Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and

26 Pages·2017·0.97 MB·English

by  Giorgia Manzo

bioRxiv preprint first posted online May. 1, 2018; doi: http://dx.doi.org/10.1101/312215. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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Minor sequence modifications in temporin B cause drastic changes in antibacterial potency and
selectivity by fundamentally altering membrane activity
Giorgia Manzo1, Philip M. Ferguson1, Vicente B. Gustilo1, Hind Ali1, Tam T. Bui2, Alex. F. Drake2, R.
Andrew Atkinson2, Giovanna Batoni3 Christian D. Lorenz4, David A. Phoenix5, A. James Mason1

1Institute of Pharmaceutical Science, School of Cancer & Pharmaceutical Science, King’s College London,
Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom; 2Centre for
Biomolecular Spectroscopy and Randall Division of Cell and Molecular Biophysics, King’s College
London, New Hunt’s House, London SE1 1UL, United Kingdom; 3Department of Translational Research
and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy; 4Department of Physics,
King’s College London, London WC2R 2LS, United Kingdom; 5School of Applied Science, London South
Bank University, 103 Borough Road, London SE1 0AA, United Kingdom
Running title: Patch-clamp to investigate antimicrobial peptides activity

Keywords: Antimicrobial peptides (AMP), Patch-clamp, molecular dynamics, peptide-membrane
interactions, structure-function, biophysics

ABSTRACT Escherichia coli. Our findings suggest even closely
Antimicrobial peptides (AMPs) are a potential related AMPs may target the same bacterium with
source of new molecules to counter the increase in fundamentally differing mechanisms of action.
antimicrobial resistant infections but a better
understanding of their properties is required for
effective translation as therapeutics. Details of the
The number of bacterial pathogens resistant
mechanism of their interaction with the bacterial
to available classes of antibiotics is increasing, with
plasma membrane are desired since damage or
the UK government estimating 10 million deaths
penetration of this structure is considered essential
due to antimicrobial resistance (AMR) by 2050 if
for AMP activity. Relatively modest modifications
no countermeasures are taken (1, 2). As a global
to AMP primary sequence can induce substantial
threat, the World Health Organization recognised in
changes in potency and/or spectrum of activity but,
2015 that discovery of new antibiotic classes is an
hitherto, have not been predicted to substantially
essential element of a global action plan to tackle
alter the mechanism of interaction with the bacterial
AMR (3).
plasma membrane. Here we use a combination of
molecular dynamics simulations, circular Antimicrobial peptides (AMPs) are a possible new
dichroism, liquid- and solid-state NMR and patch source of antibiotic molecules and attract interest
clamp to investigate the extent to which temporin B due to their multifaceted features which confer a
and its analogues can be distinguished both in vitro broad spectrum of activity and low rate of
and in silico on the basis of their interactions with resistance development (4, 5, 6). AMPs have been
model membranes. Enhancing the hydrophobicity isolated from almost all living forms, from bacteria
of the N-terminus and cationicity of the C-terminus to humans, where they act as a primary line of
in temporin B improves its membrane activity and defence against pathogenic infections (7, 8, 9). It is
potency against both Gram-negative and Gram- generally accepted that the main factor determining
positive bacteria. In contrast, enhancing the the potency of AMPs is their interaction with the
cationicity of the N-terminus abrogates its ability to bacterial plasma membrane and that they kill
trigger channel conductance and renders it pathogens by altering the permeation of the
ineffective against Staphylococcus aureus while membrane and causing cell lysis (4, 5, 6, 10) or by
nevertheless enhancing its potency against crossing the membrane to interact with intracellular
targets (4, 9, 11). To penetrate or damage the
1
bioRxiv preprint first posted online May. 1, 2018; doi: http://dx.doi.org/10.1101/312215. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

