Faculty of Science

DETERMINING ANTIMICROBIAL SUSCEPTIBILITY AND HEMOLYTIC ACTIVITY
OF INDOLICIDIN DERIVATIVES
2024 | ALIVIA LYN MONET MERCER-BRUNELLE

B.Sc. HONOURS THESIS – CHEMICAL BIOLOGY

DETERMINING ANTIMICROBIAL SUSCEPTIBILITY AND HEMOLYTIC
ACTIVITY OF INDOLICIDIN DERIVATIVES

by

ALIVIA LYN MONET MERCER-BRUNELLE

A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE (HONS.)
in the
DEPARTMENTS OF BIOLOGICAL
AND PHYSICAL SCIENCES
(Chemical Biology)

This thesis has been accepted as conforming to the required standards by:
Heidi Huttunen-Hennelly (Ph.D.), Thesis Supervisor, Dept. Physical Sciences
Naowarat (Ann) Cheeptham (Ph.D.), Secondary Supervisor, Dept. Biological Sciences
Mark Rakobowchuk (Ph.D.), External Examiner, Dept. Biological Sciences

Dated this 24th day of April, 2024, in Kamloops, British Columbia, Canada
© Alivia Mercer-Brunelle, 2024

ABSTRACT
Antibiotic-resistant microorganisms have posed significant challenges to the development of
novel antimicrobial agents. Antibiotic-resistant bacteria are increasingly being detected in humans
and animals treated with antibiotics, making commercially available antibiotics less effective than
they once were. Microbial compounds from various species are being studied extensively for
potentially better and more effective antibacterial properties that may be used as an alternative.
Antimicrobial peptides (AMPs) appear to be potential antibacterial medication candidates;
however, the development of AMPs is impeded by their cytotoxicity. In the current study,
indolicidin, a known antimicrobial originally isolated from bovine neutrophils, was modified by
changing its tryptophan content. We predicted that replacing tryptophan with a less hydrophobic
amino acid, alanine, would minimize hemolytic activity and maximize bioactivity. To address this
question, we analyzed four indolicidin derivatives in which two of its tryptophan residues were
replaced with alanine at different positions. We assessed their antibacterial and antifungal activity
against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Candida albicans,
Staphylococcus aureus, and Methicillin-Resistant Staphylococcus aureus (MRSA). Antimicrobial
testing identified two active derivatives with minimum inhibitory concentrations in the 250-500
μg mL-1 range against all microbes, except Pseudomonas aeruginosa. Notably, the substitution of
tryptophan residues at position 4 significantly affected antimicrobial activity. All derivatives
exhibited a substantial decrease or complete removal of hemolytic activity at concentrations above
their minimum inhibitory concentration (MIC). Additionally, derivatives containing the ProlineTryptophan-Tryptophan-Proline (PWWP) domain showed DNA-binding capability, while those
lacking this domain did not. These findings highlight the potential of modified indolicidin

ii

derivatives as effective and less cytotoxic antimicrobial agents, offering a potential solution for
combating antibiotic resistance.
Thesis Supervisor: Associate Professor Heidi Huttunen-Hennelly

iii

ACKNOWLEDGEMENTS
First off, I would like to express my deepest appreciation to my thesis supervisor, Dr.
Heidi Huttunen-Hennelly, for her unequivocal support throughout my research journey. Her
expertise, teaching philosophy, and support will be something I remember as I continue my
journey as a student outside of Thompson Rivers University. I would also like to thank Dr.
Naowarat Cheeptham for access to her laboratory and its resources and for her knowledge and
feedback. I am grateful to Dr. Mark Rakobowchuk for helping me with my research, being a source
of knowledge with my project and for taking the time to read and provide feedback on my thesis.
I appreciate all the help from Dr. Donald Nelson for my DNA binding assay. My sincere
appreciation goes to the TRU UREAP for funding my research journey. Kathy Baethke has been
instrumental in teaching me about all the equipment and has helped me gain confidence in the lab.
Lastly, I want to recognize Ethan Furlong as my partner for this project. His support has been
appreciated in overcoming challenges, celebrating successes, and advancing our research as a
team.

iv

TABLE OF CONTENTS
ABSTRACT.................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................... iv
LIST OF FIGURES ...................................................................................................................... vii
LIST OF TABLES ......................................................................................................................... ix
LIST OF ABBREVIATIONS ......................................................................................................... x
1 INTRODUCTION ....................................................................................................................... 1
1.1 Antimicrobial Peptides.......................................................................................................... 1
1.2 Pharmacodynamics of AMPs ................................................................................................ 2
1.3 Structures and Characteristics of AMPs ............................................................................... 4
1.4 Pathogen Specificity ............................................................................................................. 5
1.4.1 Mechanism of Action of AMPs ..................................................................................... 5
1.4.2 Gram-positive/Gram-negative Bacteria and Fungi ........................................................ 6
1.5 Indolicidin ............................................................................................................................. 9
1.5.1 Trp and Pro .................................................................................................................. 10
1.5.2 Structure and Mechanism of Action ............................................................................ 11
1.5.3 Hemolytic Activity....................................................................................................... 15
1.6 Objective ............................................................................................................................. 16
2 MATERIALS AND METHODS ............................................................................................... 17
2.1 AMP Stock Solution Preparation ........................................................................................ 17
2.2 Antimicrobial Susceptibility ............................................................................................... 18
2.2.1 Kirby-Bauer Disc Diffusion Assay .............................................................................. 18
v

2.2.2 Minimum Inhibitory Concentration Assay .................................................................. 19
2.3 Microbial Viability Assay ................................................................................................... 20
2.4 Hemolytic Activity.............................................................................................................. 20
2.5 Electrophoretic Mobility Shift Assay ................................................................................. 21
3 RESULTS .................................................................................................................................. 22
3.1 Bioactivity Characterization ............................................................................................... 22
3.1.2 Kirby-Bauer Disc Diffusion Assay .............................................................................. 22
3.1.3 MIC Assay ................................................................................................................... 23
3.1.3 Hemolysis .................................................................................................................... 26
3.2 DNA Binding Assay ........................................................................................................... 27
4 DISCUSSION ............................................................................................................................ 28
4.1 Antimicrobial Activity ........................................................................................................ 29
4.2 Hemolytic Activity.............................................................................................................. 31
4.3 DNA Binding by Inhibition of Plasmid Migration ............................................................. 32
5 CONCLUSIONS AND FUTURE WORK ................................................................................ 34
LITERATURE CITED ................................................................................................................. 35

vi

LIST OF FIGURES
Figure 1. Pharmacodynamic curves of AMPs. The curves illustrate the effects (reflected as net
bacterial growth rate) of increasing the concentrations of AMP(s). κ predicts the shape and slope
of the pharmacodynamic curve; the higher the κ value, the steeper the pharmacodynamic curve
(Yu et al. 2016). .............................................................................................................................. 3
Figure 2. Structural classes of antimicrobial peptides; (A) Mixed structured peptide; (B) βsheeted peptide; (C) α-helical peptide; (D) Extended peptide (Jenssen 2009). .............................. 5
Figure 3. Models of antibacterial mechanisms of AMPs (Zhang et al. 2021). .............................. 6
Figure 4. Schematic membrane structures of Gram-positive and Gram-negative bacteria. Grampositive bacteria have a thick layer of peptidoglycan surrounding the cytoplasmic membrane,
while Gram-negative bacteria have a thinner layer of peptidoglycan and an additional outer
membrane (Li et al. 2017). .............................................................................................................. 8
Figure 5. Fungal cell wall of Candida albicans (Aroso et al. 2021).............................................. 9
Figure 6. Theoretical shape of indolicidin with red indicating hydrophobic regions and blue
indicating positively charge residues; the molecule is oriented from N-terminus (left) to amidated
C-terminus (right) (Kumar et al. 2018). ........................................................................................ 11
Figure 7. Charge distribution on the surface of indolicidin. Positive and neutral potentials are
coloured blue and white, respectively. (A) End view looking down from the N-terminus (B) Side
view with the N-terminus pointed to the top (Rozek et al. 2000). ................................................ 12
Figure 8. Illustration of indolicidin's mode of action following brief membrane disruption: (a)
proteins and nucleic acids are intracellular potential targets; (b) the indolicidin–LPS interaction;
(c) indolicidin anion carrier mechanism (Araujo et al. 2022). ...................................................... 14

