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Antimicrobial Peptide Kinetics and Macromolecular Crowding
Devin Porter1, Melanie Juba1 and Barney Bishop1
1 Department of Chemistry and Biochemistry, George Mason University, Manassas VA, USA
Conclusions
Future Directions
Works Cited
Antimicrobial Peptide Kinetics Assay Results
•Kinetic performance assays show that NA-CATH can rapidly destroy a bacterial cell
population in a low ionic-strength environment, while L- and D-ATRA-1A take 15-
20 minutes to reach 100% killing.
•The L- and D-ATRA-1A kinetic results correlate with what was expected from their
original EC50 concentrations.
•Ficoll 70 significantly increases the potency of CAMPs, while in the presence of
PEG, potency is decreased. This suggests that the increase of potency of CAMPs,
found in Ficoll 70, is associated with an interaction with polysaccharide moieties
and not a factor of molecular crowding.
•Perform kinetics studies on NA-CATH, L- and D-ATRA-1A in a high ionic-strength
environment and in the presence of macromolecules.
•Further expand molecular crowding studies to include more crowding agents, as
well as fluorescent microscopy studies to explain the relationship found between
polysaccharide moieties and CAMPs.
•Kinetic assays were performed with the parent peptide, NA-CATH, as well as the
truncated ATRA-1A isomers against E.coli (ATCC: 25922) and B. Cereus (ATCC:11778).
•CAMP kinetic performance was analyzed by observing the percent killing of the
peptide at 200, 2, and 0.2 µg/mL at 0.5, 2, 4, 10, and 20 minutes.
•All experiments shown were performed in a low ionic-strength environment (10mM
phosphate buffer, pH7.4)
•Each experiment used 1x105 CFU/mL of bacteria and were performed in true-
triplicates.
Macromolecular Crowding Results
1. Zasloff, M., Nature, 2002. 415(6870): p. 389-95.
2. Zhao, H., et al., Peptides, 2008. 29(10): p. 1685-91.
3. F.A. de Latour, et al., Biochem Biophys Res Commun, 2010. 396(4): p. 825-30.
4. Flicker. 2008.
http://www.flickr.com/groups/ark_hongkong_reptiles/discuss/72157610056756818/?search=cobra.
(accessed:April 18,2012)
5. Zimmerman S.B. and Minton,A.P. (1993). Annu. Rev. Biophys. Biomol. Struct., 22, 27–75.
Abstract
Introduction
• The ATRA peptides are a series of 11-residue
peptides designed based on a repeated
pattern found in the sequence of NA-CATH, a
34-residue cathelicidin identified in the venom
glands of the Chinese cobra, Naja atra [3].
• ATRA-1A is a relatively short, 11-residue C-
terminally amidated CAMP with the sequence
KRAKKFFKKLK-NH2 and a nominal charge of
+8 at pH 7. The ATRA-1A sequence contains 7
basic residues: 6 lysines and 1 arginine.
Peptide Sequence
NA-CATH KRFKKFFKKLKNSVKKRAKKFFKKPKVIGVTFPF
ATRA-1A KRAKKFFKKLK-NH2
• Previous studies have shown that CAMPs can cause destruction of a bacterial cell
membrane through depolarization or permeabilization[5].
• CAMP kinetic studies provide a direct quantitative approach for determining the speed
at which CAMPs exert their antimicrobial effect.
• The physiological properties of CAMPs are effected by environmental factors, such as
macromolecular crowding, which is referred to as the volume macromolecules occupy
in the intracellular (20-30% in cytoplasm) and extracellular space (8% plasma) [6].
• Ficoll 70 (a synthetic cross linkage of repeating sucrose monomers) and polyethylene
glycol (a polyether,) are frequently used to simulate the effects of macromolecular
crowding found in vivo.
• The effects macromolecular crowding has on CAMP kinetic/performance properties
could provide valuable insights on how environmental factors impact CAMP
performance.
Figure 1. Chinese Cobra [4]
NA-CATH Against E. Coli (ATCC:25922)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
D-ATRA-1A Against E. Coli (ATCC:25922)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
L-ATRA-1A Against E. Coli (ATCC:25922)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
NA-CATH Against B. Cereus (ATCC:11778)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
D-ATRA-1A Against B. Cereus (ATCC:11778)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
L-ATRA-1A Against B. Cereus (ATCC:11778)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
200 g/mL against B. Cereus (ATCC:11778)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
200 g/mL against E. Coli (ATCC:25922)
0 2 4 6 8 10 12 14 16 18 20
0
25
50
75
100
Time, minutes
%Killing
Cationic Antimicrobial Peptides (CAMPs) represent an ancient defense mechanism
against pathogenic bacteria and are essential elements of innate immunity in higher
organisms. Although the mechanism of CAMPs are known to involve the interaction with
the anionic lipid membrane in bacteria, the exact mechanism remains unclear [1]. Part of
this ongoing conversation, is being able to identify the mechanism by which CAMPs are
able to attack bacterial cell walls. Studies investigating bactericidal kinetics provide
valuable insight into the speed with which CAMPs exert their antimicrobial affect,
destroying bacterial populations. Environmental factors, such as macromolecular crowding,
could impact bactericidal CAMP kinetics. With large proteins and polysaccharides
occupying significant portions of extracellular (8% in plasma) and intracellular space (20-
30%), macromolecular crowding is a significant factor in these environments [2]. While
macromolecular crowding has been shown to impact protein structural and functional
properties, CAMPs have not generally been the subjects of such investigations. The kinetic
and macromolecular crowding studies reported here focus on a series of CAMPs that
includes NA-CATH, from the elapid Naja atra, and truncated NA-CATH variants. It was found
that the kinetic properties of NA_CATH were superior then the truncated L- and D-ATRA
variants. Additionally, macromolecular crowding was found to dramatically impact CAMP
performance, both in a positive and negative capacity.