bacterial plasma membrane, AMPs must pass on its ability to insert and disrupt model membranes
through the lipopolysaccharide or lipoteichoic and the effect this has on bactericidal activity
layers in Gram-negative and Gram-positive against representatives of Gram-negative and
bacteria, respectively. Experimental proofs that Gram-positive pathogens. Temporin B (TB)
describe these events and subsequent membrane belongs to the temporin family, initially isolated
damage in bacteria are considered lacking (4). from the skin mucus of the European common frog
Nevertheless, damage of model membranes by Rana temporaria (15, 16), and one of the largest
AMPs is well documented with alteration of family of AMPs (16). Temporins are mainly active
membrane permeability by AMPs generally against Gram-positive bacteria, with minimal
explained using mainly three hypothetical models. inhibitory concentration (MIC) values ranging from
The “barrel-stave” or “toroidal” pore formation 2.5-20 µM (16, 17). They are less active against
models respectively involve peptides self- Gram-negative bacteria, with the exception of
associating or associating with lipids in the temporin L, which showed MIC ranging from 2-20
membrane causing opening of pores and leakage of µM (16, 18). Temporin B L1FK (TB_L1FK) and
cytoplasm material. The third model, the “carpet- temporin B KKG6A (TB_KKG6A) are two
like” mechanism involves the aggregation of high analogues derived from the temporin B sequence,
concentrations of peptide on the top of the designed to improve activity against Gram-negative
membrane causing its destabilization and bacteria. Temporin B L1FK was obtained from a
dissolution in a detergent-like fashion (4, 10). computational statistical model designed to predict
the toxicity of the peptide as well as the
The main approach adopted to improve the
antimicrobial activity. Temporin B L1FK maintains
antimicrobial properties of native AMPs, involves
the physicochemical properties of the native
the modification of the physicochemical properties
peptide, such as hydrophobicity and amphipathic
of the peptide sequence, including the net positive
profile, but it has a broader spectrum of activity at
charge, hydrophobicity, hydrophobic moment and
low concentration, including against Gram-
potential to adopt helical conformation to balance
negative bacteria, as well as a higher potency in
antimicrobial activity and host cell toxicity (12).
eradication of biofilms (19, 20). Temporin B
Such studies often proceed on the basis that the
KKG6A was instead obtained by further
overall mechanisms of bactericidal action are not
optimization of an analogue obtained through
expected to alter following minor sequence
alanine scanning of the temporin B sequence. The
modification but that the ability of the AMP to
properties of temporin B KKG6A differ from
perform its function is enhanced. Recently we have
temporin B, with a higher positive charge, a
compared the mechanism of action of seemingly
different amphipathic profile and a higher helical
closely related AMPs from the same class and
content, but is reported to have improved
found substantial differences in both the bacterial
antimicrobial activity against Gram-negative and
response to challenge and the ability of each peptide
Gram-positive bacteria, a low haemolytic activity
to penetrate model membranes (13, 14). As a result,
and a strong tendency to bind to lipopolysaccharide
we hypothesised that even relatively minor
(19, 21).
alterations to the primary sequence of an AMP can
result in fundamentally different behaviour when To distinguish between the mode of interaction of
binding to models of the bacterial plasma the three temporin B analogues we combined all
membrane leading to substantial changes in anti- atom molecular dynamics simulations, with
bacterial potency and/or selectivity. circular dichroism and liquid- and solid-state NMR
to determine conformational and topological
To test our view we report here our investigation of
differences during the initial stages of membrane
the effect of modifying the sequence of temporin B
interaction and in the steady state. In addition,
2
bioRxiv preprint first posted online May. 1, 2018; doi: http://dx.doi.org/10.1101/312215. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

electrophysiology measurements of the current bacteria (19, 21) and 16-fold against P. aeruginosa
transitions on model membranes obtained with the (19) when compared to temporin Be. However
patch-clamp technique reveal how these impact on other bacterial strains and protocols were applied in
the ability of each peptide to permeate analogous the previous works. Although limited in scope, the
model membranes and enable understanding of the antibacterial susceptibility testing indicates that,
antibacterial outcome. indeed relatively small changes in the primary
sequence can have substantial impacts on
Results antimicrobial activity and, further, that these
impacts cannot be easily related to changes in
Primary sequence and antibacterial activity –
physicochemical properties such as nominal charge
Comparison of the primary sequences of temporin
or hydrophobicity. Specific effects related to the
B (TB) and its two analogues, temporin B L1FK
peptide conformation or ability to effectively
(TB_L1FK) and temporin B KKG6A
penetrate membranes can be suspected.
(TB_KKG6A) indicates the extent of the
modifications to the primary sequence and their Conformation and structure of temporin B
effects on nominal charge, hydrophobicity and peptides in SDS micelles and in model membranes
hydrophobic moment, assuming each peptide in the steady state - The secondary structures of the
adopts an idealised α-helix (Table 1). All three three temporin B peptides were analysed and
temporin B peptides are relatively hydrophobic and characterised with far-UV CD and NMR
short and even single amino acid substitutions will spectroscopy in the presence of SDS micelles to
be expected to have some impact on the mimic the negative surface of a bacterial membrane
physicochemical properties. Temporin B L1FK and (Fig. 1). Structures for temporin B and temporin B
temporin B KKG6A were both designed (19, 21) to KKG6A have been solved previously in LPS
broaden the spectrum of activity of temporin B. For micelles (21, 22) and in the presence of E. coli and
temporin B L1FK, the optimization involved the Staphylococcus epidermidis cells (23, 24) although
substitution of Leu1 with Phe1 to render the N- coordinates are not available in the Protein Data
terminus more hydrophobic, the addition of a lysine Bank.
residue at the C-terminus and the deletion of Asn7
In Tris-buffer alone, far-UV CD spectroscopy
(19). These modifications improved the activity of
reveals that none of the three peptides adopts any
the peptide inducing a 4.5-fold increase in potency
ordered structure. However, in the presence of SDS,
against uropathogenic Escherichia coli NCTC9001
NMR spectroscopy reveals that all three peptides
and a two-fold increase against Staphylococcus
do adopt some order (Fig. 1) with temporin B and
aureus Newman. To create temporin B KKG6A,
temporin L1FK both adopting a single β-turn
two lysines are added to the N-terminus, increasing
conformation (~20% of the whole sequence) in the
the nominal positive charge and Gly6 is substituted
–LLK- region. Temporin B KKG6A adopts a more
with alanine (21). These modifications improved
extended double β-turn (~40% of the whole
the activity of the peptide against E. coli
sequence) in the -IVANLK- region. In all three
NCTC9001 with slightly more than a two-fold
peptides there is little evidence of order from the N-
improvement in potency, but the peptide loses
terminus to the glycine residue (temporin B and
activity against S. aureus Newman. None of the
temporin B L1FK) or from the N-terminus to the
three temporin B peptides were effective against
proline residue (temporin B KK6A). In support, the
Pseudomonas aeruginosa PAO1 in the conditions
far-UV CD spectra indicate that the Ramachandran
used here. The results reported here for temporin B
angles adopted by the ordered regions of the
KKG6A are in disagreement with those reported
peptides are similar to those of an α-helix and hence
previously, which showed up to 8-fold
improvement in the activity against Gram-positive an α-helix like spectrum. The greater magnitude of
3
bioRxiv preprint first posted online May. 1, 2018; doi: http://dx.doi.org/10.1101/312215. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

