Download Proposal Summary - Clarkson University

Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
Identification of a Peptide with Antimicrobial
Activity in the Venom of Conus
Honors Thesis Proposal
April 2005
Sarah Morris
Department of Biology
Clarkson University, Potsdam, NY 13699-7165
Mentor: Dr. Jon-Paul Bingham, Ph.D.
Research Assistant Professor, Department of Biology
Clarkson University, Potsdam, NY 136995-5805
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
Goal
To identify a peptide(s) that possesses antimicrobial behavior in the venom,
Conus
Purpose
In the past few years, there has been increasing interest in the interaction between
peptides and bacterial channels. Just recently, in 2003, Roderick MacKinnon was
awarded the Nobel Prize in Chemistry for his research on potassium ion channels. He
determined that an exquisitely specific toxin isolated from scorpion venom binds to
specific potassium ion channels just as tightly as it does to bacterial potassium channels.
This suggests that these channels share the same structure and may be useful for
screening potential new drugs (MacKinnon, 2004).
Along with MacKinnon, several other scientists have found interactions between
scorpion toxins and bacterial channels. Some of the findings include: interaction between
KcsA potassium ion channel and the pore-blocking toxin Agitoxin2 (Takeuchi et al.,
2003), binding mechanism of voltage-sensitive potassium channel and toxin kappaconotoxin PVIIA (Scanlon et al., 1997), and binding of Kaliotoxin and Hongotoxin 1 to
KcsA-Kv1.3 chimeric potassium channels (Legros et al. 2000). These are just a few
examples of the research that is currently being conducted on the binding mechanisms of
peptides and bacterial channels.
Therefore, interactions of several scorpion toxins and potassium channels have
been shown and it is of interest to continue this research to find other peptides, which
may interact specifically with bacterial channels. This paper breaks down this project
into several sections. The first section presents the background knowledge of the topics
being investigated. The second section shows proposed work and the paper is concluded
with preliminary results and a timetable for the completion of this work.
Background
This section serves as a brief overview of each topic in this project. Topics
include the types of bacteria that will be used to examine the antimicrobial activity of the
toxins, the precedent set by 2scorpion toxins and their interaction with bacterial channels,
and potassium channels and their selectivity.
Gram-Positive and Gram-Negative Bacteria
Bacteria are either classified as gram-positive or gram-negative depending on
their response to the gram stain. Gram-positive bacteria have a thick cell wall consisting
of multiple layers of peptidoglycan. This is because it has no outer membrane. Teichoic
acids are attached to the peptidogylcan layer, which are antigenic and serve as the
receptors for bacteriophages. On the other hand, gram-negative bacteria have a much
thinner cell wall consisting of a single layer of peptidoglycan which is between the inner
and outer lipid bilayer membranes. The outer membrane of gram-negative bacteria is
coated with a highly complex lipopolysaccharide, which consists of a lipid group joined
2
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
to a polysaccharide made up of long chains with many different and characteristic
repeating structures. These different units determine the antigenicity of the bacteria.
These antigenic determinants are called the O antigens and the variation in these antigens
has a role in the recognition of one type of cell by another and in evasion of the host
immune cell (Garrett and Grisham, 2005). The research being performed will focus on
four different types of bacteria, two being gram-positive and two being gram-negative.
Staphylococcus aureus
Staphylococci are gram-positive spherical bacteria that occur in microscopic
clusters.
Figure 1: Electron micrograph of Staphylococcus aureus (Todar, 2004).
They are facultative anaerobes that grow by aerobic respiration or by fermentation that
yields lactic acid. They are catalase-positive and oxidase-negative. Staphylococcus
aureus forms a fairly large yellow colony on rich medium. This bacterium produces the
enzyme coagulase and is considered a potential pathogen. S. aureus causes a wide range
of suppurative infections and toxinoses in humans. It causes superficial skin lesions such
as boils, styes and furunculosis. It causes more serious infections including pneumonia,
mastitis, phlebitis, meningitis, urinary tract infections, osteomyelitis, and endocarditis. S.
aureus is also a major cause of hospital acquired infection of surgical wounds and
infections, food poisoning, and toxic shock syndrome by release of superantigens into the
blood stream (Todar, 2004).