vii

Figure 9. Predicted Key Tryptophan Residues for Bacterial Lysis and Hemolysis (Ando et al.,
2010) ............................................................................................................................................. 16
Figure 10. Kirby-Bauer assay of the different indolicidin derivatives against Escherichia coli. 22
Figure 11. Serial dilution results with decreasing concentrations of Δ6,8 (from left to right: 500
μg/mL to 0 μg/mL) against Candida albicans. ............................................................................. 25
Figure 12. Electrophoretic mobility assay of DNA binding to peptides. Different amounts of
indolicidin (A), Δ4,11 (B), Δ4,6 (C), Δ9,11 (D), and Δ6,8 (E) was incubated with 200 ng of
pDN5 plasmid DNA. The binding affinity was assessed by the inhibition of the electrophoretic
migration of the DNA by the peptides. The peptide:DNA mass ratios for the different lanes from
left to right. are 0:1, 0.25:1, 0.5:1, 2:1, 5:1. .................................................................................. 28

viii

LIST OF TABLES
Table 1. Sequence and physicochemical properties of indolicidin. ............................................. 10
Table 2. Sequence of indolicidin and other derivative peptides with abbreviations. ................... 17
Table 3. Bacteria and fungi species used for antimicrobial susceptibility assays, cell type, and
antibiotic used for positive control. .............................................................................................. 19
Table 4. Zone of inhibition diameter measurements of the indolicidin derivatives tested on
various bacteria and fungi. ............................................................................................................ 23
Table 5. MICs of peptide derivatives against a variety of bacterial and fungal pathogens, where
active concentrations are determined by exhibiting at least 90% reduced optical density
compared to each control culture incubated without peptide; a dash (—) indicates that the peptide
was not active at the concentrations tested. .................................................................................. 24
Table 6. MICs of peptide derivatives against a variety of bacterial and fungal pathogens, where
active concentrations are determined by exhibiting at least 50% reduced optical density
compared to each control culture incubated without peptide; a dash (—) indicates that the peptide
was not active at the concentrations tested. .................................................................................. 25
Table 7. Bacteriostatic/Bactericidal (BS/BC) activity of indolicidin derivatives. ....................... 26
Table 8. Minimum hemolytic concentrations (MHCs) of indolicidin and derivatives compared to
the AMP hemolytic activity predictor. In cases of high MHCs, the highest concentration tested is
given with its percent hemolysis at that concentration. Hemolytic Index (HI) is defined here as
the MHC divided by the lowest MIC derived. .............................................................................. 27

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LIST OF ABBREVIATIONS
Abbreviation

Meaning

AMPs

Antimicrobial peptides

MIC

Minimum inhibitory concentration

MHC

Minimum hemolytic concentration

TBE

Tris/Borate/EDTA

RBCs

Red blood cells

TA

Teichoic acid

EMSA

Electrophoretic mobility shift assay

PWWP

Proline-Tryptophan-Tryptophan-Proline

MSW

Mutant selection window

HDGF

Hepatoma-derived growth factors

LPS

Lipopolysaccharides

PBS

Phosphate buffer saline

Trp

Tryptophan

Ala

Alanine

Pro

Proline

MH

Mueller-Hinton

MRSA

Methicillin-resistant Staphylococcus aureus

mL

Millilitre

µL

Microlitre

CaM

Calmodulin

ASTM

American Society for Testing and Materials

HI

Hemolytic Index

x

1 INTRODUCTION
The discovery of penicillin started the current era of antibiotics (Lee, 2015). Since then,
antibiotics have revolutionized contemporary medicine and saved millions of lives. With the
introduction of antibiotic therapy, infectious bacteria have long been kept in check. In the
conventional medical industry, antibiotics have been utilized for illness treatment and infection
prevention in recent decades (Wang et al. 2019). However, the absence of early detection methods
to identify the specific microorganisms causing infections and their susceptibility to antibiotics led
to the excessive and often unnecessary use of broad-spectrum antibiotics (Frieri et al. 2017).
Antibiotic-resistant bacteria are increasingly being identified in humans and animals treated with
antibiotics, and resistance to commonly used medicines has become one of the world's most
important public health issues (Wang et al. 2019). The World Health Organization estimates that
over 95% of Staphylococcus aureus strains worldwide are resistant to penicillin (Briethaupt,
1999). Due to growing resistance and the rise of multi-drug resistant bacteria, the development of
new antimicrobial treatments is critical to preserving reduced morbidity and mortality rates
associated with harmful bacterial infections (Frieri et al. 2017). Fortunately, antimicrobial peptides
(AMPs) offer a solution.

1.1 Antimicrobial Peptides
AMPs are small peptides that represent an important component of the innate immune
system, serving as the first-line of defense against invading pathogens for many organisms
(Pasupuleti et al. 2011) Found in all species such as bacteria, fungi, animals and plants, insects,
amphibians, and all other mammalian species, these small peptides are between 12–50 amino acids
long and 2–9 kDa in size (Zhang, 2022). They effectively kill a wide range of microorganisms

1

such as viruses, fungi, and both Gram-negative and Gram-positive bacteria (Swithenbank and
Morgan 2017; Cunha et al. 2017).
Animals express AMPs in a wide variety of cells, including epithelial cells of the stomach,
skin, and respiratory tracts, mast cells, monocytes, and neutrophils (Swithenbank and Morgan
2017). These peptides play a critical role in defending against pathogens at epithelial surfaces and
in the immune system's cellular response to infection (Ganz 2003). Animal tissues exposed to
microbes or cell types involved in host defense have the highest concentrations of AMPs.
Epithelial surfaces secrete AMPs from both barrier epithelia and glandular structures. In
invertebrates, AMPs are found in the fluid portion of blood (hemolymph) as well as in the granules
of phagocytic cells (hemocytes), which contain various microbicidal substances and digestive
enzymes (Ganz 2003). During phagocytosis, granules fuse to phagocytic vacuoles containing
ingested microbes, exposing them to very high concentrations of microbicidal and digestive
substances. Other granules are secreted into the extracellular fluid, where their contents kill
microbes or inhibit their growth. Both types of granules contain abundant AMPs (Ganz 2003).
AMPs are effective against multiple pathogenic bacteria, targeting both plasma membranes and
intracellular components and demonstrate high effectiveness against drug-resistant strains (Zhang.
2021). As a result, AMPs represent a novel alternative to conventional antibiotics.
1.2 Pharmacodynamics of AMPs
Antibiotic resistance is common and occurs quickly. According to Yu et al. (2018),
resistant strains often appear two years after a new antibiotic is introduced in the clinical setting.
One of the alleged advantages of AMPs is that microbial resistance would occur much more slowly
than against antibiotics (Yu et al. 2018). This reduced susceptibility of microbial resistance can be
attributed to the rapid bactericidal action and broad-spectrum effects on microbial membranes and
2

intracellular processes, without targeting specific molecules or pathways (Zhang, 2021).
Additionally, microorganisms treated with AMPs are less susceptible to evolution compared to
those treated with antibiotics, largely due to the steepness of the pharmacodynamic curves (Figure
1). The concept of pharmacodynamics gives information about how drug dosage relates to
bacterial death rates (El Shazely et al. 2020). Pharmacodynamic curves, particularly time-kill
curves, play a crucial role in understanding the efficacy of antimicrobial agents against bacterial
populations (El Shazely et al. 2020).