the temporin B KKG6A CD spectrum compared observed using CD for the binding of the temporin
with the temporin B CD spectrum reflects the B analogues to model membranes in the steady
increase from ~27% to ~40% whole molecule state, in the first 100 ns of the interaction between
order. In contrast, the CD spectrum of temporin small groups of each temporin B analogue, clear
L1FK is more compatible with the extended α-helix differences in the preferred conformation adopted
often found in membrane proteins located in by temporin B and its analogues could be
membranes as opposed to just water (25). These determined as well as in conformational disorder
results are in broad agreement with the predicted (Fig. 2). Averaged psi dihedral angles for each
effects of the design of temporin B L1FK (19) residue in the eight temporin B peptides are similar
where the aim was to largely retain the in the presence of both POPE/POPG (Fig. 2A) and
physicochemical properties of the ancestor peptide. POPG (Fig. 2B) membranes; as in previous work
(14), phi dihedral angles were not affected and are
The conformation of the peptides in lipid bilayers
not shown. The values for these psi dihedral angles
composed of either POPE/POPG (75:25 mol:mol)
do not lie in the range of values accepted for α-helix
or POPG was investigated in the steady state
conformations but instead correspond to more
through far-UV CD spectroscopy (Fig. S1). Small
extended conformations (26). Temporin B displays
amounts of peptide stock solution were added to
considerable conformational flexibility during
small unilamellar vesicles (SUVs) to decrease the
these first 100 ns which increases from N- to C-
lipid-to-peptide ratio and investigate the effect of
terminus. Modifications made to generate temporin
peptide concentration on the secondary structure.
B L1FK have a noticeable impact on the preferred
Measurements were performed in the presence of
conformation adopted in both membrane types; the
100 mM NaCl, to maintain as similar environment
deletion of Asn7, such that Leu7 now follows Gly6,
as possible to those used for other experimental
induces a shift in local conformation with psi angles
procedures. All three peptides adopted a disordered
for residues 6-8 (GLL) consistent with the β-turn
structure when in buffered solution and adopted
(single turn α-helix) conformation (Fig. 2C/D). The
ordered conformations, of the type described for
conformational flexibility of temporin B is however
temporin B KKG6A in SDS. The titration indicates
retained throughout the length of the modified
that effectively all peptide molecules in solution
peptide. The modifications made to generate
bind to the vesicles. A notable exception in the
temporin B KKG6A have a much more substantial
series is illustrated for temporin B itself in the
impact. Except for the five residues at the N-
presence of POPE/POPG (75/25) and POPG, with
terminus, there is a shift in psi angles associated
a less pronounced negative 220 nm feature which
with the replacement of a glycine (Gly6 in temporin
indicates a more ordered tail or a less ordered β-turn
B) with alanine (Ala9 in temporin B KKG6A) such
(Fig. S1A/D). Otherwise, differences are small and
that residues 6-12 (PIVANLK) adopt a two turn α-
the CD spectra can be said to be broadly similar.
helix conformation (Fig. 2E/F). Conformational
MD simulations reveal different conformational flexibility is also reduced throughout the length of
flexibilities on initial binding to model membranes the peptide with only the two residues at the C-
– To characterise the interaction of temporin B terminus unaffected (Fig. 2E/F).
analogues with model membranes with atomic
Modification of temporin B affects its penetration
resolution, duplicate all atom simulations were run
of lipid bilayers and aggregation at their surface –
for 100 ns with, in each case, eight peptides for each
In addition to changes in preferred secondary
temporin B analogue placed in water above bilayers
structure, MD simulations allow visualisation of the
comprising either 512 POPG lipids or a mixture of
ability of the three temporin B peptides to penetrate
384 POPE and 128 POPG lipids. Although only
either POPE/POPG or POPG bilayers (Fig. 3). All
small differences in secondary structure were
peptides start the simulation in water with no
4
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It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