Staphylococcus aureus expresses many potential virulence factors including
surface proteins that promote colonization of host tissues; invasions that promote
bacterial spread in tissues; surface factors that inhibit phagocytic engulfment;
biochemical properties that enhance the survival in phagocytes; immunological disguises;
membrane-damaging toxins that lyse eukaryotic cell membranes; exotoxins that damage
host tissues; and inherent and acquired resistance to antimicrobial agents (Todar, 2004).
Most strains are resistant to all clinically useful antibiotics except vancomycin; however,
vancomycin strains are increasingly being reported. In addition, S. aureus exhibits
resistance to antiseptics and disinfectants.
Bacillus Subtilis
Bacilli are a diverse group of bacteria that include several species that synthesize
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
important antibiotics. They are rod-shaped, gram-positive, sporulating, aerobes or
facultative anaerobes.
Figure 2: Individual Cell of B. Subtilis photographed on nutrient agar (15,000X
magnification) (Todar, 2003).
Each bacterium creates only one spore, which is resistant to heat, cold, radiation, and
disinfectants. B. Subtilis causes various infections ranging from ear infections to
meningitis, and urinary tract infections to septicemia. For the most part, they occur as
secondary infections in immunodeficient hosts (Todar, 2003). It is resistant to most
antibiotics making treatment difficult.
Enterococcus faecalis
Enterococcus faecalis, also known as E. faecalis, is the most common, clinically
relevant intestinal species. E. faecalis is nonmotile gram-positive cocci in pairs or short
chains.
Figure 3: Vancomycin Resistant Enterococcus faecalis photgraphed with a scanning
electron microscope (Todar, 2003).
It is catalase negative and most are facultative anaerobes. It inhabits the intestines of
humans and animals and is capable of surviving high concentrations of bile and sodium
chloride. It colonizes the large intestine and urinary tract, where most infections
originate. E. faecalis is responsible for several infections in humans: nasocomial
infections, urinary tract infections, bacteremia, subacute endocarditis, wound infections,
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
foodborne disease, and meningitis. Treatment is difficult since it is resistance to most
antibiotics (Todar, 2003).
Escherichia Coli
E. coli is part of a large bacterial family, Enterobacteriaceae, which are
faculatively anaerobic gram-negative rods that live in the intestinal tracts of animals in
health and disease.
Figure 4: E. Coli photographed with a scanning electron microscope (Todar, 2002).
E. coli is among the most important bacteria medically. Over seven hundred antigenic
types are recognized based on O, H, and K antigens. These types are important in
distinguishing which strains actually cause disease. E. coli is responsible for three types
of infections in humans: urinary track infections, neonatal meningitis, and intestinal
diseases (Todar, 2002). These diseases depend on a specific array of pathogenic
determinants. Although E. Coli is multi-drug resistant, it is sensitive to a combination of
penicillin or ampicillin plus aminoglycoside.
Scorpion Toxins
Scorpion toxins are short peptides that affect ion channel function. The following
is the structure of a scorpion toxin from Androctonus austalis.
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
Figure 5: Scorpion Toxin from Androctonus australis
It is a 64-residue protein that binds to mammalian sodium channels, causing paralysis and
death at high enough doses. The alpha helix is shown in pink, while the beta sheet is
shown in orange. Most of the residues indicated to be crucial to binding to sodium
channels are located on a knob at the lower left of the image. Positively charged amino
acids are shown in red. Arg 62 and His 64 protrude furthest from the core of the protein.
It is reasonable to expect such residues to be involved in binding to sodium channels,
which usually handle positively-charged Na+ ions. Mutational analysis has shown that
Asp 8 (blue) and Lys 58 (green) are likely to be critical to binding (Figure 5). Arg 18 is
not in the same region as the rest of the binding residues, but probably also has a major
role in binding. The purple segment is residues 9 through 12, which together with residue
8, form a structurally conserved loop found in many scorpion toxins.
Some scorpion toxins have been found to block potassium ion and chloride ion
channels. These particular toxins consist of 29-41 amino acid residues and are stabilized
by three or four disulfide bridges. They have a segment of alpha helix and at least two
segments of antiparallel beta sheet structure. These scorpion toxins block these channels
through direct obstruction of the channel outer vestibule, therefore preventing ion
permeation (Chagot et al., 2005). Other toxins, such as this toxin from Androctonus
austalis have been found to block sodium ion channels. These types of toxins are
composed of 60-76 amino acids and are stabilized by four disulfide bridges. They have
one alpha-helix and three segments of beta-sheet structure. These toxins block the
sodium ion channel function by modifying the channel gating mechanism (Possani et al.,
1999).