Figure 1. Pharmacodynamic curves of AMPs. The curves illustrate the effects (reflected as net
bacterial growth rate) of increasing the concentrations of AMP(s). κ predicts the shape and slope
of the pharmacodynamic curve; the higher the κ value, the steeper the pharmacodynamic curve
(Yu et al. 2016).

The pharmacodynamic properties of both susceptible and resistant bacterial strains can be
used as indicators for predicting the emergence of resistance (Yu et al., 2018). These predictions
are based on a principle known as the 'mutant selection window' (MSW). The width of the MSW
3

is determined in part by the steepness of the pharmacodynamic curve. The concentration range
within which AMPs exhibit no killing to maximal killing is much narrower compared to that of
antibiotics, resulting in a much steeper curve (see red curve in Figure 1) (Yu et al., 2018). The
maximum killing rate of AMPs is much higher than that of antibiotics, as reflected in quicker
killing time (Yu et al. 2018). Consequently, AMPs have a narrower MSW compared to antibiotics,
making the occurrence of resistance toward AMPs less likely (El Shazely et al., 2020).

1.3 Structures and Characteristics of AMPs
Typically, AMPs are composed of cationic and hydrophobic amino acids, giving them both
positively charged and amphiphilic characteristics (Lei et al., 2019). These peptides contain
hydrophobic and hydrophilic regions, contributing to their amphiphilic nature. Cationic AMPs are
often short polypeptides, typically less than 100 amino acid residues, and are rich in positively
charged amino acids such as lysine and arginine, which favours the interaction between these
peptides and microbial cytoplasmic membranes (Lei et al., 2019; Cunha et al. 2019). In eukaryotes,
AMPs are encoded by specific genes, which are expressed at basal levels and rapidly transcribed
after induction by contact or exposure to invading pathogens (Cunha et al. 2019).
AMP molecules exhibit α-helical and β-sheet structures characterized by both hydrophobic
and hydrophilic regions, which contribute to their amphiphilicity (Figure 2) (Lei et al., 2019). This
enables them to interact effectively with microbial cell membranes. Such interactions play a crucial
role in the mechanism by which AMPs exert their antimicrobial activity. Positively charged AMPs
engage with negatively charged cell membranes through electrostatic interactions, leading to
membrane adsorption and conformational changes (Lei et al., 2019). The peptides anchor onto
membrane surfaces with their hydrophobic sides embedded within the lipid core of the bilayer (Lei

4

et al., 2019). Notably, these peptides have a high concentration of basic amino acids at their Nterminal ends, contributing to their strong alkalinity, while their C-terminal ends are amidated. The
antimicrobial activity of specific peptides correlates with the number of cationic charges they
possess, whereas their hydrophobicity aligns with their hemolytic activity (Lei et al., 2019).

Figure 2. Structural classes of antimicrobial peptides; (A) Mixed structured peptide; (B) βsheeted peptide; (C) α-helical peptide; (D) Extended peptide (Jenssen 2009).
1.4 Pathogen Specificity
1.4.1 Mechanism of Action of AMPs
AMPs have diverse mechanisms of action, primarily focusing on disrupting bacterial
membranes or intracellular targets (Cuhan et al. 2019). The interaction between cationic AMPs
and anionic bacterial membranes is the initial step, driven by electrostatic forces. Gram-positive
and Gram-negative bacteria respond differently due to variations in cell wall composition (Cuhan
et al. 2019). Several models have been proposed to explain the membrane targeting mechanism
including the Toroidal pore, Carpet, Aggregate, and Barrel-Stave model, as shown in Figure 3

5

(Cuhan et al. 2019). Another mechanism of action involves non-membrane targeting. In this case,
AMPs enter the cell and interact with its interior components, for example, disrupting DNA, RNA,
and protein synthesis, affecting protein folding, enzyme function, and cell wall formation,
ultimately resulting in cell death (Mazurkiewicz-Pisarek et al. 2023).

Figure 3. Models of antibacterial mechanisms of AMPs (Zhang et al. 2021).
1.4.2 Gram-positive/Gram-negative Bacteria and Fungi
AMPs are essential components of the innate immune system, playing crucial roles in
defending against bacterial infections. Bacteria are categorized as either Gram-positive or Gramnegative, each characterized by distinct differences in their cell envelopes (Li et al. 2017). While
the inner or cytoplasmic membranes of both bacterial groups exhibit similarities, their outer cell
envelopes differ significantly. In Gram-positive bacteria, AMPs encounter a layer of cross-linked
peptidoglycan. Anionic glycopolymers called teichoic acids (TAs) are covalently bound to the

6

peptidoglycan and surround the cytoplasmic membrane (Swoboda et al. 2009). This matrix
provides a robust barrier that maintains cellular rigidity, yet nano-sized pores within the
peptidoglycan layers allow AMP diffusion (Li et al. 2017).
Gram-negative bacteria, on the other hand, have a thinner and less cross-linked
peptidoglycan layer, with an outer membrane beyond this layer (Figure 4) (Li et al. 2017). The
inner leaflet of this membrane comprises predominantly phosphate lipids, while the outer leaflet
is primarily composed of lipopolysaccharides (LPS). LPS molecules contain negatively charged
phosphate groups that form salt bridges with divalent cations, creating an electrostatic network.
This charged region serves as a significant barrier to most hydrophobic antibiotics, resulting in
low permeability (Mazurkiewicz-Pisarek et al. 2023). Consequently, AMPs have different
mechanisms for penetrating Gram-positive and Gram-negative bacteria due to their distinct cell
envelope structures. In Gram-positive bacteria, AMPs first cross the peptidoglycan matrix before
acting on the cytoplasmic membrane. Conversely, targeting Gram-negative bacteria involves
disrupting both the outer and cytoplasmic membranes (Mazurkiewicz-Pisarek et al. 2023). Failure
to permeabilize or penetrate the outer membrane results in the loss of antimicrobial activity (Li et
al. 2017).
Moreover, AMPs are generally "electrostatically specific" for prokaryotic cells as the
negatively charged membrane components (LPSs and TAs) are absent in mammalian cells (King
et al. 2014). This electrostatic interaction between the positively charged AMPs and the negatively
charged microbial membranes, facilitated by the absence of similar components in mammalian
cells, enhances the selectivity of AMPs for targeting bacterial cells while minimizing cytotoxic
effects on host cells.

7

Figure 4. Schematic membrane structures of Gram-positive and Gram-negative bacteria.
Gram-positive bacteria have a thick layer of peptidoglycan surrounding the cytoplasmic
membrane, while Gram-negative bacteria have a thinner layer of peptidoglycan and an
additional outer membrane (Li et al. 2017).

While peptidoglycan forms the cell wall in bacteria, fungal cell walls have more complex
structures primarily composed of β-1,3-glucan, β-1,6-glucan, and chitin (Figure 5) (Lozančić et
al., 2021). Mannoproteins and fibrous β-1,3-glucan are the predominant components of the walls
(Lipke and Ovalle, 1998), with branched β-1,6-glucan linking other wall components. Chitin,
although a minor component, contributes to the insolubility of the fibers. The β-1,3-glucan–chitin
complex constitutes the main component of the inner wall, while β-1,6-glucan connects the inner
and outer wall components. Mannoproteins, heavily O and N glycosylated, are located on the outer
surface of the cell wall, closely spaced to restrict solute permeability (Lipke and Ovalle, 1998).
Recent studies found that AMPs can bind to chitin and induce cell wall fragility (Peng et al., 2022).
Moreover, certain AMPs can penetrate the fungal cells and interact with nucleic acids and fungal
mitochondria, resulting in cell death.

8

Figure 5. Fungal cell wall of Candida albicans (Aroso et al. 2021).

