 – 50 times with

ACN:H2O:CH3COOH:TFA (20:72:8:0.01). Samples were mixed and directly analyzed

by LCMSMS. Standards were prepared in ACN:H2O:CH3COOH:TFA (20:72:8:0.01).

The method was linear over 4 – 1440 µg/L.

LCMSMS method

LCMSMS-equipment comprised a Waters Alliance 2695 HPLC equipped with column

heater (set at 30 oC) and sample cooler (set at 5 oC). The mass spectrometer was a

Quattro Micro (Waters/Micromass, Manchester, UK) operating in ESI+ mode. Both

instruments were controlled by MassLynx 4.0 (Waters/Micromass, Manchester, UK).

13

The column used was a Biosuite PA-A C18, 3 µm particles, 100 x 2.1 mm (I.D.)

(Waters, Milford, MA). Elution was done using a gradient from 5% acetonitrile to 50%

in 9.5 min followed by an increase to 80% acetonitrile in 3 min. This was held constant

for 4.5 min before returning to initial conditions. The concentration of modifier (8%

acetic acid, 0.01% TFA) was held constant during the run. Injection volume: 10 µL.

MS-setup: The triple quadropole instrument was run in ESI+ using the following

conditions: capillary voltage: 3.5 kV, cone voltage: 32 V, source temp: 150°C,

desolvation gas temp: 350 oC, desolvation gas flow (N2): 650 L/hr, collision energy: 32

eV, collision gas: Ar. Data acquisition was done in SRM mode using the transitions:

881.4 > 120.1 amu.

Mouse infection studies

The antimicrobial efficacy of plectasin was tested in two murine infection models32,33

against three Streptococcus pneumoniae isolates with serotypes 2 (strain D39), serotype

3 (strain 68034) and serotype 6A (strain 6A).

Outbred, NMRI female mice, 25-30 g (Harlan Scandinavia Aps) were used in both the

peritonitis and pneumonia infection models. Both animal models were approved by the

Animal Experiments Inspectorate under the Danish Ministry of Justice.

A pneumococcal suspension of fresh overnight colonies was inoculated

intraperitoneally one hour before start of treatment. A single dose of 10 mg/kg plectasin

i.v. or 70 mg/kg vancomycin (Alpharma A/S, Oslo, Norway) s.c. was tested in groups

of three mice (the two drugs were tested in independent studies). Control groups of

untreated mice were also included. Colony counts from peritoneal fluid were

determined at 0, 3 and 5 hours after start of treatment. The mice were sacrificed and 2

14

ml sterile 0.9% saline was injected i.p. and the abdomen gently massaged before the

abdomen was opened and fluid sampled with a pipette.

In addition, the survival of mice infected i.p. one hour before start of treatment with

plectasin was investigated. Mice were treated s.c. with plectasin 10 mg/kg/dose in

groups of 8 mice. The following dose regimens were tested in mice infected with the

D39 strain; vehicle at 0, 5, 24 and 29 hrs, plectasin at 0 hr, 0 and 5 hrs or 0, 5, 24 and 29

hrs. The dose regimens tested in mice infected with strain 68034 were: vehicle at 0, 5,

24 and 29 hrs, plectasin at 0 and 5 hrs or 0, 5, 24 and 29 hrs. The mice were inspected 2-

3 times daily during the study period and each mouse was clinically scored based on its

behaviour and clinical signs. The mouse was sacrificed if severe pain and disease was

observed to minimize the suffering of the animal.

In the pneumonia model, the mice were anaesthetized (mixed solution of tiletamin,

zolazepam, narcosyl and buthorphanol) and hung by their front teeth on a steel wire.

The mice were then inoculated intranasally with 50 µl bacterial suspension of fresh

overnight colonies. After 10 minutes, mice were removed from the wire and left in their

cages. A single dose of 10 mg/kg plectasin was given the following day (approximately

24 hours after inoculation) in a group of 6-7 mice. A control group of untreated mice

were also included. In a similar study, mice were treated with two s.c. doses of 15

mg/kg penicillin (Leo Pharma A/S, Ballerup, Denmark). Colony counts from lung

homogenates were determined 24 hours after start of treatment. The mice were

sacrificed and lungs removed and homogenized in a total of 3 ml 0.9 % saline. Colony

counts from lung homogenates were determined and total number of colonies/lungs per

mouse was calculated.

The Mann-Whitney test was used to compare groups of plectasin-treated mice and none

plectasin-treated mice and the survival fractions were calculated by the Kaplan-Meier

15

method and curves compared using the logrank test (GraphPad Prism, GraphPad

Software, CA, US).

References

47. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

48. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6 (1997).

49. Smith, T. F. & Waterman, M. S. Identification of common molecular subsequences. J. Mol. Biol. 147, 195–197 (1981).

50. Borenfreund, E. & Puerner, J. A. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119–124 (1985).