Potassium Ion Channels
Potassium channels are designed to allow the flow of potassium ions across the
membrane, but to block the flow of other ions, in particular sodium ions. These channels
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
are typically composed of two parts: the filter, which selects and allows potassium but
not sodium to pass, and the gate, which opens and closes the channel based on
environmental signals. The filter is comprised of four identical protein molecules that
span the width of the membrane, forming a selective pore down the center.
Figure 6: Ribbon representation of the tetramer illustrating the 3-D fold of the KcsA
tetramer viewed from the extracellular side. The ribbons represent the pore helices and
four identical protein molecules form the selectivity filter, which is shown down the
center (Doyle et al., 1998).
Potassium ions flow freely through this filter, at rates of up to one hundred million ions
per second. However, it is also remarkably selective; it allows only one sodium ion to
pass for every ten thousand potassium ions. The gating domains open and shut the
channel through structural mechanisms based on different signals, such as voltage or the
presence of key signaling molecules (Shealy et al., 2003).
The ability of the potassium channel to pass only potassium ions is accomplished
through the filter. Under normal conditions, potassium ions are encased by eight water
molecules. In order to pass through the filter, the ion must shed these water molecules.
The filter works through the following process: the dimensions of the channel are
designed to mimic this shell of water. Oxygen atoms that line the pore are oriented
toward the center of the channel. Eight of the oxygen atoms surround each ion and act as
a replacement for the water molecules. During transport, the ions move from one site to
the next along the pore; once across the filter, they are enclosed by water molecules
again. Sodium ions, on the other hand, are smaller in size, which results in a failure to
interact with the oxygen atoms lining the pore wall. They prefer their normal shell of
water so they are not efficiently moved across the membrane (Doyle et al., 1998).
Potassium channels play a critical role in signaling nerves and therefore blockage
of these channels can have serious effects. As stated above, scorpion venom contains
neurotoxins that bind to ion channels and block the flow of ions. An example of a toxin,
which has been investigated is Charybdotoxin, a small 37 amino acid peptide, which has
been found to attack potassium channels and block their function if signaling nerves. The
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
positively-charged amino acids covering the surface of the protein are thought to “glue”
the toxin over the exposed mouth of the pore. The small and highly stable structure of
Charybdotoxin consisting of three disulfide linkages help to hold the peptide in its proper
3-D configuration to block the channel (Gao and Garcia, 2003).
Proposed Work
The research will be broken into five stages. The preamble stage included
learning techniques such as preparation of the materials needed, transfer of bacteria using
inoculation, the spread plate technique (bacterial lawn), maintaining bacteria, reversephase high pressure liquid chromatography (RP-HPLC), and measuring growth
inhibition. The first stage involves the set-up of solution phase assay and confirmation
through testing with antibiotics. In addition, peak(s) will be identified that may have
direct biological activity. The second stage involves the isolation of an adequate quantity
of peaks that will allow for the identification of the peptide(s) of interest. Third, the
peptide(s) of interest will be characterized using classical Edman techniques and/or
advanced techniques in mass spectrometry. The fourth stage will be to synthetically
make the peptide(s) of interest using Fmoc solid phase peptide synthesis. The final stage
will include the reconfirmation of the biological activity originally observed using the
synthetic material.
Stage 1 (B): Solution Phase Assay
Microorganisms: Escherichia coli and Staphylococcus aureus
The bacteria will be grown in Brain Heart Infusion at 37C and after 4 hours, the
suspension will be undergo a serial dilution to approximately 10-10 ml bacteria. The
bacteria will be incubated in a 96-well plate in a spectrophotometer with continuous
shaking for 36 hours. Readings will be taken every hour at of a wavelength of 620 nm to
locate the time of maximum growth. Once this peak is determined, the experiments will
be tested with known antibiotics to confirm activity and reference the quantification.
This approach follows the method as described by Moerman et al. (2002).