1.5 Indolicidin
Indolicidin, a natural cationic peptide found within the cathelicidin family, is found in
cytoplasmic granules of bovine neutrophils (Friedrich et al., 2001). With a composition of 13
amino acid residues, it has a unique amino acid sequence (ILPWKWPWWPWRR-NH2),
containing 39% tryptophan (Trp) and 23% proline (Pro), with an amidated carboxyl terminus
(Friedrich et al., 2001). Approximately half of its amino acid residues are hydrophobic, including
the five Trp residues (Araujo et al., 2022). Remarkably, it has the highest Trp content among all
known AMPs (Falla et al., 1996). Additionally, its broad-spectrum antimicrobial activity against
various gram-positive and gram-negative bacteria and fungi has drawn significant interest for
further research (Friedrich et al., 2001). The sequence and key physicochemical characteristics of
indolicidin are detailed in Table 1.
9

Table 1. Sequence and physicochemical properties of indolicidin.
Sequence

ILPWKWPWWPWRR-NH2

Structure

Multiple spatial conformation

Residue number

13

Molecular weight

1906.3 Da

Net charge*

+4

Isoelectric point

14

Solubility

1 mg/mL (water)

*Net charge at pH 7: Lys (K), Arg (R), and C-terminal amidation (NH2) were assigned with +1
charge (Araujo et al. 2022).
1.5.1 Trp and Pro
Trp and Pro are significant in indolicidin’s sequence as they are both important in the
assembly and structure of membrane proteins (Ladokhin et al. 1997). Trp is one of the most
hydrophobic among the 20 canonical amino acids, as assessed by the Wimley-White scale for
peptides (Wimley and White 1996). This hydrophobicity impacts indolicidin’s interactions with
the hydrophobic interior of phospholipid membranes (Mishra et al. 2018). Additionally, Trp has a
strong tendency to embed itself within membranes and localize near the interface between the
membrane and water phases, showing a preference for the lipid-carbonyl region (Mishra et al.
2018). Because of its aromatic indole side chain, Trp readily forms hydrogen bonds with
components of the lipid bilayer and the NH group on the indole ring. The unique structure of the
indole side chain in Trp leads to a specific orientation preference when it resides at the membrane
interface (Mishra et al. 2018). The bulky indole side chain, resembling a large paddle and
approximately one-third the thickness of a phospholipid monolayer, disrupts the cohesive
hydrophobic interactions among the lipid acyl chains when it penetrates deep into the hydrocarbon
10

core of the bilayer (Mishra et al. 2018). The presence of Trp in indolicidin residues facilitates its
insertion and partitioning with biological membranes, contributing to its hemolytic activity
(Subbalakshmi et al., 1996).
Moreover, Pro residues tend to introduce bends or kinks in peptides, which can interfere
with the formation of secondary structures (Figure 6) (Podorieszach and Huttunen-Hennelly,
2010). Consequently, despite having five Trp residues that contribute to its high hydrophobicity,
indolicidin's hydrophilic regions (with a net charge of +4) are not easily segregated into distinct
amphipathic regions. As a result, indolicidin tends to adopt a disordered conformation or contains
β-turn structures when free or bound to bilayers (Podorieszach and Huttunen-Hennelly, 2010).

Figure 6. Theoretical shape of indolicidin with red indicating hydrophobic regions and blue
indicating positively charge residues; the molecule is oriented from N-terminus (left) to
amidated C-terminus (right) (Kumar et al. 2018).
1.5.2 Structure and Mechanism of Action
The charge distribution of indolicidin reveals a hydrophobic core flanked by positively
charged areas positioned near both ends of the peptide, with three out of four charges oriented in
the same direction (Rozek et al., 2000). This molecular arrangement creates a wedge shape,

11

facilitated by four of the five Trp indole rings being closely packed against the peptide backbone
(Figure 7). Such a shape is well-suited for insertion between lipid molecules within a bilayer
(Rozek et al., 2000).
There are two possible membrane orientations that are proposed: (i) insertion into one
leaflet of the bilayer, resembling a "boat" structure with the positively and negatively charged
regions directed toward one side of the membrane, and (ii) a transmembrane orientation where the
two pairs of charges project onto opposite sides of the membrane (Figure 7b). Indolicidin
predominantly binds at the membrane interface, as demonstrated by Rozek et al. (2000),
suggesting penetration into one leaflet of the bilayer. This preference is likely due to the strong
affinity of the Trp indole rings for this arrangement.

Figure 7. Charge distribution on the surface of indolicidin. Positive and neutral potentials are
coloured blue and white, respectively. (A) End view looking down from the N-terminus (B) Side
view with the N-terminus pointed to the top (Rozek et al. 2000).

12

It has been demonstrated that indolicidin functions as an antimicrobial agent via a different
mechanism than the well-researched lytic peptides (Subbalakshmi and Sitaram 1998). Although
its main mode of action is by interaction with bacterial membranes, specifically the cytoplasmic
membrane, recent studies indicate other aspects of its mode of action (Figure 8). Indolicidin
permeabilizes both the outer and cytoplasmic membranes of bacteria by displacing divalent cations
from their binding sites on surface LPS (Falla et al. 1996). To achieve this, it uses a self-promoted
uptake pathway, in which indolicidin penetrates bacterial membranes directly, facilitating its
internalization into the cells (Falla et al. 1996). Inner membrane permeabilization by indolicidin
occurs rapidly following outer membrane permeabilization and is characterized by the formation
of discrete channels with relatively stable conductance (Falla et al. 1996).
Indolicidin disrupts fungal membranes similarly to bacterial membranes. Initially, it binds
to the membrane via electrostatic interactions, which are influenced by salt concentrations and
stabilized by Na⁺ and Mg²⁺ ions (Lee et al. 2003). This binding is followed by the formation of ion
channels in the membrane, leading to membrane destabilization and increased permeability.
Overall, indolicidin's fungicidal effect is driven by its ability to disrupt fungal membranes and
disrupt their integrity (Lee et al. 2003).
Indolicidin’s mode of action extends beyond membrane disruption. The mechanism of
action of indolicidin has been determined to primarily function through intracellular targets. The
most noteworthy action of indolicidin may be its capacity to inhibit uridine and thymidine from
being incorporated into RNA and DNA, respectively, hence preventing the synthesis of RNA and
DNA (Subbalakshmi and Sitaram 1998). Furthermore, this inhibition of DNA synthesis leads to
filamentation of the treated cells, suggesting that indolicidin's mechanism of action is aimed at
inhibiting cell replication (Hsu et al. 2005). Researchers also discovered that indolicidin binds to
13

a calcium-binding protein called Calmodulin (CaM), which is widely distributed in all eukaryotic
cells and plays a crucial role in Ca2+-mediated cellular processes like cell division, cycle,
cytoskeletal organization, and ion channel regulation (Sitaram et al. 2003). Bacteria have also been
found to express a CaM-like mutant protein called CalM, which is the protein product of the CaMlike gene (Michiels et al. 2002). This protein shares similar properties to CaM and binds Ca²⁺.
Although the exact mechanism by which indolicidin works remains unclear, it is reasonable to
assume that the peptide enters the cytoplasm to exert its action with intracellular targets. Its
multifaceted mechanisms underscore its potential as an effective antimicrobial agent with broad
applications in combating microbial infections.

Figure 8. Illustration of indolicidin's mode of action following brief membrane disruption: (a)
proteins and nucleic acids are intracellular potential targets; (b) the indolicidin–LPS interaction;
(c) indolicidin anion carrier mechanism (Araujo et al. 2022).