contact with the membrane, after the simulation had proportion of α-helix conformation and the reduced
run for 100 ns, all the peptides were interacting with conformational disorder is that temporin B KKG6A
the lipids regardless the composition of the has a much lower tendency to form higher order
membrane. In general, the temporin B peptides aggregates in either membrane, though may still
showed deeper insertion in POPG membranes, as form dimers and trimers (Fig. S6/7). Penetration
observed previously (27), compared with mediated by the previously hydrophobic N-
POPE/POPG membranes, with contact and terminus is halted by interactions between the two
insertion beginning at the N-terminus in all cases. lysines and anionic lipid headgroups.
The temporin B peptides were also frequently were
Peptide effects on membrane order – the relative
self-associated (Fig. 4) and assembled as dimers,
differences in the ability of the temporin B peptides
trimers or even tetramers (Fig. S2-6). Aggregation
to penetrate lipid bilayers is expected to impact on
was consistently lower in POPG membranes
their ability to disorder, disrupt or permeate
compared with POPE/POPG membranes.
membranes. In the MD simulations described
Temporin B is known to act in synergy with above, temporin B and temporin B KKG6A had a
temporin L with the latter acting to inhibit the greater tendency to cluster POPG lipids in mixed
aggregation of the former (28). Acting alone, membranes (Fig. 5A). This could be related either
temporin B struggles to penetrate far into to the greater nominal positive charge of these
POPE/POPG membranes, with residues 1-7 only peptides relative to temporin B (Table 1) or to their
inserting and these reaching only the interfacial inability to penetrate easily beyond the interfacial
region of the bilayer (Fig. 3A), with most peptides region such that cationic residues remain
residing in an aggregated state (Fig. 4A; Fig. S2). concentrated at the bilayer surface and induce
In POPG membranes the aggregation of temporin lateral separation of anionic and zwitterionic lipids.
B is much less pronounced (Fig. 4B) and
The interaction of cationic AMPs with anionic
penetration proceeds more rapidly, is deeper –
lipids has been demonstrated previously, in vitro
beginning to enter the hydrophobic core - and more
(14, 29, 30) and in silico (14, 31, 32), to induce
of each peptide becomes buried (Fig. 3B; Fig. S3).
disordering of the lipid acyl chain and is associated
Temporin B L1FK behaves similarly to temporin B with antimicrobial activity. Here, a good qualitative
(Fig. 3CD; Fig. 4C/D). However, its initial binding comparison was observed between the peptide
and the insertion were more effective than observed induced membrane disordering obtained
for the parent peptide. Temporin B L1FK inserted experimentally, in the steady-state, using 2H solid
almost flat in both lipid compositions enabling state NMR (Fig. 5B-D) and from disordering of
more of the peptide to be buried (Fig. 3C/D) and, lipids determined to be within a 4 Å ring of each
notably in POPE/POPG, had a much lower peptide in MD simulations (Fig. S8). In the steady
tendency to self-associate than temporin B (Fig. state, the average difference (S ) between the
CD
4A/C). Again, in POPG bilayers the tendency to order parameter in the presence and absence of 2
self-associate is reduced (Fig. 4D) and penetration mol% of each temporin B peptide on the POPG-d31
into the hydrophobic interior of the membrane is (Fig. 5B) and POPE-d31 (Fig. 5C) component in
greater than in POPE/POPG bilayers (Fig. 3D; Fig POPE/POPG membrane and POPG-d31 in POPG
S4/5). membranes (Fig. 5D) allows comparison of any
differing ability to disorder different components of
In contrast with temporin B and temporin B L1FK,
each membrane. Notably, temporin B L1FK has a
temporin B KKG6A fails to insert into either
significantly (p < 0.05) greater disordering effect on
bilayer beyond the first two residues of the N-
the anionic component in mixed membranes than
terminus (Fig. 3E/F). The result of additional
temporin B KKG6A (Fig. 5B) while the latter has a
positive charges at the N-terminus, the greater
5
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It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