Stage 2 (C): Isolation of Peaks for Identification of Peptide(s) of Interest
Using reverse phase HPLC, in combination with solution phase assay, peak(s)
will be isolated to allow for the identification of peptide(s) that have biological activity
(in this case, kill the bacteria). Lyophilized whole venom will be dissolved in 0.1%
trifluoroacetic acid and fractionated in a two-step reserve phase HPLC. 0.1%
trifluoroacetic acid in water will be used as buffer A and 0.1% trifluoroacetic acid in
acetonitrile will be used as buffer B. A linear gradient from 0 to 100% acetonitrile will
be applied for 25 minutes at a flow rate of 1 ml per minute. At this time, fractionation
will begin. After determination of the active peak, a subsequent purification will be
performed using a linear gradient from 0 to 60% 0.1% trifluoroacetic acid (TFA) in
acetonitrile for 17.5 minutes (Moerman et al., 2002). The determination and
confirmation of this active peak will then allow for the identification of peptide(s) that
have biological activity.
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
Stage 3 (D): Characterization of the peptide(s) of interest
The physical characteristics of the peptide(s) identified in stage 2 will be
determined including the molecular mass and amino acid sequence of the peptide(s). The
molecular mass will be determined through mass spectrometry. The peptide will be
dried, redissolved in an appropriate solution, and loaded into nanoelectrospray needles.
All fractions will be investigated by precursor ion scanning performed on a Thermo
Finnigan ion-trap mass spectrometer, equipped with a nanoelectrospray ion source.
Precursor ion scans will be acquired with a dwell time of 50 milliseconds (Steen and
Mann, 2002). The m/z values resulting will be compared to those acquired by a
theoretical peptide, performed by a computer program.
The amino acid sequence of the peptide will be determined using Edman
degradation and/or advanced mass spectrometry techniques (collision-induced
dissociation (CID)). For Edman degradation, the peptide will be dissolved in a mixture
of acetonitrile, water, and trifluoroacetic acid. Two microliters of the peptide will be
loaded on a glass fiber and subjected to N-terminal amino acid sequencing on a protein
sequencer running in the pulsed liquid mode. If the complete sequence cannot be
determined this way, the peptide will be enzymatically digested with trypsin for a
determined amount of time. Subsequently, the mixture will be separated by RP-HPLC
with 0.1% trifluoroacetic acid in water and acetonitrile as the buffer solutions (Moerman,
2002). These fractions will then undergo CID to determine amino acid linkage and result
in sequence composition. This will be assisted by a number of computer programs
(Wolfender et al., 1999).
Stage 4 (E): Synthesis of peptide(s) of interest
In this stage, solid phase peptide synthesis will be used to synthetically make the
peptide of interest. Solid phase peptide synthesis (SPPS) is a cyclic process in which
reactions take place in a small container with activating reagents. This can be used since
the 9-fluoroenylmethoxycarbonyl (Fmoc) group is an excellent orthogonal blocking
group for the alpha-amino group of amino acids during organic-synthesis because it can
be readily removed under basic conditions. These conditions do not affect the linkage
between the insoluble resin and the alpha-carboxyl group of the growing peptide chain.
N,N’-diisopropylcarbodiimide (DIPCDI) is one agent of choice for activation carboxyl
groups to condense with amino groups to form peptide bonds. In SPPS, the carboxyl
group of the first amino acid (the carboxyl-terminal amino acid of the peptide to be
synthesized) is chemically attached to an insoluble resin particle. The second amino acid,
with its amino group blocked by a Fmoc group and its carboxyl group activated by
DIPCDI, can be reacted with the aminoacyl-resin particle to form a peptide linkage, with
elimination of DIPCDI as diisopropylurea. Then, basic treatment can remove the Nterminal Fmoc blocking group, exposing the N-terminus of the dipeptide for another
cycle of amino acid addition. Any reactive side chains on amino acids can now be
blocked by the addition of acid-labile tertiary butyl groups. After each step, the peptide
product can be recovered by collection of the insoluble resin beads by filtration.
Following cyclic additions of amino acids, the completed peptide chain can be
hydrolyzed from linkage to the insoluble resin by treatment with TFA (Garrett and
Grisham, 2005).