•NA-CATH shows superior killing over the truncated L- and D-ATRA-1A isomers, with
100% killing achieved at 200 µg/mL in 30 seconds.
•D-ATRA-1A shows superior killing over NA-CATH at 2 µg/mL after 20 minutes,
which is contradictory to what previous EC50 values indicate.
•No significant difference in the kinetic performance of the CAMPs are seen
between E. Coli or B. Cereus, other then the decreased effectiveness of L-ATRA-1A
at 200 µg/mL against B. Cereus, which is supported by the overall poor
performance of L-ATRA-1A against B. Cereus in earlier studies.
•Performance assays were performed to
determine EC50 concentrations in the
presence of Ficoll 70 and PEG.
•Ficoll 70 showed a dramatic increase in
potency as the molecular crowding agent
increased, while PEG showed a decrease
in potency.
L-ATRA-1A EC50 Vs. % PEG
0 5 10 15 20
0
2
4
6
8
10
12
% PEG, m/v
EC50g/mL
D-ATRA-1A EC50 Vs. % PEG
0 5 10 15 20
0
2
4
6
8
10
12
% PEG, m/v
EC50g/mL
D-ATRA-1A EC50 Vs. % Ficoll 70
0 5 10 15 20
0.0
0.5
1.0
1.5
% Ficoll 70, m/v
EC50g/mL
L-ATRA-1A EC50 Vs. % Ficoll 70
0 5 10 15 20
0
1
2
3
4
% PEG, m/v
EC50g/mL
•Other studies using BSA and Dextran
70 as the crowding agent show similar
potency effects in a negative and
positive manner, respectively.
•In the absence of CAMPs, Ficoll 70
stimulates bacterial growth, while PEG
showed no change. No evidence was
seen for an antibacterial effect.

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Poster for COS Symposium 2013

  • 1. Antimicrobial Peptide Kinetics and Macromolecular Crowding Devin Porter1, Melanie Juba1 and Barney Bishop1 1 Department of Chemistry and Biochemistry, George Mason University, Manassas VA, USA Conclusions Future Directions Works Cited Antimicrobial Peptide Kinetics Assay Results •Kinetic performance assays show that NA-CATH can rapidly destroy a bacterial cell population in a low ionic-strength environment, while L- and D-ATRA-1A take 15- 20 minutes to reach 100% killing. •The L- and D-ATRA-1A kinetic results correlate with what was expected from their original EC50 concentrations. •Ficoll 70 significantly increases the potency of CAMPs, while in the presence of PEG, potency is decreased. This suggests that the increase of potency of CAMPs, found in Ficoll 70, is associated with an interaction with polysaccharide moieties and not a factor of molecular crowding. •Perform kinetics studies on NA-CATH, L- and D-ATRA-1A in a high ionic-strength environment and in the presence of macromolecules. •Further expand molecular crowding studies to include more crowding agents, as well as fluorescent microscopy studies to explain the relationship found between polysaccharide moieties and CAMPs. •Kinetic assays were performed with the parent peptide, NA-CATH, as well as the truncated ATRA-1A isomers against E.coli (ATCC: 25922) and B. Cereus (ATCC:11778). •CAMP kinetic performance was analyzed by observing the percent killing of the peptide at 200, 2, and 0.2 µg/mL at 0.5, 2, 4, 10, and 20 minutes. •All experiments shown were performed in a low ionic-strength environment (10mM phosphate buffer, pH7.4) •Each experiment used 1x105 CFU/mL of bacteria and were performed in true- triplicates. Macromolecular Crowding Results 1. Zasloff, M., Nature, 2002. 415(6870): p. 389-95. 2. Zhao, H., et al., Peptides, 2008. 29(10): p. 1685-91. 3. F.A. de Latour, et al., Biochem Biophys Res Commun, 2010. 396(4): p. 825-30. 4. Flicker. 2008. http://www.flickr.com/groups/ark_hongkong_reptiles/discuss/72157610056756818/?search=cobra. (accessed:April 18,2012) 5. Zimmerman S.B. and Minton,A.P. (1993). Annu. Rev. Biophys. Biomol. Struct., 22, 27–75. Abstract Introduction • The ATRA peptides are a series of 11-residue peptides designed based on a repeated pattern found in the sequence of NA-CATH, a 34-residue cathelicidin identified in the venom glands of the Chinese cobra, Naja atra [3]. • ATRA-1A is a relatively short, 11-residue C- terminally amidated CAMP with the sequence KRAKKFFKKLK-NH2 and a nominal charge of +8 at pH 7. The ATRA-1A sequence contains 7 basic residues: 6 lysines and 1 arginine. Peptide Sequence NA-CATH KRFKKFFKKLKNSVKKRAKKFFKKPKVIGVTFPF ATRA-1A KRAKKFFKKLK-NH2 • Previous studies have shown that CAMPs can cause destruction of a bacterial cell membrane through depolarization or permeabilization[5]. • CAMP kinetic studies provide a direct quantitative approach for determining