greater disordering effect on the zwitterionic POPE The membrane activity of the peptides can be
when compared with both temporin B and temporin distinguished on the basis of 1) the concentration
B L1FK (p < 0.05) (Fig. 5.C). No effect of any of required for membrane activity to be recorded
the three peptides can be detected on the order (Table 2), 2) the latency – the time taken for
parameter for POPG-d31 in POPG membranes conductance to begin after addition of peptide, 3)
(Fig. 5D) though the measured order parameter here the duration of conductance activity, 4) the
is more likely to be reporting on an average of those amplitude and number of each conductance event
lipids interacting with the peptides and those and 5) whether the AMP ultimately caused the
located in the remaining bulk than in the analogous membrane to break.
experiments on mixed POPE/POPG-d31
The concentration used for the experiments for each
membranes where a higher probability of the
peptide is the minimum concentration necessary to
labelled anionic lipid associating with cationic
observe activity and was obtained with a peptide
AMP is expected.
titration of the model membrane (Table 2). Notably
Temporin B analogues have drastically different there is an apparent relationship between the ability
abilities to induce channel activity in model of each temporin B peptide to insert rapidly into
membranes – The whole-cell patch-clamp model membranes, as determined by the MD
technique allows the measurements of current simulations, and the concentration required to
transitions on a phospholipid membrane. It has been induce conductance. Less than half as much
largely used to study conductance and kinetics of temporin B L1FK as temporin B was required to
membrane channels in cells and in lipid bilayers induce conductance in DPhPE/DPhPG membranes
(33). The recent development of the Port-a-Patch while approximately seven times more temporin B
automated patch-clamp system from Nanion than temporin B L1FK is required to induce
Technologies (Munich, Germany), enables rapid conductance in DPhPG membranes. Temporin B
testing of membrane active molecules with an KKG6A, for which no more than the two N-
automated system, bringing this technique into the terminal residues penetrated either POPE/POPG or
drug discovery pipeline (34, 35, 36). Previous POPG bilayers, required nearly eighteen and over
works have applied this technique to investigate the twenty times as much peptide as temporin B L1FK
mode of action of AMPs (37, 38, 39, 40, 41, 42, 43, to induce conductance in, respectively,
44). In particular, the three main membrane-activity DPhPE/DPhPG and DPhPG membranes.
models described in the introduction can be
Characteristic channel like activity - well-defined
distinguished (37). This protocol involved the use
events with discrete opening levels – has been
of a microperfusion system on a standard pipette
observed previously by others for alamethicin,
patch-clamp set up and mammalian cells (37). We
gramicidin A, cecropin B and dermicidin (38, 39,
report here that the same aim was achieved with the
40) and for other peptides in our hands. However,
Port-a-Patch® system and the use of model
Temporin B presented an irregular activity in both
membranes, which enabled the peptides to be
mixed and negatively charged membranes (Fig.
distinguished on the basis of their disruption of
6A/B). Temporin B caused a perturbation of the
either DPhPE/DPhPG (60:40 mol:mol) and
membrane characterized by bursts of current (fast
DPhPG, mimicking Gram-negative and Gram-
events seen mostly as spikes) with a wide
positive bacteria, respectively (45, 46, 47, 48, 49).
distribution of amplitudes, ultimately resulting in a
Diphytanyl chains are used here since membranes
destabilization of the membrane. Interestingly,
composed of these lipids are in a fluid phase at
temporin B activity started relatively soon after
room temperature, irrespective of headgroup.
peptide addition, always causing an immediate
rupture of the membrane (Fig. 7).
6
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It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

