Antimicrobial activity of GN peptides and
their mode of action
Troels Godballe1, Biljana Mojsoska 1, Hanne M. Nielsen2, Håvard Jenssen1,*
1
Dept. of Science, Systems & Models, Roskilde University, Universitetsvej 1, DK-4000 Roskilde,
Denmark.
2
Dept. of Pharmacy, Biologics, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen,
Denmark.
*Corresponding Author: jenssen@ruc.dk Tel +45 4674 2877
Supplementary data
Matherial and methods
Flow cytometry
Flow cytometry experiments were performed to investigate the peptide effect on both bacterial
DNA content and change in cell size after exposure with peptides. In order to be able to follow the
changes over time, samples were extracted at 0, 20, 40, 80, 120 and 180 minutes. Overnight culture
of E. coli ATCC 25922 was diluted 1:50 in fresh Mueller Hinton broth and allowed to grow until an
OD600 of 0.1 had been reached. The suspension was then diluted 1:10 in fresh Mueller Hinton broth
and re-grown to an OD600 of 0.1 to ensure a uniform bacterial population. A total of 90 μL of
bacterial suspension was loaded into a flat bottomed 96-well plate for peptide and antibiotic
exposure. After extraction of the zero sample, 10 μL of peptide solution corresponding to 10× and
40× the MIC concentration was added for a total volume of 100 μL. Two wells were loaded for
each treatment to ensure enough bacteria for the flow cytometry analysis. Immediately after
extraction, the contents of two wells were transferred to a single E-tube and put on ice. The plate
was placed at 37°C between extractions. The samples were, whenever appropriate during the
experiment, centrifuged at 10.000 g for 5 min at 4°C. The supernatant was then carefully removed
and the cells were resuspended in 100 μL 10 mM Tris HCL pH 7.4 and 1000 μL 77% Ethanol was
added before storing the samples at 4° C for later analysis. The Rifampicin/Cephalexin samples
were prepared in the same way as the other samples with the exception that the 200 μL from the
wells were transferred to an E-tube with 45 μL mixture of Rifampicin (300 μg/mL) and Cephalexin
(36 μg/mL) and placed in a 37°C water bath for 2½ hours. Simultaneously, the OD600 was measured
approximately every 25 minutes in a multi-detection microplate reader Synergy HT to monitor
bacterial growth. The following day, the samples were centrifuged at 10.000 g for 5 min before
gently removing the supernatant and adding 140 μL staining solution (90 μg/mL Mitramycin and 20
μg/mL Ethidium Bromide in 10 mM Tris pH 7.4, 10 mM MgCl2). The samples where run on an
A10 Bryte Flow Cytometer and when possible, 20.000 events were included in the analysis.
Circular dichroism (CD) spectroscopy assay
CD spectroscopy measurements were conducted to identify secondary structure of peptides, using
both the prokaryotic membrane mimicking liposomes and mammalian mimicking liposomes
described previously and also with 10 mM Tris buffer. Spectra were obtained in the far UV
spectrum (195-250 nm) with a Jasco J-715 CD spectrophotometer at room temperature with a N2
flow of 40 L/min using a 0.1 cm path-length quartz cuvette at a scan rate of 50 nm/minutes at 1.0
nm intervals. The spectrophotometer was calibrated to read +190.4 ± 1 millidegrees (mdeg) at a
wavelength of 291 nm with a 1 cm path-length quartz cuvette using a 0.6 mg/mL solution of
ammonium d-Camphor 10-sulfonate, as prescribed by the manufacturer. Liposomes was added at
concentration of 2 mM and peptides at 20 µM to obtain a lipid to peptide ratio of 100:1 as
previously described (1). The displayed CD spectra are the mean of 5 scans which are smoothed
using a Savitzky-Golay routine and corrected for the relevant background contribution.
Results
Peptide quality
The peptides was purified on a reverse phase column to a purity greater than 95% and the precise
mass was verified using Dionex Ultimate 3000 RP-UHPLC (C18 Kinetex 100 x 2.1 mm, 100 Å)
electrospray ionization mass spectrometry (Finnigan LTQ) (Figure S1, Table S1).
A
B
C
D
E
F
G
H
I
J
Figure S1. Mass spectrum and LC-MS chromatogram of the peptides GN-2 (A), GN-3 (B), GN-4
(C), GN-5 (D), GN-6 (E), GN-8 (F), GN-9 (G), GN-11 (H), GN-12 (I) and GN-14 (J).