the speed at which CAMPs exert their antimicrobial effect. • The physiological properties of CAMPs are effected by environmental factors, such as macromolecular crowding, which is referred to as the volume macromolecules occupy in the intracellular (20-30% in cytoplasm) and extracellular space (8% plasma) [6]. • Ficoll 70 (a synthetic cross linkage of repeating sucrose monomers) and polyethylene glycol (a polyether,) are frequently used to simulate the effects of macromolecular crowding found in vivo. • The effects macromolecular crowding has on CAMP kinetic/performance properties could provide valuable insights on how environmental factors impact CAMP performance. Figure 1. Chinese Cobra [4] NA-CATH Against E. Coli (ATCC:25922) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing D-ATRA-1A Against E. Coli (ATCC:25922) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing L-ATRA-1A Against E. Coli (ATCC:25922) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing NA-CATH Against B. Cereus (ATCC:11778) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing D-ATRA-1A Against B. Cereus (ATCC:11778) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing L-ATRA-1A Against B. Cereus (ATCC:11778) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing 200 g/mL against B. Cereus (ATCC:11778) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing 200 g/mL against E. Coli (ATCC:25922) 0 2 4 6 8 10 12 14 16 18 20 0 25 50 75 100 Time, minutes %Killing Cationic Antimicrobial Peptides (CAMPs) represent an ancient defense mechanism against pathogenic bacteria and are essential elements of innate immunity in higher organisms. Although the mechanism of CAMPs are known to involve the interaction with the anionic lipid membrane in bacteria, the exact mechanism remains unclear [1]. Part of this ongoing conversation, is being able to identify the mechanism by which CAMPs are able to attack bacterial cell walls. Studies investigating bactericidal kinetics provide valuable insight into the speed with which CAMPs exert their antimicrobial affect, destroying bacterial populations. Environmental factors, such as macromolecular crowding, could impact bactericidal CAMP kinetics. With large proteins and polysaccharides occupying significant portions of extracellular (8% in plasma) and intracellular space (20- 30%), macromolecular crowding is a significant factor in these environments [2]. While macromolecular crowding has been shown to impact protein structural and functional properties, CAMPs have not generally been the subjects of such investigations. The kinetic and macromolecular crowding studies reported here focus on a series of CAMPs that includes NA-CATH, from the elapid Naja atra, and truncated NA-CATH variants. It was found that the kinetic properties of NA_CATH were superior then the truncated L- and D-ATRA variants. Additionally, macromolecular crowding was found to dramatically impact CAMP performance, both in a positive and negative capacity. •NA-CATH shows superior killing over the truncated L- and D-ATRA-1A isomers, with 100% killing achieved at 200 µg/mL in 30 seconds. •D-ATRA-1A shows superior killing over NA-CATH at 2 µg/mL after 20 minutes, which is contradictory to what previous EC50 values indicate. •No significant difference in the kinetic performance of the CAMPs are seen between E. Coli or B. Cereus, other then the decreased effectiveness of L-ATRA-1A at 200 µg/mL against B. Cereus, which is supported by the overall poor performance of L-ATRA-1A against B. Cereus in earlier studies. •Performance assays were performed to determine EC50 concentrations in the presence of Ficoll 70 and PEG. •Ficoll 70 showed a dramatic increase in potency as the molecular crowding agent increased, while PEG showed a decrease in potency. L-ATRA-1A EC50 Vs. % PEG 0 5 10 15 20 0 2 4 6 8 10 12 % PEG, m/v EC50g/mL D-ATRA-1A EC50 Vs. % PEG 0 5 10 15 20 0 2 4 6 8 10 12 % PEG, m/v EC50g/mL D-ATRA-1A EC50 Vs. % Ficoll 70 0 5 10 15 20 0.0 0.5 1.0 1.5 % Ficoll 70, m/v EC50g/mL L-ATRA-1A EC50 Vs. % Ficoll 70 0 5 10 15 20 0 1 2 3 4 % PEG, m/v EC50g/mL •Other studies using BSA and Dextran 70 as the crowding agent show similar potency effects in a negative and positive manner, respectively. •In the absence of CAMPs, Ficoll 70 stimulates bacterial growth, while PEG showed no change. No evidence was seen for an antibacterial effect.