Conductance induced by temporin B L1FK was technique (38, 39, 40), while many show irregular
also unaffected by membrane composition. membrane activity which is hard to define (38, 50).
Temporin B L1FK acted more slowly than Nevertheless, a rationale has been suggested in
temporin B (significantly so in DPhPG; p < 0.05) order to distinguish between the “barrel-stave”,
and induced a mixture of membrane activities, “toroidal” pores or “carpet-like” mechanisms on the
characterised by some channel-like events, with base of the current traces (37). Events showing a
defined levels of amplitude, and irregular, fast single-channel nature would characterise a pore,
events, similar to those induced by temporin B (Fig. while an irregular current trace would be
6C). In DPhPG, more pore-like events were visible, representative of a carpet mechanism. Moreover,
however the distribution of the opening and closing through the application of a perfusion system which
levels was broad and no single populations could be allows addition and removal of the peptide from the
defined (Fig. 6D). Temporin B L1FK often broke membrane, the different mechanisms could be
DPhPE/DPhPG membranes, while it rarely broke distinguished on the basis of shorter or longer time
DPhPG membranes (Fig. 7C/D), perhaps since constants of current activation and recovery,
conductance was achieved with a lower active indicating a “barrel-stave” or a “toroidal” pore,
concentration. respectively, and the irreversibility of the
permeabilization process that would be the
Membrane conductance induced by temporin B
signature of a “carpet” like mechanism (detergent-
KKG6A was not detectable by electrophysiology
like) (37, 41). This characterisation is based on the
unless a high concentration was reached. At these
predominant models currently used to describe
high concentrations, considerable destabilization of
AMPs activity (4, 10). However, more recent
the membrane is observed with more numerous
studies indicate many events at the bacterial plasma
high amplitude events than observed for temporin
membranes are poorly described by these existing
B, particularly in DPhPG (Fig. 6E/F). Although
models and refinements or entirely new models are
conductance appears relatively soon after temporin
required to satisfactorily explain the behaviour of
B KKG6A addition, the membrane remains
some AMPs; for instance the “snorkelling
resilient and either does not break (Fig. 7C) or takes
mechanism” (51, 52), the “dynamic peptide-lipid
significantly longer (p < 0.05) to break than when
supramolecular pore” (50, 53, 54, 55) or the
challenged with much less temporin B (Fig.7D).
“disordered toroidal pore” (27, 32, 56).
Discussion
Here we investigate whether the patch-clamp
The utility of patch-clamp for providing insights technique is capable of distinguishing between the
into the differing mechanisms of membrane membrane activities of closely related AMPs. We
disruption by the three temporin B peptides - developed an approach using the Port-a-Patch®
Patch-clamp has been mainly used to study tool from Nanion Technologies (Munich,
membrane channels formed by proteins in Germany) and, in order to associate the current
mammalian cells or reconstituted in model trace to a specific mechanism, we correlated the
membranes and the typical current trace obtained in findings obtained from patch-clamp analysis to the
such experiments shows well-defined square- primary sequence and the biophysical behaviour of
shaped events indicating single channels with the peptides. In the present work, we applied this
discrete opening and closing events (33, 35). The approach to the analysis of temporin B and its two
study of membrane active molecules, such as analogues. These peptides are poorly studied, and
AMPs, with electrophysiology is complicated by little information is known about their mode of
their irregular activity when interacting with the action. Hydrophobic AMPs such as temporin B
bilayers. Indeed, only few AMPs apparently form have been proposed to go through an aggregation
regular single-channels when analysed with this process and form fibrils as a step in the bactericidal
7
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It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

process (57). As with amyloid peptides, this Newman. We ascribe this improvement in
mechanism would form a cytotoxic intermediate membrane activity, and consequently in anti-
which would cause the death of the cell (bacterial bacterial potency, to increased insertion into the
in this case) through poration of the plasma membrane associated with reduced self-
membrane. Evidence is indeed provided here for association. This is achieved with greater
substantial aggregation of temporin B and its hydrophobicity at the N-terminus by substituting
analogues but there is no evidence of any phenylalanine for leucine, the increased positive
conformational shift, from -helix to -sheet, as charge and the reduced conformational flexibility
characteristic of amyloid-like peptides as was between residues six and eight. For both temporin
predicted (57). We performed far-UV CD B and temporin B L1FK, patch-clamp revealed no
spectroscopy in several environment conditions difference in activity according to membrane
(different lipid compositions, presence of metals, composition. In neither case could the observed
different pH, ionic strength etc. only the data activity be defined as regular single-channel
relative to POPE/POPG and POPG are reported formation, but rather a perturbation of the
here) and no conformational change suggestive of permeability of the membrane due to defects of the
β-sheet conformations was ever observed. A self- lipid bilayer that cause spikes of current. Unlike
association of temporin B monomers through temporin B, temporin B L1FK, in addition to the
hydrophobic interactions was also reported on spikes also caused some square-shaped events
lipopolysaccharide (22, 58); this was suggested to resembling channels or pores but characterized by
be the cause of the poor activity of this peptide disordered openings in which an uneven
against Gram-negative bacteria. Further, an distribution of levels can be detected. Such
increase in activity was observed for temporin B conductance activity is well removed from that
when used in combination with temporin L with the reported for “barrel-stave” or “toroidal” pores, as
latter reported to inhibit the self-association. described previously for alamethicin, gramicidin A,
Presumably, temporin L can stabilise the monomer cecropin B and dermicidin (38, 39, 40). Instead, the
conformation of temporin B and/or cause structural current traces obtained for both temporin B and
changes in lipopolysaccharide when bound, which temporin B L1FK are comparable to the ones
prevents temporin B aggregation. When in obtained for gramicidin S (38), for which they
combination, the two peptides showed a higher suggest a mechanism similar to the so called “in-
potency in lipopolysaccharide disintegration (16, plane diffusion” model (59, 60). In this model the
22, 58). Primary sequence modification strategies peptides insert into the phospholipid bilayer
that seek to reduce self-association of temporin B disordering the packing and causing defects that
may therefore enhance its activity against Gram- can increase the membrane permeability. However,
negative bacteria. in this model the aggregation of peptides is
considered minimal and unfavourable, while we
Modification of the temporin B primary sequence
observed considerable self-association for temporin
to create temporin B L1FK – Temporin B L1FK
B and, although reduced, also temporin B L1FK.
was computationally designed with a statistical
The immediate membrane rupture observed for
model considering the cytotoxicity of the sequence,
temporin B in all the electrophysiology
but also a close physicochemical pattern to the
measurements is consistent with the “carpet-like”
parent peptide (19). Temporin B L1FK behaves
mechanism, where peptides disintegrate the
similarly to temporin B in inducing conductance
membrane following a detergent-like process (41,
albeit with a greater number of channel-like events
61). However, such behaviour was not observed for
and at somewhat lower concentrations and this
temporin B L1FK. Current traces similar to those
correlates with the improved potency obtained
obtained for temporin B L1FK, characterised by
against both E. coli NCTC 9001 and S. aureus
8
bioRxiv preprint first posted online May. 1, 2018; doi: http://dx.doi.org/10.1101/312215. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

