Supplementary Information
Studies on the assembly of a leucine zipper antibacterial peptide and its analogs
onto mammalian cells and bacteria
Aqeel Ahmad, Sarfuddin Azmi and Jimut Kanti Ghosh*
Methods
Peptide Synthesis, their Fluorescent Labeling and Purification
Stepwise solid phase syntheses of all the peptides, their labeling at the N-terminus by
fluorescent probes, their cleavage from resins and purification by reverse phase HPLC
were achieved by standard procedures as described earlier [Ahmad et al., 2006; Fields
and Noble, 1990; Ghosh et al., 1997; Yadav et al., 2003]. The purified peptides were
~95% homogeneous and experimental molecular mass of the peptides, detected by
MALDI-TOF analysis, corresponded very close to the desired values as reported [Ahmad
et al., 2006]. The concentrations of the peptides were determined by measuring their
absorbance at 280 nm in 6 M guanidine hydrochloride with the help of tryptophan
extinction coefficients of 5690.
Preparation of Large Unilamellar Vesicles (LUVs)
LUVs were prepared by a standard procedure [Ahmad et al., 2006; Asthana et al.,
2004; Ghosh et al., 1997; Yadav et al., 2003] with required amounts of either of the
PC/cholesterol (8:1 w/w), or PC/PG (1:1 w/w) as follows [Hawrani et al., 2008]. Dry
lipids of required amounts were dissolved in CHCl3/MeOH (2:1 v/v) in small glass vials.
Solvents were evaporated under a stream of nitrogen, which resulted in the formation of a
thin film on the wall of glass vial. The glass vials containing lipid films were further kept
under vacuum for ~ 24 hours in order to remove even a trace amount of organic solvents.
The thin film was then re-suspended in PBS at a concentration of 8.2 mg/ml by vortex
mixing. The lipid dispersions were then sonicated in a bath-type sonicator (Laboratory
Supplies Company, New York) for 10-20 min until it became transparent. The lipid
concentration was determined by phosphorus determination[Bartlett, 1959].
Assay of cytotoxicity of peptides against 3T3 cells
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was done to
check the cytotoxic activity of the peptides against 3T3 cells by a standard procedure as
described earlier [Nordahl et al., 2005]. 10000 cells per well were seeded in 96 well
plates and overnight incubation was done in CO2 incubator for adherence. The complete
media were discarded from the plate and incomplete media were supplemented. After
that, different concentration of peptides were added and incubated for 2 hrs. Now 10 l
of MTT (conc. 5mg/ml) solution were added in each well and again incubated for 3 hrs.
Incomplete media were discarded from 96 well plates and 200 l of DMSO were mixed
with the crystal in each well to dissolve them. The well in which no peptide was added
was considered as the control cells of 100% viability. Readings of these samples were
taken at 550 nm by an ELISA reader [Nordahl et al., 2005]. The viability of the peptidetreated cells was calculated with respect to the control cells of 100% viability.
Hemolytic activity assay:
Hemolytic activity of these designed peptides was assayed against fresh hRBCs that were
collected in the presence of an anti-coagulant from a healthy volunteer by a standard
procedure [Ahmad et al., 2006] as reported earlier except the final concentration of the
hRBCs was (0.6% final in v/v equivalent to ~3.0x 107 cells/ml) in PBS. This assay was
performed at least thrice at different point of time during the course of the whole study
with blood from different healthy voluntary blood donors in order to avoid any effect of
their blood cholesterol.
Assay of peptide-induced depolarization of mammalian and bacterial cell membrane
Peptide-induced depolarization of the hRBC, 3T3, E. coli and S. aureus cell
membrane was detected by their efficacy to dissipate the potential across these cell
membranes [Papo et al., 2004; Papo and Shai, 2005; Yadav et al., 2008]. Fresh human
red blood cells were collected in the presence of an anti-coagulant from a healthy
volunteer and washed three times in PBS and re-suspended in the same buffer with a final
cell density of ~ 3.0x 107 cells/ml. While for 3T3, final cell density was ~105 cells/ml.
E. coli and S. aureus were grown at 37°C until it reached to its midlog phase and
centrifuged followed by washing with buffer (20 mM glucose, 5 mM HEPES pH 7.3).
Then bacteria were re-suspended (final ~2x105 CFU/ml) in the similar buffer containing
0.1M KCl[Senol et al., 2007; Suarez et al., 2005; Wiegand et al., 2008] [Senol et al.,
2007; Suarez et al., 2005; Wiegand et al., 2008]. The mammalian cells and bacteria were
incubated with diS-C3-5 probe for 1hr. When the fluorescence level (excitation and
emission wavelengths set at 620 and 670 nm respectively) of the hRBCs or bacterial
suspension became stable, different amounts of each of the peptides were added to these
suspensions in order to record the peptide-induced membrane depolarization of either
mammalian or bacterial membrane.
Membrane depolarization as measured by the
fluorescence recovery (Ft) was defined by the equation [Ghosh et al., 1997; Yadav et al.,
2003].
Ft = [(It - I0)/(If - I0)] x 100 %.
Where If, the total fluorescence, was the fluorescence levels of cell suspensions
just after the addition of diS-C3-5; It, was the observed fluorescence after the addition of a
peptide at a particular concentration either to hRBCs, 3T3, E. coli or to S. aureus
suspensions, which were already incubated with diS-C3-5 probe for 1hr and I0, was the
steady-state fluorescence level of the cell suspensions after one hr incubation with the
probe.