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
This method can be used to synthesize peptides manually. To cleave the peptide,
resin complexes will be performed by treatment with an appropriate mixture of
trifluoroacetic acid/1,2-ethandithiol/anisole/phenol/water using 10 ml per gram at room
temperature. Next, the mixture will be filtered to remove the resin and diethyl ether will
be added which causes precipitation of the crude peptides. These peptides will be
collected as a pellet after a centrifugation at 1000g for 15 minutes. The crude peptides
will then be solubilized in water and chromatographed under reverse-phase HPLC. The
elution will be monitored at a determined wavelength and each fraction eluted will be
collected into glass vials. The homogeneity and correct sequence of the synthetic
peptides will be assessed by analytical HPLC and ESI-MS analysis (Mendes et al., 2004).
Stage 5 (F): Reconfirmation of Biological Activity
In this final stage, the biological activity will be reconfirmed using the synthetic
material. Using solution phase assay, the synthetic material will be tested to reconfirm
that the peaks identified in stage 2 and 3 still show biological activity.
Preliminary Results
Several preliminary experiments were performed to practice techniques learned
for this project. Plate assay experiments were performed on bacteria using bleach and
antibiotics as an alternative to the toxins. These experiments are described below.
Tests with bleach were performed through plate assay in which plates were
inoculated to form bacterial lawns with E. Coli, S. aureus, E. faecalis, and B. Subtilis.
Bleach, in concentrations of 10%, 20% and 50%, was either placed directly on the lawn
or was placed on filter paper, which was then placed on the lawn. The plates were
incubated overnight and results were observed. The bleach placed directly on the lawn
did not inhibit the growth of the bacteria, while the 50% bleach on the filter paper
produced the most growth inhibition (data not shown).
To test the effectiveness of the antibiotics against the bacteria, the Kirby-Bauer
test, also known as the disk diffusion test, was performed. In this experiment, antibiotic
impregnated paper disks were placed on a plate inoculated to form a bacterial lawn. The
bacteria placed on the plates were: S. aureus, E. coli, E. faecalis, and B. Subtilis. The
antimicrobial agents used were the following antibiotics: neomycin 30 mcg,
chloramphenicol 30 mcg, and erythromycin 15 mcg. The plates were incubated to allow
growth of the bacteria and time for the antibiotic to diffuse into the agar. A clear zone
appeared around the disks in which the growth was inhibited, showing the organism’s
susceptibility to the antibiotic. The size of this inhibition depended upon the sensitivity
of the bacteria to the specific antibiotic. The results showed that chloramphenicol and
erythromycin inhibited the growth of all four types of bacteria, while neomycin caused
very little to no growth inhibition.
Once these initial experiments were completed, techniques were mastered, and
results were produced, the effectiveness of toxins in inhibiting bacterial growth was
tested through this plate assay. Tests were completed on all four types of bacteria using
several different toxins including: C. miles, C. victoria, C. virgo, C. tulipa, C. episcopus,
C. anemone, C. omaria, C. geographus, C. textile, and C. californicus. These toxins are
pictured below:
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
C. anemone
C. textile
C. tulipa
Sarah Morris
C. victoriae
C. virgo
C. ppiscopus
C. miles
C. omaria
Figure 7: Conus shells of toxins used in plate assays (Poppe and Poppe, 1996-2005)
Pictures were taken of all plates and the rings of inhibition were measured. Although
many resulted in inhibition of the toxins, C. miles was the most notable; it inhibited the
growth of S. aureus the most (Figure 5).
Figure 8: Growth of inhibition caused by the crude venom of C. miles and C. geographus
on S. aureus
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
As seen in the figure, the ring of inhibition around the C. Miles saturated disk is
quite large compared to C. geographus (as well as other bacteria not shown). This large
ring of inhibition indicates that C. miles possesses antimicrobial activity. This activity
was further examined using HPLC.
HPLC purification was used to fractionate the venom. Fractionation was
performed on several venom including: C. miles (figure 9), C. victoria, C. virgo, C.
tulipa, C. episcopus, C. anemone, C. omaria, C. geographis, C. textile, and C.
californicus.
1.70
1.60
Crude Duct venom of C. miles (A215)
1.50
1.40
1.30
1.20
1.10
1.00
AU
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
-0.10
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
Minutes
45.00
50.00
55.00
60.00
65.00
70.00
Figure 9: Plot of the RP-HPLC fractionation of crude venom from C. miles.