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1.5.3 Hemolytic Activity
The hemolytic activity of indolicidin poses a challenge to its potential use as a
pharmaceutical. Rokitskaya et al. (2011) found that indolicidin induces erythrocyte lysis by
disrupting the metabolic regulation of osmotic balance, potentially leading to increased erythrocyte
permeability. This disruption could occur through the induction of either anionic metabolite efflux
or the influx of the peptide itself into the cytoplasm, ultimately resulting in hemolysis (Rokitskaya
et al., 2011). Previous research demonstrated that indolicidin's hemolytic activity decreases when
the number of Trp residues is reduced, attributed to the peptide's reduced hydrophobicity and
overall

membrane-interface

propensity

(Podorieszach

and

Huttunen-Hennelly,

2010;

Subbalakshmi et al., 2000). This reduction in hydrophobicity may result in a decreased affinity for
lipid membranes and lower the tendency to disrupt them, leading to reduced hemolytic effects
(Subbalakshmi et al., 2000).
Numerous studies have explored the role of specific residues in increasing the activity of
indolicidin through the synthesis of analogous peptides. For instance, Ando et al. (2010)
demonstrated that analogs with a single Trp at positions 4, 8, or 11, and others replaced by leucine,
effectively reduced hemolytic activity while maintaining antibacterial efficacy (Figure 9).
Additionally, Podorieszach and Huttunen-Hennelly (2010) found that replacing more than two Trp
residues, starting from the C-terminus in indolicidin sequences, eliminated the peptide's
antimicrobial activity. These studies indicate that (i) Trp residues or large aromatic moieties play
important but complex roles in acting as AMPs, and (ii) some specific Trp analogs can exhibit
lower hemolytic activities than indolicidin. Despite these findings, no comprehensive investigation
has been conducted to develop indolicidin derivatives with high antimicrobial activity against both
Gram-positive and Gram-negative bacteria and fungi, while minimizing hemolytic effects.

15

Figure 9. Predicted Key Tryptophan Residues for Bacterial Lysis and Hemolysis (Ando et
al., 2010)

1.6 Objective
The significance of Trp and hydrophobicity must be considered when designing novel
AMPs. Hydrophobicity plays a critical role in the peptide's membrane transience and hemolytic
activity. Thus, in the present study, the impact of hydrophobicity will be evaluated by testing
indolicidin derivatives. These derivatives substitute two Trp residues with the less hydrophobic,
non-polar amino acid alanine (Ala) at different positions, thereby reducing the peptide's
hydrophobicity. This approach will enable investigation of variations in the hydrophobic residue
position within the peptide with respect to antimicrobial activity and hemolysis. Consequently,
indolicidin derivatives listed in Table 2 were designed and studied to hopefully optimize
bioactivity and reduce hemolysis.

16

Table 2. Sequence of indolicidin and other derivative peptides with abbreviations.
Sequence of Designed Peptides

Abbreviations

ILPWKWPWWPWRR-NH2

Indolicidin

ILPAKAPWWPWRR-NH2

Δ4,6

ILPAKWPWWPARR-NH2

Δ4,11

ILPWKAPAWPWRR-NH2

Δ6,8

ILPWKWPWAPARR-NH2

Δ9,11

Note: Single-letter amino acid abbreviations include: Isoleucine (I), leucine (L) proline (P),
tryptophan (W), lysine (K), arginine (R), and alanine (A). Bolded red alanine residues are the
position at which Trp is being replaced with Ala.

2 MATERIALS AND METHODS
2.1 AMP Stock Solution Preparation
Each peptide was synthesized and analyzed using MS and HPLC by Biomatik Corporation.
All peptides were guaranteed to have a purity of approximately >95% and were synthesized using
solid-phase synthesis. The AMP stock solutions were prepared by dissolving lyophilized AMPs
into sterile deionized water (18 mΩ) and their concentrations were determined by the NanoDrop
One Spectrophotometer. To create a 1000 μg mL-1 AMP stock, a 15 mL centrifuge tube was used,
into which 1 mL of sterilized deionized water was added. A small amount of AMP powder was
added to the deionized water, followed by vortexing. The NanoDrop One Protein A205 Scopes
method was selected. After zeroing the NanoDrop with deionized water, 2 µL of the AMP solution
was transferred onto the pedestal and the concentration was read using the A205 Scopes method.
This method monitors the absorbance of the peptide bond at 205 nm. The scopes method provides

17

a more accurate ε205 value, as it considers the substantial absorbance at 205 nm attributed by the
aromatic side chains of Trp.
2.2 Antimicrobial Susceptibility
2.2.1 Kirby-Bauer Disc Diffusion Assay
Each peptide's bioactivity was qualitatively confirmed using the Kirby-Bauer assay. At
various concentrations, 25 µL of each peptide was pipetted and absorbed into 6 mm sterilized
paper discs three times, totaling 75 µL, with an hour of drying time between each interval.
Additionally, bacterial and fungal species (Table 3) were incubated for approximately 16 hours to
grow to a turbidity of 0.600–1.000 in Muller Hinton (MH) broth. The turbidity was determined
using a spectrophotometer set to a wavelength at 600 nm. From the MH broth containing the
microbes, 0.2 mL of the broth was pipetted into 20 mL of MH agar, to get a bacterial/fungal
concentration of 1%. Immediately after, the bacteria/fungi containing agar was poured into Petri
dishes. Once the agar solidified, the peptide-concentrated paper discs were evenly placed on the
agar plates, alongside the negative control (water) and a positive control (antimicrobial disc). The
plates were then incubated at 37°C for 24 hours and then were examined for the presence of clear
zones, indicating inhibition of bacterial growth around the antibiotic discs. Antimicrobial activity
was evaluated by measuring the diameter of inhibition zone against each bacterial/fungal strain.

18

Table 3. Bacteria and fungi species used for antimicrobial susceptibility assays, cell type, and
antibiotic used for positive control.
Microbe

Microbe type

Antibiotic Control

Pseudomonas aeruginosa

Gram-negative

Tetracycline

Salmonella typhimurium

Gram-negative

Tetracycline

Escherichia coli

Gram-negative

Ampicillin

MRSA

Gram-positive

Ampicillin

Staphylococcus aureus

Gram-positive

Ampicillin

Candida albicans

Yeast

Nystatin

2.2.2 Minimum Inhibitory Concentration Assay
The minimum inhibitory concentrations (MICs) for the five AMPs were measured against
various bacterial and fungal species (Table 3) using the broth microdilution method in triplicate.
The test organisms were inoculated into a 2-mL microcentrifuge tube containing 1.5 mL of MH
nutrient broth by touching three to four different isolated cultured colonies with a flame sterilized
loop. The organisms were then grown to mid-logarithmic phase by incubating at 37°C with
constant mixing at 200 rpm for 16–18 hours on an orbital shaker. The bacterial and fungal cultures
were measured using the spectrophotometer set at 621 nm wavelength and a 1-cm light path. The
cultures were adjusted by diluting with MH broth to get an absorbance reading within the range of
0.08 to 0.10, indicative of the McFarland 0.5 standard (~1 x 108 CFU mL-1). The bacterial and
fungal cultures were diluted 100-fold to get an approximate cell density of ~1 x 106 CFU mL-1. In
a standard tray containing 96-wells, 20 µL of the 1 x 106 CFU mL-1 culture was transferred to each
well and the appropriate AMP concentration of 10–500 μg mL-1 was added and diluted to a final
volume of 200 µL. The final inoculum size was ~5 x 105 CFU mL-1. The 96-well plates were
19