abrupt transient current spikes, were reported for visible with technique as patch-clamp. Adoption of
poly-arginine peptides, known to act as cell- this mechanism against E. coli is consistent with the
penetrating peptides (CPP) (62). The deep and flat loss of activity against Gram-positive S. aureus, the
insertion, observed for this temporin B during the plasma membrane of which is mainly composed of
MD simulations, might suggest the ability of this negatively charged lipids (45, 46, 47, 48, 49).
peptide to better penetrate the bilayer and act inside Whether this or other mechanisms more accurately
the cell – activity not captured by techniques describe the behaviour and anti-bacterial activity of
applied in the present study. temporin B KKG6A, it is clear that, following the
optimization process, its mechanism of action
Modification of the temporin B primary sequence
differs substantially from the native temporin B
to create temporin B KKG6A – Modification of
peptide.
temporin B through addition of two lysines at the
N-terminus and replacement of a glycine with an Conclusion - Since cecropin, the first AMP, was
alanine ensures that self-association of the resulting isolated from the hemolymph of the silk moth
peptide, temporin B KKG6A, is substantially Hyalophora cecropia in 1981 (65), AMPs have
reduced. This is achieved though at cost of been studied as a new interesting class of molecules
preventing insertion of the peptide, beyond the first for the development of new antibiotics. The
two residues at the N-terminus, into either attraction of these short, cationic peptides is related
membrane and a very large increase in the amount to their ability to defeat infections from many
of peptide required to obtain any conductance different pathogens (bacteria, fungi, viruses) and, in
activity. This is a reminder that not only the net particular, the low rate of bacterial resistance onset
charge, but also its distribution along the sequence (4, 5, 6, 9). Despite the efforts made to characterise
is important for AMPs activity (56, 63). However, different AMPs from different sources, there is still
while this might explain the loss of activity against a lack of understanding of their specific modes of
S. aureus, activity against E. coli was actually action and variation thereof due to sequence
improved compared to the original temporin B modifications (4). The activity of temporin B is
peptide. Notably however, and in contrast to likely improved in vivo through its synergistic
temporin B and temporin B L1FK that disorder the interaction with temporin L which acts to prevent
anionic phosphatidylglycerol component, temporin self-association. Both sequence modification
B KKG6A disorders the zwitterionic strategies described here also act to reduce self-
phosphatidylethanolamine component of association of the AMP in the target membrane but
membranes designed to mimic the plasma lead to fundamentally different behaviour which
membrane of Gram-negative bacteria. This, was revealed by a combination of MD simulations
together with a clear interaction with the headgroup and patch-clamp conductance recordings but not by
region of the phospholipid bilayer observed in the e.g. circular dichroism. Therefore, in characterising
MD simulations, suggests that the peptide is the membrane interaction of temporin B and its
interacting with the membrane, but that the effect of analogues, the present study shows that even minor
this interaction does not readily induce channel alterations to AMP primary sequences may cause
conductance detectable through patch-clamp. The drastic changes in the mechanism of interaction
disorder observed in phosphatidylethanolamine with bacterial plasma membranes which in turn
lipids is related to the observation, in MD affect antibacterial potency and selectivity. Future
simulations, that temporin B KKG6A is particularly attempts to enhance the potency of designed or
effective at clustering anionic lipids. This can be naturally occurring AMP sequences will be aided
toxic to bacteria by disrupting membrane domains by as sophisticated as possible understanding of the
essential for specific interactions with other crucial peptide-peptide and peptide-membrane
molecules (64) and might not cause a leakage interactions and their outcomes.
9
bioRxiv preprint first posted online May. 1, 2018; doi: http://dx.doi.org/10.1101/312215. The copyright holder for this preprint (which
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY 4.0 International license.
Patch-clamp to investigate antimicrobial peptides activity