Conditions: RP-HPLC C18 column (narrowbore); Detector: PDA; Solvent A: 0.1% v/v
TFA/Aq. Solvent B 90/10 v/v MeCN/0.08% TFA/Aq. Flow rate:250uL/min.; Gradient:
0-5 min 5% Solvent B; 5-65 min, 5-65% Solvent B; 65-70 min, 65-80% Solvent B; 70-75
min 80% Solvent B; 75-80 min, 5% Solvent B.
Once fractionated, the plate assay technique was used to test the effectiveness of
C. miles against the bacteria, S. aureus. This particular combination was chosen since it
gave the best results with the crude venom. Fifteen fractions were taken with hopes that
the ones containing the venom would produce large rings of inhibition. However, the
results of this fractionation did not match those of the crude venom; the rings of
inhibition were very minimal. Therefore, it is necessary to repeat initial experiments to
verify the activity seen in C. miles. To confirm the activity seen, solution phase assay
will be used, which produces better results than the plate assay used in these preliminary
experiments.
In order to set-up and use solution phase assay, the peak of maximum activity
must be determined. The procedure mentioned in Stage 1 of the proposed research was
completed with S. aureus and E. coli. Readings were taken every hour for 36 hours with
continuous shaking. Several different serial dilutions were tested to determine which
dilution gives the best results. The following figure shows the results of the experiment
75.00
80.00
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
performed with S. aureus at dilutions of 0.1 mL bacteria per 1 mL broth, and 1E-04 mL
bacteria per 1 mL broth.
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.1 Dilution
24
21
18
15
12
9
6
1E-04 Dilution
3
0
Optical Density
Optical Density vs. Time
Time (hours)
Figure 10: Graph of the optical density of S. aureus versus time at 620 nm.
This figure (fig. 9) shows that the 0.1 dilution of S. aureus is not dilute enough because
growth occurs too rapidly in the beginning and then begins to level off. The 1E-04
dilution has a lag phase and then a phase where the bacteria is grown before maximal
growth. This maximal growth occurs around 8 hours. Therefore, when an inhibitor is
added to the solution, the growth of the control and the inhibited will be compared at this
time mark.
Timetable
A
B
C
D
E
F
G
H
I
Milestones
Preamble (preliminary results), literature review
Set up solution phase assay, confirm assay by testing with
antibiotics, in combination with assay, identify peak(s) that may
have direct biological activity
Isolate adequate quantity of peaks that will allow the
identification of the peptide(s) of interest
Characterize the peptide(s) of interest using classical Edman
techniques and/or advanced techniques in mass spectrometry
Synthetically make peptide(s) of interest
Reconfirm biological activity originally observed using the
synthetic material
Thesis Preparation
Thesis Write-up
Presentation Preparation
Time effort*
4 months
3 months
1 month
2 months
3 months
1-2 months
8 months
5 months
1 month
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
*Timetable reflects time effort but as shown below, many can be done simultaneously to
maximize time efficiency.
Timeline and Milestones
Thesis Write-Up
Lab Work
Thesis Work
H
Presentation
Preparation
I
Thesis Preparation
G
Isolation
Synthesis
C
E
Characterization
Assay Development
B
D
Preamble
A
0
1
2
3
4
Months
5
6
Confirmation
F
7
8
9
Proposal Summary
Recently, there has been increasing interest in the field of toxins, their
antimicrobial activity, and their interaction with bacterial channels. The aim of this thesis
work is to identify peptide(s) that have antimicrobial behavior in the venom, Conus. Two
types of bacteria, E. coli and S. aureus, will be used in testing the antimicrobial behavior
of several different toxins, including C. miles, C. victoria, C. virgo, C. tulipa, C.
episcopus, C. anemone, C. omaria, C. geographus, C. textile, and C. californicus.
Preliminary research has shown that these toxins possess some antimicrobial activity, but
further testing is needed to confirm these results. This testing will be completed through
several different stages including: solution-phase assay, isolation of peaks for
identification of peptide(s), characterization and synthesis of the peptide(s) of interest,
and reconfirmation of the biological activity originally observed.