incubated overnight for 16–18 hours at 37°C on an orbital shaker, providing sufficient time for
bacterial growth and observation of the antimicrobial effect of the peptides. The optical density
was measured using the ThermoFisher Multiskan Ascent plate reader at 621 nm. The instrument
was calibrated using MH broth as a blank. Bacterial/fungal cultures incubated without peptide
were used as negative controls, whereas antimicrobial activity exhibited by unaltered indolicidin
was taken as a positive control to demonstrate pathogen death with a known AMP. All samples
were prepared in triplicate. The lowest concentration of the derivatized AMP that prevented
growth represented the MIC. The MIC was defined as ≥85% killing of the initial inoculum.
2.3 Microbial Viability Assay
To elucidate the antimicrobial properties (bactericidal or bacteriostatic) of indolicidin and
its derivatives, 10 µL aliquots from the MIC wells were transferred into a 96-well plate and
incubated overnight for observable growth. The optical density was measured again using the
ThermoFisher Multiskan Ascent plate reader at 621 nm. If bacteria failed to resume growth in MH
broth after overnight incubation, the AMPs were bactericidal in the mechanism of cell death,
otherwise, it was considered bacteriostatic.
2.4 Hemolytic Activity
All horse whole blood samples were donated from the TRU Veterinary Technician
program. All peptides were prepared at in 1X phosphate-buffer saline (PBS). Whole blood samples
were centrifuged at 3000 RCF for 10 minutes at 4°C and the supernatant was poured off to isolate
the pellet containing the red blood cells (RBCs). The pellet was resuspended in 1X PBS at ten
times the volume of the pellet and centrifuged again, with the same conditions. The blood samples
were washed three times. Samples were then brought back up to whole blood volume and diluted
ten-fold to a final cell concentration of ~5 x 108 cells/mL, which was confirmed with a
20

hemocytometer. Of this, 200 µL was added to 800 µL of 1X PBS (pH 7.4) containing known
peptide concentrations in 10-fold serial dilutions. Triton X-100 (1% final concentration) was added
as a positive control, whereas 1X PBS was used as a negative control. Samples were then inverted
several times to ensure homogeneity and incubated for one hour at 37°C on the orbital shaker at
50 RPM, with further inversions after 30 minutes. After incubation, samples were centrifuged at
14000 RPM for five minutes at 4°C and the absorbance of the supernatant was measured on the
VWR® M4, UV/Visible Spectrophotometer at 541 nm to detect free hemoglobin in solution. The
instrument was blanked against 1X PBS, and the amount of hemolysis exhibited by 1% Triton X100 was taken to be 100% hemolysis (positive control). Thus, the percent hemolysis was taken to
be:
(𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎) − (𝑝𝑝ℎ𝑜𝑜𝑜𝑜𝑜𝑜ℎ𝑎𝑎𝑎𝑎𝑎𝑎 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎)
𝑥𝑥 100%
(𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 − 𝑋𝑋 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎)

The minimum hemolytic concentration was taken to be the concentration of peptide that caused
100% hemolysis (relative to Triton X-100).
2.5 Electrophoretic Mobility Shift Assay
To evaluate the DNA binding capabilities of indolicidin and its derivatives, an
electrophoretic mobility shift assay (EMSA) was performed. The plasmid pDN5, derived from
pRc/CMV, was purified using a QIAfilter plasmid midi kit. The plasmid concentration was
determined by UV spectrophotometry on the Nanodrop at 260 nm and quantitative analysis on an
agarose gel. The plasmid DNA pDN5 (200 ng) was mixed with increasing amounts of peptides
(50-1000 ng) with 2 μL of 10X digestion buffer and sterilized deionized water such that the final
volume of the reaction was 20 μL. For the negative control, 200 ng of pDN5 was used. The

21

mixtures were incubated at room temperature for 30 minutes. The peptide:DNA mass ratios were
0:1, 0.25:1, 0.5:1, 2.5:1, 5:1, respectively. After adding 5 μL of loading buffer (5X loading dye),
complexes of DNA and peptides were resolved by electrophoresis on a 1% agarose gel in 1X
Tris/Borate/EDTA (TBE) buffer (40 mM tris borate and 1 mM EDTA, pH 8.0) containing
ethidium bromide for visualization of the DNA bands. The gel was run for 60 minutes at 80 V and
the migrated DNA was visualized under UV light.
3 RESULTS
3.1 Bioactivity Characterization
3.1.2 Kirby-Bauer Disc Diffusion Assay
Kirby-Bauer disc diffusion assays were performed on nutrient agar with all target
organisms listed (Figure 10). Zones of inhibition occurred one day post-inoculation and ranged
from 5.5 mm to 8 mm in diameter (Table 4). All derivatives demonstrated activity against
Escherichia coli, except for indolicidin and ∆4,11. ∆4,11 demonstrated activity against Salmonella
typhimurium. Indolicidin was not active in the Kirby-Bauer assay against any microbes tested.

Figure 10. Kirby-Bauer assay of the different indolicidin derivatives against Escherichia coli.

22

Table 4. Zone of inhibition diameter measurements of the indolicidin derivatives tested on various
bacteria and fungi.
Escherichia
coli

Pseudomonas
aeruginosa

Salmonella
typhimurium

MRSA

Staphylococcus
aureus

Candida
albicans

∆0

–

–

–

–

–

–

∆4,6

++

–

–

–

+

–

∆4,11

–

–

++

–

+

–

∆6,8

++

–

–

–

++

–

∆9,11

++

–

–

–

++

–

Note: – indicated no zones of inhibition, + indicates inhibition zones < 7 mm, ++ indicates zones
≥ 7 mm.
3.1.3 MIC Assay
The MIC values at a 90% reduction in optical density for indolicidin and the analogs are
shown in Table 5. All the peptides listed in Table 4, except for Δ4,6, exhibited antibacterial
activities against Gram-positive bacteria. Interestingly, compared to indolicidin, all peptide
derivatives demonstrated a >10-fold decrease in antimicrobial activity against both Gram-positive
and Gram-negative bacteria and fungi. Substitution of Trp residues at 4,11 resulted in the loss of
activity against Gram-positive bacteria. Substitution of the Trp residues at 4,6 resulted in a loss of
activity against Gram-positive and Gram-negative bacteria, only being active against Candida
albicans. The derivative Δ4,11 was only active against the Gram-negative bacteria Salmonella
typhimurium. The assay was conducted in triplicate, resulting in consistent findings with variations
of less than 5% between replicates.

23

Table 5. MICs of peptide derivatives against a variety of bacterial and fungal pathogens, where
active concentrations are determined by exhibiting at least 90% reduced optical density compared
to each control culture incubated without peptide; a dash (—) indicates that the peptide was not
active at the concentrations tested.
Active Concentration/μg mL-1
Peptide

Escherichia
coli

Pseudomonas
aeruginosa

Salmonella
typhimurium

Candida
albicans

MRSA

Staphylococcus
aureus

Gramnegative

Gramnegative

Gramnegative

Yeast

Gram-positive

Gram-positive

Indolicidin

50

250

50

50

50

50

Δ 4,11

—

—

500

—

—

—

Δ 4,6

—

—

—

500

—

—

Δ 9,11

500

—

500

500

250

500

Δ 6,8

250

—

500

500

250

500

The MIC values at a 50% reduction in optical density for indolicidin and its analogs are
presented in Table 6. Interestingly, when tested for a 50% reduction instead of 90% in optical
density, Δ4,11 exhibited activity against Candida albicans at 500 μg mL-1, showing a significant
reduction in optical density. Additionally, Δ4,6 demonstrated activity at 50 μg mL-1 against
Candida albicans. Similarly, Δ9,11 displayed activity at 50 μg mL-1, reducing the optical density
by 58%. Moreover, Δ6,8 was the only analog that showed activity against Pseudomonas
aeruginosa at 500 μg mL-1.

24

Table 6. MICs of peptide derivatives against a variety of bacterial and fungal pathogens, where
active concentrations are determined by exhibiting at least 50% reduced optical density compared
to each control culture incubated without peptide; a dash (—) indicates that the peptide was not
active at the concentrations tested.
Active Concentration/μg mL-1
Peptide

Escherichia Pseudomonas Salmonella Candida
coli
aeruginosa typhimurium albicans

MRSA

Staphylococcus
aureus

Gramnegative

Gramnegative

Gramnegative

Yeast

Gram-positive

Gram-positive

Indolicidin

50

250

50

50

50

10

Δ 4,11

—

—

500

500

—

—

Δ 4,6

—

—

—

50

—

—

Δ 9,11

250

—

500

500

250

50

Δ 6,8

250

500

250

250

250

250

Figure 11. Serial dilution results with decreasing concentrations of Δ6,8 (from left to right: 500
μg/mL to 0 μg/mL) against Candida albicans.