Experimental procedures obtained was fitted with a sigmoidal curve to obtain
the concentration necessary to inhibit the growth of
Materials - All the peptides (Table 1) were
50% of bacteria (MIC ). The MIC reported for
purchased from Pepceuticals (Enderby, UK) and 50 50
each peptide in Table 1 is the average of at least
Cambridge Research Biochemicals (Cleveland,
three independent repeats.
UK) as desalted grade and were further purified
using water/acetonitrile gradients using a Waters NMR spectroscopy and structure determination -
SymmetryPrep C8, 7 mm, 19 × 300 mm column. Liquid state NMR spectroscopy experiments and
They were all amidated at the C-terminus. The data analysis were performed as described before
lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (14). The samples solution consisted of 2 mM of
(1′-rac-glycerol) (POPG), 1-palmitoyld31-2- peptide and 100 mM of deuterated sodium dodecyl
oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sulphate (SDS-d25) micelles. 10% D O containing
2
(POPG-d31), 1-palmitoyl-2-oleoyl-sn-glycero-3- trimethylsilyl propanoic acid (TSP) was added for
phosphoethanolamine (POPE), 1-palmitoyld31-2- the lock signal and as internal chemical shift
oleoyl-sn-glycero-3-phosphoethanolamine (POPE- reference. NMR spectra were acquired on a Bruker
d31), 1,2-diphytanoyl-sn-glycero-3-phospho-(1'- Avance 500 MHz spectrometer (Bruker, Coventry,
rac-glycerol) (DPhPG) and 1,2-diphytanoyl-sn- UK) equipped with a cryoprobe. Standard Bruker
glycero-3-phosphoethanolamine (DPhPE) were TOCSY and NOESY pulse sequences were used,
purchased from Avanti Polar Lipids, Inc. with water suppression using an excitation sculping
(Alabaster, AL) and used without any purification. sequence with gradients (mlevesgpph and
All other reagents were used as analytical grade or noesyesgpph). The 1H 90-degree pulse length was
better. 8.0s. One TOCSY with a mixing time of 60 ms, and
two NOESY with mixing time of 100 and 200 ms
Antibacterial activity assay - Bacteria (Escherichia
were acquired for each sample. The relaxation
coli NCTC 9001, Pseudomonas aeruginosa PAO1
delay was 1 s. 2048 data points were recorded in the
and Staphylococcus aureus Newman) were grown
direct dimension, and either 256 or 512 data points
overnight in 5 mL of Mueller-Hinton (MH) broth
in the indirect dimension. CARA (version 1.9.1.2)
without shaking at 37°C. The antibacterial activity
and Dynamo software (66, 67) were used for the
of the five peptides studied was assessed through a
structure calculation. Inter-proton NOEs
modified two-fold broth microdilution assay, as
interactions were used as distance restraints in the
described before (13). Briefly, a two-fold dilution
structure calculation. CARA software generated a
of peptides’ stock solutions was performed in MH
total of 200 structures on UNIO’08 (version 1.0.4)
broth in a 96-well polystyrene microtiter (Greiner)
(68) and XPLOR-NIH (version 2.40), (69, 70) after
plate. Then, 50 µL of a bacteria suspension (A of
620
7 iterations, using the software’s simulated
0.0001) were added to 50 µL of peptide solution in
annealing protocol. 20 structures with the lowest
each well of the plate reaching a final volume of
energy were chosen to produce a final average
100 µL. Growth and sterility controls were present
structure. In the case of ambiguous NOEs
for each experiment. The plate was sealed with a
assignments by CARA software, Dynamo
sticker and incubated at 37°C for 18 hours without
software’s annealing protocol was applied. Only
shaking. Bacterial growth was observed with a
unambiguous NOEs were used in this case after
colorimetric assay (63). 20 µL of a resazurin
being classified as strong, medium and weak on the
solution (0.2 g/L) were added to each well and the
base of the relative intensity of the cross-peaks in
plate was incubated for further 2 hours. The results
the NOESY spectra. On the base of this
were read at A and A (pink and blue colour,
570 600
classification an upper limit of 0.27, 0.33 and 0.50
respectively) and plotted as difference in
nm have been applied, respectively, as restraint on
absorbance versus peptide’s concentration. The plot
the corresponding inter-proton distance, as
10