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
References
Chagot, B., Pimentel, C., Dai, L., Pil, J., Tytgat, J., Nakajima, T., Corzo, G., Darbon, H.
and Ferrat, G. (2005). An unusual fold for potassium channel blockers: NMR
structure of three toxins from the scorpion opisthacanthus madagascariensis. J.
Biochem.: [Epub ahead of print].
Doyle, D., Cabral, J., Pfeutzner, R., Kuo, A., Glubis, J., Cohen, S., Chait, B., and
MacKinnon, R. (1998). The Structure of the Potassium Channel: Molecular Basis
for K+ Conduction and Selectivity. Science 280: 69-76.
Garrett, R. and Grisham, C. 2005. Biochemistry, Third Edition. University of Virginia:
pp.129-131, 229-232.
Gao, Y. and Garcia, M. (2003). Interaction of agitoxin2, charybdotoxin, and iberiotoxin
with potassium channels: selectivity between voltage-gated and Maxi-K channels.
Proteins 52(2):146-54.
Legros, C., Pollmann, V., Knaus, H., Farrell, A., Darbon, H., Bougis, P. MartinEauclaire, M., and Pongs, O. (2000). Generating a High Affinity Scorpion Toxin
Receptor in KcsA-Kv1.3 Chimeric Potassium Channels. J. Biol. Chem.
275(22):16918-16824.
MacKinnon, R. (2004). Potassium Channels and the Atomic Basis of Selective Ion
Conduction. Angew. Chem. Int. Ed. 43, 4265-4277.
Mendes, M., Souza, B. Marques, M., and Palma, M. (2004). Structural and biological
characterization of two novel peptides from the venom of the neotropical social
wasp Agelaia pallipes pallipes. Toxicon 44: 67-74.
Moerman, L, Bosteels, S. Noppe, W., Willems, J. Clynen, E., Schoofs, L., Thevissen, K.,
Tytgat, J., Eldere, J., Van der Walt, J., and Verdonck, F. Antibacteria and
antifungal properties of alpha-helical, cationic peptides in the venom of scorpions
from southern Africa. Eur. J. Biochem. 269: 4799-4810.
Poppe, G. and Poppe, P. 1996-2005. Conchology, Inc.
Possani, L., Becerril, B., Delepierre, M. and Tytgat, J. (1999). Scorpion toxina specific
for Na+-channels. Eur. J. Biochem. 264: 287-300.
Scanlon, M., Naranjo, D., Thomas, L., Alewood, P., Lewis, R., and Craik, D. (1997).
Solution structure and proposed binding mechanism of a novel potassium channel
toxin kappa-conotoxin PVIIA. Structure, 5(12): 1585-97.
Shealy, R., Murphy, A., Ramarathnam, R., Jakobsson, E., and Subramaniam, S. (2003).
Sequence-Function Analysis of the K+-Selective Family of Ion Channels Using a
Identification of a Peptide with Antimicrobial Activity in the Venom of Conus
Sarah Morris
Comprehensive Alignment and the KcsA Channel Structure. J. Biophysics 84:
2929-2942.
Steen, H., and Mann, M. (2002). Analysis of Bromotryptophan and Hydroyproline
Modifications by High-Resolution, High-Accuracy Precursor Ion Scanning
Utilizing Fragment Ions with Mass-Deficient Mass Tags. Anal. Chem. 74:62306236.
Takeuchi, K., Yokogawa, M., Matsuda, T., Seiko, K., Kohno, T., Nakamura, H.,
Takahashi, H., and Shimada, I. (2003). Structural Basic of the KcsA K+ Channel
and Agitoxin2 Pore-Blocking Toxin Interaction by Using the Transferred CrossSaturation Method. Structure 11, 1381-1392.
Todar, K. 2002-2004. Online Textbook of Bacteriology. University of WisconsinMadison: Department of Bacteriology.
Wolfender, J., Chu, F., Ball, H., Wolfender, F., Fainzilber, M., Baldwin, M., and
Burlingame, A. (1999). Identification of tyrosine sulfation in Conus pennaceus
conotoxins alpha-PnIA and alpha-PnIB: Further investigation of labile sulfo- and
phosphopeptides by electrospray, matrix-assisted laser desorption/ionization
(MALDI) and atmospheric pressure MALDI mass spectrometry. J. Mass
Spectrom. 34: 447-454.