3.1.2 Microbial Viability Assay
The bacteriostatic (BS) and bactericidal (BC) activities of the indolicidin derivatives were
assessed by subculturing the broths used for MIC determination into fresh MH broth. Among the
derivatives, Δ4,11 exhibited BS activity against Salmonella typhimurium at its MIC of 500 μg
mL-1, while Δ4,6 demonstrated BS activity against Candida albicans at its MIC of 500 μg mL-1
(Table 7). Additionally, both Δ6,8 and Δ9,11 displayed BS activity at their respective MICs for all
tested microorganisms. However, Δ6,8 demonstrated BC activity above its MIC at 500 μg mL-1

25

against Escherichia coli and MRSA. Similarly, Δ9,11 exhibited BC activity against MRSA above
its MIC at 500 μg mL-1.
Table 7. Bacteriostatic/Bactericidal (BS/BC) activity of indolicidin derivatives.
Peptide

Δ 4,11

Δ 4,6

Δ9,11

Δ 6,8

MIC
(μg mL-1)

BS/BC
action

MIC
(μg mL-1)

BS/BC
action

MIC
(μg mL-1)

BS/BC
action

MIC
(μg mL-1)

BS/BC
action

Escherichia
coli

—

—

—

—

500

BS

250

BS

Pseudomonas
aeruginosa

—

—

—

—

—

—

—

—

Salmonella
typhimurium

500

BS

—

—

500

BS

500

BS

Candida
albicans

—

—

500

BS

500

BS

500

BS

MRSA

—

—

—

—

250

BS

250

BS

Staphylococcus
aureus

—

—

—

—

500

BS

500

BS

3.1.3 Hemolysis
The hemolytic activity of the five structurally related peptides was evaluated in
erythrocytes from horses. The subsequent release of hemoglobin was used to assess hemolytic
activity as function of peptide concentration by measuring to absorbance of the supernatant at 541
nm to detect free hemoglobin in solution. Comparing the analogues to indolicidin, the hemolysis
test findings show more than 16-fold reduction in hemolysis, or decreased cytotoxicity (Table 8).
However, since the hemolytic concentrations for these derivatives were beyond the measurable
range, the hemolytic index (MHC/MIC) could not be calculated (NA). These results indicate
significantly lower hemolytic activity when the two Trp’s were substituted for Ala in all

26

derivatives compared to indolicidin. According to the database, DBAASP, it was predicted that all
peptides, except for the peptide Δ9,11, would have substantial hemolytic activity. However, the
data in table 8 indicates that this prediction is only valid for indolicidin.
Table 8. Minimum hemolytic concentrations (MHCs) of indolicidin and derivatives compared to
the AMP hemolytic activity predictor. In cases of high MHCs, the highest concentration tested is
given with its percent hemolysis at that concentration. Hemolytic Index (HI) is defined here as the
MHC divided by the lowest MIC derived.
Hemolytic concentration
(μg mL-1)

Hemolytic Index
(MHC/MIC)

Database Hemolytic
Concentration
Prediction
(μg mL-1)

Indolicidin

30

39

<25

Δ4,6

700 (2.92%)

NA*

<25

Δ4,11

700 (0.84%)

NA*

<25

Δ9,11

700 (4.2%)

NA*

>100

Δ6,8

700 (2.2%)

NA*

<25

Note: *Not available; For hemolytic activity predictions, the DBAASP was used
(https://dbaasp.org/tools?page=property-calculation).
3.2 DNA Binding Assay
To determine Trp’s effect on indolicidin’s mechanism, DNA binding was indirectly
measured by monitoring the effect of increasing peptide amount on the migration of purified pDN5
plasmid DNA. Specifically, indolicidin and its derivatives were tested to see if they could interact
with DNA molecules, which would lead to altered mobility in agarose gel electrophoresis. The
plasmid migration of the derivatives is shown in Figure 12. The assay revealed distinct binding
patterns among the tested peptides. Δ9,11 and Δ6,8 demonstrated the weakest ability to inhibit
plasmid migration since strong DNA bands were still observed at peptide:plasmid weight ratios of
27

5:1. As shown in Figure 12, Δ4,11 and Δ4,6 appeared to inhibit plasmid migration at a
peptide:plasmid ratio of 2.5:1 and 5:1, as the bands travelled less far compared to the control.
Indolicidin had the highest DNA affinity, inhibiting plasmid migration at a weight ratio as low as
0.5:1. These findings suggest that certain structural modifications within indolicidin and its
derivatives, particularly with Trp, influence their ability to interact with DNA.

A

Indolicidin

D

Δ9,11

B

Δ4,11

E

C

Δ4,6

Δ6,8

Figure 12. Electrophoretic mobility assay of DNA binding to peptides. Different amounts of
indolicidin (A), Δ4,11 (B), Δ4,6 (C), Δ9,11 (D), and Δ6,8 (E) was incubated with 200 ng of pDN5
plasmid DNA. The binding affinity was assessed by the inhibition of the electrophoretic migration
of the DNA by the peptides. The peptide:DNA mass ratios for the different lanes from left to right.

4 DISCUSSION

28

The relationships of the structural features of indolicidin and functional properties of Trp
with cell lysis activities can be derived from the evaluations of a series of indolicidin derivatives:
their antimicrobial activities, and DNA binding capacity. This study assessed both the changes in
antimicrobial and hemolytic activity of indolicidin and its derivatives, when Trp was replaced with
the less hydrophobic amino acid Ala, as well as the DNA binding capabilities of indolicidin and
its derivatives. By replacing Trp in indolicidin’s sequence, its antimicrobial activity decreased by
>10-fold, however its hemolytic activity was almost completely removed, decreasing by >16-fold.
Furthermore, indolicidin, Δ4,11, and Δ4,6 were found to bind DNA, while Δ9,11 and Δ6,8 did not.

4.1 Antimicrobial Activity
In the Kirby-Bauer assay, indolicidin was not active even though indolicidin has known
activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Candida
albicans, Staphylococcus aureus, and MRSA. A possible explanation for why indolicidin may not
have shown any zones of inhibition is that perhaps indolicidin was too hydrophobic to diffuse well
from the disc into the aqueous agar. One known shortcoming of the Kirby-Bauer assay is that the
antimicrobial agent on the disc needs to be able to diffuse from the disc into the agar plate. Due to
the inconsistencies with the Kirby-Bauer assay and due to its qualitative nature, we moved on to
the MIC assay.
According to the MIC results of indolicidin and the analogs, there was no antibacterial
activity observed against Gram-negative and Gram-positive bacteria in the Trp analog substituted
at positions 4,6. Δ4,6 was only microbially active against the eukaryotic fungi Candida albicans.
Conversely, the Trp analog substituted at 4,11 was no longer active against Candida albicans and
only against the Gram-negative bacteria Salmonella typhimurium. On the other hand, the Trp