Table S1. Peptide characteristics, antimicrobial activity and toxicity
Peptide
Sequence
RWKRWWRWI-CONH2
GN-2
KWWRWRRWW-CONH2
GN-3
RWKKWWRWL-CONH2
GN-4
KKRWWWWWR-CONH2
GN-5
RKRWWWWFR-CONH2
GN-6
IWKRWWWKR-CONH2
GN-8
RIWKIWWKR-CONH2
GN-9
IKWKRWWWR-CONH2
GN-11
KWWKIWRWR-CONH2
GN-12
RLWKRWWIR-CONH2
GN-14
Indolicidin ILPWKWPWWPWRR-CONH2
HPLC retention
Observed mass
Theoretical
time (min)
[M+2H]2+ (Da)
mass (Da)
6.33
6.62
6.54
7.10
7.08
4.54
4.63
5.88
6.13
4.43
ND
736.75
773.58
723.00
759.50
754.00
722.58
686.25
722.67
772.67
700.17
ND
1471.78
1544.83
1443.77
1516.82
1505.80
1443.77
1370.71
1443.77
1443.77
1398.73
1906.28
Peptide effect on bacterial morphology
Insights into the effect of the GN peptides on cell size and DNA content was investigated by the use
of flow cytometry. The assay was done in microtitter format over the course of 3 hours with a
starting inoculum of OD600 0.1 to ensure sufficient bacterial numbers for analysis. By treating
bacterial cells with a mixture of the nucleic acid stains Mitramycin and Ethidium Bromide,
fluorescence detected by the flow cytometry apparatus, corresponds to DNA content in cells and
detected lightscatter is fairly proportional to cell size. The control samples decreases gradually in
both size and DNA over the course of 3 hours (Figure S2), in agreement with previous observations
(2), ascribed to the bacterial population approaching stationary phase. For both GN-2 and GN-6, the
addition of peptides at 1×MIC seem to induce a period of somewhat constant cell size up until 80
minutes, where a slight decrease in cell size sets in (Figure S2). Addition of either peptide at
1×MIC have no pronounced effect on cell size, except for a slight indication of delaying the
decrease in cell size which is also observed in the control samples. A more distinct effect on cell
size is observed when the peptides are added at concentrations that correlate to 4×MIC, displaying a
clear and rapid increase. The rather dramatic increase in cell size takes effect already after 20
minutes and reveals a slight increase up until the last measurement at 180 minutes.
B
80
80
60
60
60
40
Lightscatter
Fluorescense
Lightscatter
A
40
20
30
60
90
120
150
20
20
0
0
0
0
40
180
0
0
30
30
60
60
Control
90
90
120
120
150
150
180
180
Time (min)
(min)
Time
Time (min)
Control
GN-2 25 µg/ml
GN-2 6.25 µg/ml
C
GN-2 25 µg/ml
GN-2 6.25 µg/ml
D
80
60
50
Fluorescense
Lightscatter
60
40
40
30
20
20
10
0
0
0
30
60
90
120
150
180
0
30
Time (min)
Control
GN-6 12.5 µg/ml
60
90
120
150
180
Time (min)
GN-6 50 µg/ml
Control
GN-6 12.5 µg/ml
GN-6 50 µg/ml
Figure S2. Flow cytometry investigation of exponentially growing E. coli exposed to peptides GN2 (A-B) and GN-6 (C-D) at concentrations corresponding to 1× and 4×MIC. Lightscatter is
equivalent to bacterial size and the measured fluorescence corresponds to bacterial DNA content.
The experiment was performed over the course of 3 hours in a microtitter plate format with an
inoculum of OD600 of 0.1 and incubated at 37°C between sample extractions. Whenever possible,
20.000 events were included in the analysis.
Determination of peptide secondary structure in membrane models.
Secondary structure has in many contexts been shown to be important for activity of antimicrobial
peptides and consequently, secondary structure was investigated using circular dichroism
spectroscopy. Secondary structure of this set of GN peptides was investigated, both in Tris buffer
pH 7.4, and LUVs consisting of POPC:POPG:Cholesterol (5:2:3 molar ratio) and POPC:POPG (3:7
molar ratio), mimicking mammalian and bacterial membranes, respectively (Figure S2).
210
220 (nm)
Wavelength
0
-10
-10
-20
20
Melittin
GN-2
10
GN-3
0
-20
210
GN-4
GN-5
220
230
240
GN-6
GN-8
200
GN-9
210
0
220
230
240
-10
Bacterial membrane mimic
-20
GN-3
GN-4
GN-5
GN-12
GN-14
Mel
Melittin
GN-
GN-2
GN-
GN-3
GN-
GN-
GN-4
GN-
GN-5
GN-6
Melittin
GN-
GN-2
GN-4
GN-3
GN-9
200
GN210
220
230
Wavelength (nm)
GN-11
GN-6
GN-8
GN-9
GN-5 GN-
GN-8
Wavelength (nm)
GN-11
GN-2
10
-20
Wavelength (nm)
Melittin
20
-10
-10
-20
200
C
Mammalian membrane mimic
Molar Elipticity [q] deg cm2 dmol-1 / 103
10
240
B
0
Tris buffer
20
230
Molar Elipticity [] deg
200
Molar Elipticity [q] deg cm2 dmol-1 / 103
Molar Elipticity [] deg cm2 dmol-1 / 103
Molar Elipticity [q] x 10-3 (deg cm2 dmol-1)
A
10
0
GN-12
GN-11
240
GN-
GN-
GN-12
GN-14
GN-14
Figure S3. CD spectra of peptides in (A) Tris buffer, (B) POPC:POPG:Cholesterol (5:2:3 molar ratio), (C) POPG:POPC (3:7 molar ratio).
Peptide concentration is kept 20 μM and LUV concentration to 2 mM for a 100:1 of a lipid to peptide ratio. Results are the mean of 5 scans
and processed using Savitsky-Golay algorithm.
GN-12
GN-14
References
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2.
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