Analysis of peptide-induced membrane damage of mammalian and bacterial cells
Peptide-induced phospholipid asymmetry or damage of phospholipid membrane
organization of hRBCs was determined by staining the cells (~3.0x 107 cells/ml) with
FITC-annexin V [Kuypers et al., 1996; Yadav et al., 2008] after the treatment with the
peptides at r.t. for 5 min. Peptide-induced membrane damage of 3T3 cells (final cell
density ~105 cells/ml) was probed by propidium iodide staining of the cells after the
peptide treatment at 370C for 15 min. The cells were then analyzed by flow cytometry in
the form of dots plot with respect to the control cells, not treated with any peptide. In
order to check the membrane integrity of E. coli, E coli ATCC 10536 and S. aureus
after peptide treatment, the cells at mid-log phase were incubated with LZP, SASA or
DASA for 30 min at 37°C with constant shaking [Park et al., 2006]. The cells were
centrifuged, washed two times with PBS, and incubated further with propidium iodide at
4°C for 30 min, followed by removal of the unbound dye through washing with an excess
of PBS and re-suspended in buffer. Peptide-induced damage of bacterial cells was then
analyzed by flow cytometry.
Supplementary Figure 1 Determination of cytotoxic activity of LZP and its analogs
SASA and DASA against murine 3T3 cells. Dose-dependent MTT assay with 3T3 cells
in the presence of LZP (a), SASA (b) and DASA (c). Final cell density was 10000
cells/well.
% of Hemolytic Activity
70
a
60
50
40
b
30
20
10
0 0
c
5
10
15
20
25
30
Peptide (M)
Supplementary Figure 2: Dose-dependent hemolytic activity of the LZP, a; SASA [LZP
(L8A)], b; and DASA [LZP (L8,11 A), c; against the human red blood cells. The final
concentration of hRBCs was 0.6% v/v (~3x 107 cells/ml). The hRBC samples were
centrifuged after the treatment and incubation with peptides for 10 min at 2000 r.p.m. and
the release of hemoglobin was monitored by measuring the absorbance of the supernatant
at 540 nm. Hemolytic activity of the peptides was calculated by usual procedure as
reported before (Ahmad et al., J. Biol. Chem. 281,22029-22038).
Supplementary Figure-3: Highlights of quadrant Statistics of flow cytometric data
presented in Figure- 1.
Suplimentary Figure-4: Determination of peptide-induced membrane damage of E. coli
and S. aureus cells by flow cytometric studies. The left (A to D) and right (E to H) hand
side respectively depict the PI staining of E. coli and S. aureus cells after the treatment
with different peptides. The panels in the left hand side show PI staining of E. coli either
without any peptide treatment (A) or treated with ~ 8.0 M of LZP (B), SASA (C) and
DASA (D). Panels E to H show the PI staining of S. aureus cells without any peptide
treatment and treated with ~8.0 M of LZP, SASA and DASA respectively. On Y axis,
FSC-Height is forward scattered height which shows the distribution of the cells. In X
axis, FL2-Height means fluorescence recorded by fluorescent filter 2 (red channel).
10,000 events were counted for each experiment.
Supplementary Figure-5: Highlights of quadrant Statistics of flow cytometric data
presented in Supplementary Figure- 4.
Supplementary Fig. 6: Determination of peptide-induced membrane damage of E. coli
ATCC 10536 by flow cytometric studies. The left hand side (A to D) depict the PI
staining of cells without any peptide treatment (A) and treated with ~ 8.4 M of LZP (B),
SASA (C) and DASA (D) respectively.
The right hand side shows the detection of self-assembly of the LZP, SASA and
DASA onto the E. coli ATCC 10536 cells by FRET experiments with the help of flow
cytometry. Equimolar amounts of donor- (NBD) labeled peptides (D) and either the
corresponding unlabeled peptide (U) or acceptor- (Rho) labeled peptide (A) incubated
with E. coli ATCC 10536 at 370C and then analyzed by flow cytometry. Black (thinner)
and gray (thicker) lines represent (D+U) and (D+A) respectively. (E), (F) and (G) show
the FRET experiments onto bacteria with LZP, SASA and DASA respectively with ~7.2
M of each of the peptides. 10000 events were counted for each experiment.
Supplementary Figure 7:
Detection of NBD-labeled LZP, SASA and DASA and their unlabeled version induced
membrane damage of S. aureus by flow cytometric studies. In each panel quadrants a, b,
c and d represent the PI labeled cells, unlabeled cells, cells labeled with PI and NBD both
and NBD labeled cells respectively. At left hand side panels A to D depict the PI staining
of cells without any peptide treatment and treated with ~ 8.0 M of NBD-LZP, NBDSASA and NBD-DASA respectively. E, F and G depict the similar experiments as in the
panels B, C and D; only bacteria were treated with unlabeled LZP, SASA and DASA
respectively instead of their NBD-labeled versions. 10000 events were counted in each
case.
Supplementary Figure 8:
Left hand side shows the dose-dependent peptide-induced transmembrane depolarization
of S. aureus by NBD-labeled LZP, SASA and DASA and their unlabled versions at
different concentrations. Panels A, B and C show the fluorescence profiles of membrane
depolarization of bacteria induced by two different concentrations (a and b, 9.6 M and c
and d, 6.6 M) of NBD-labeled (dashed line, b and d) and the corresponding unlabeled
(solid line, a and c) LZP, SASA and DASA respectively.
The right hand side shows the dose-dependent peptide-induced transmembrane
depolarization of hRBCs by Rho-labeled and unlabeled LZP, SASA and DASA at
different peptide concentrations. Panels D, E and F show the fluorescence profiles of
membrane depolarization of hRBCs at two different peptide concentrations (a and b, 30.6
M and c and d, 6.0 M) of labeled (dashed line, b and d) and unlabeled (solid line, a and
c) LZP, SASA and DASA respectively.
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