29

analogs substituted at positions 6,8 and 9,11 exhibited the highest activity among the analogs. This
demonstrates the important role of the Trp residue at position 4 for the antimicrobial activity of
indolicidin, while indicating that position 6 is not crucial for activity.
The absence of antibacterial activity observed in the Trp analogs substituted at position 4
suggests the critical importance of the Trp residue at that specific position for effective
antimicrobial action. This is consistent with previous findings by Ando et al. (2010), which also
highlighted the significance of the Trp residue at position 4 in indolicidin's activity. However, they
found the Trp residue at position 11 was important to the antimicrobial activity of indolicidin
(Ando et al. 2010). Conversely, the derivative in which the Trp analog was substituted for Ala at
positions 9,11 had strong antimicrobial activity against Gram-negative and Gram-positive bacteria,
as well as fungi, consistent with the findings of Podorieszach and Huttunen-Hennelly (2010). The
current results further highlight the significance of the Trp residue at position 4 for antimicrobial
activity, as its replacement led to a significant decrease or complete loss of antimicrobial efficacy.
While Δ6,8 and Δ9,11 exhibited antimicrobial activity against most of the microbes tested,
both had a reduction in activity by ~10-fold, compared to indolicidin. The decrease in antimicrobial
activity observed in all derivatives compared to indolicidin can be attributed to the reduction in
overall hydrophobicity by removing two of the Trp residues. Hydrophobicity plays a crucial role
in the interaction of AMPs with bacterial membranes. When hydrophobicity is reduced, AMPs
may have decreased affinity for the lipid bilayer, leading to less efficient membrane disruption and
ultimately reduced antimicrobial activity. Additionally, Trp residues are known to contribute to
the amphipathic nature of AMPs, allowing them to insert into the lipid bilayer and disrupt
membrane integrity. The substitution of Trp residues in our derivatives may have disrupted this

30

amphipathic structure, impairing their ability to interact with and permeabilize bacterial
membranes effectively.
A 90% reduction in optical density was stated to be the MIC. At concentrations of 500 μg
mL-1, above their MIC, for Δ6,8 and Δ9,11, cell membranes were completely solubilized, resulting
in a 100% decrease in optical density. These Δ6,8 and Δ9,11 experimental cultures were reinoculated, and no growth was observed as confirmed by no absorbance at 621 nm, supporting
completed bacterial lysis and complete cell death. However, at lower concentrations near the MIC,
growth was severely limited by over 90%, yet the initial inoculum often remained intact, yielding
a slight absorbance. Additionally, the initial inoculum remained viable, as demonstrated by reinoculation of the pathogens in peptide-free media. The re-inoculated solutions appeared clear, but
still exhibited a slight absorbance, indicating their bacteriostatic and bacteriolytic properties.
Growth inhibition that does not compromise membrane integrity would also suggest an
intracellular mode of action, which may be related to the peptide derivatives' reduced
hydrophobicity (Podorieszach and Huttunen-Hennelly 2010) and hence preference to be inside the
aqueous cytosol. While studies have reported that the primary mechanism of action involves selfpromoted uptake through electrostatic interactions with bacterial/fungal cell wall and LPS
components (Falla et al. 1996), our study suggests that other factors, such as hydrophobicity and
Trp substitution location, also play crucial roles.

4.2 Hemolytic Activity
In this study, we observed a reduction in hemolysis with each Trp substitution (Table 5).
This reduction in hemolysis can be attributed to Trp’s significant contribution to the
hydrophobicity of the peptides and their positioning at the membrane interface, as well as its

31

involvement in protein/DNA binding. These results provide important insights into the hemolytic
nature of cationic AMPs.
The reduction in hemolytic activity observed in our derivatives indicates that the
mechanisms of membrane interaction are more complex than previously thought. Rather than
solely relying on electrostatic associations, peptides with reduced Trp content may interact
differently with cell membranes, leading to decreased cytotoxicity. This suggests that AMPs can
potentially be designed or modified to minimize hemolytic effects while maintaining antimicrobial
efficacy.
According to the American Society for Testing and Materials (ASTM), hemolysis of less
than 5% is considered negligible (Luna-Vázquez-Gómez et al. 2021). All the peptide derivatives
exhibited ≤5% hemolysis, indicating their minimal cytotoxicity. This, coupled with their
antimicrobial activity, suggests that these peptides have significant potential as pharmaceutical
agents, particularly Δ6,8 and Δ9,11. These two peptides demonstrated broad-spectrum activity
against Gram-negative and Gram-positive bacteria, as well as eukaryotic yeast cells, all while
having minimal hemolytic effects.

4.3 DNA Binding by Inhibition of Plasmid Migration
To evaluate the DNA binding abilities of indolicidin and its derivatives, plasmid migration
was monitored in an agarose gel with increasing peptide concentrations. Indolicidin exhibited the
strongest affinity for DNA binding, followed by Δ4,6 and Δ4,11, which also displayed significant
DNA binding capabilities. Conversely, Δ9,11 and Δ6,8 showed no detectable DNA binding. This
differential binding pattern suggests that specific structural features and ordering of the amino
acids in a sequence influence the DNA binding activity and mechanisms of these peptides.
32

Several possible factors may impact the DNA binding activity of these peptides. This may
be explained by the presence of the PWWP motif present in indolicidin’s sequence. Indolicidin,
Δ4,6, and Δ4,11, which include the PWWP domain in their sequence, bind DNA more strongly
than Δ6,8 and Δ9,11, which lack the PWWP domain. PxxP motifs consist of a proline (P) followed
by any two amino acids (x) and another proline (P) (Lee et al. 2023). Studies have shown that
central PxxP sequences are crucial for bacterial selectivity and efficient interaction with negatively
charged membranes (Lee et al. 2023). Specifically, PWWP motifs are known to recognize and
bind DNA and form a helical turn structure (Ghosh et al. 2014). PWWP motifs are found in nuclear
proteins such as hepatoma-derived growth factors (HDGF) and DNA methyltransferase proteins
(Marchand et al. 2006). Another example of a PWWP domain-containing protein is the lens
epithelium-derived growth factor (LEDGF/p75), which directly binds HIV-1 and facilitates its
tethering to chromosome (Marchand et al. 2006). The results of this study demonstrate that the
removal of this PWWP motif from the Δ6,8 and Δ9,11 derivatives, affected their capacity to bind
to plasmid DNA. This suggests that the presence of the PWWP domain enhances the DNA binding
affinity of indolicidin and its derivatives.
Although the derivatives Δ4,11 and Δ4,6 exhibited DNA binding capacities, this study
found no correlation between the peptide’s ability to bind DNA and its antimicrobial activity.
Surprisingly, peptides Δ6,8 and Δ9,11, lacking the PWWP domain, exhibited better antimicrobial
activity compared to Δ4,11 and Δ4,6 that retained the PWWP motif and bound DNA. This suggests
that mechanisms other than DNA binding, such as membrane disruption or intracellular targeting,
may play a significant role in determining the antimicrobial efficacy of these peptides. Once these
analogs enter the cell, they may disrupt various intracellular lipid-bound organelles in eukaryotic
cells, including the nucleus (exposing DNA to cytosolic DNases), the endoplasmic

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reticulum/Golgi body (disrupting protein synthesis), mitochondria (impairing cellular respiration),
or vesicles (releasing proteolytic enzymes or disrupting storage compartments). An
apoptosis/necrosis assay can help distinguish between these possibilities in animal cells, where
DNA fragmentation or mitochondrial disruption would indicate apoptosis, while cellular
permeabilization would suggest necrosis. This assay would also assess the cytotoxicity against
nucleated animal cells.
5 CONCLUSIONS AND FUTURE WORK
This study demonstrates the significant impact of Trp substitution on the antimicrobial and
hemolytic activities of indolicidin and its derivatives. The findings reveal that while substitutions
at specific Trp positions led to a loss or reduction in antimicrobial activity, they also resulted in a
significant decrease in hemolytic activity, suggesting a more nuanced role for Trp in both
functions. Furthermore, the substitution of Trp for Ala at position 4 resulted in a significant loss
or complete removal of antimicrobial efficacy against Gram-positive and Gram-negative bacteria
and fungi, which is consistent with previous studies. Furthermore, it was found that derivatives
lacking the PWWP domain, known for DNA binding, exhibited better antimicrobial activity than
those with strong DNA binding capacity. This suggests that mechanisms other than DNA binding
may play a significant role in determining the antimicrobial efficacy of these peptides. Further
studies are needed to elucidate the specific mechanisms involved in the antimicrobial action of
these peptides. Additional future work should include cytotoxicity testing against nucleated animal
cells to ensure safety and testing the effects of indolicidin derivatives on biofilm formation to
provide insights into their potential as anti-biofilm agents.

34

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