Antimicrobial peptides are polypeptide substances between 12-50 amino acids in length that have antimicrobial properties. They are classified based on their secondary structure as either beta-sheets, alpha-helices, extended coils or loop structures. They are found in many organisms and body locations where they participate in the innate immune system and have multifunctional roles beyond direct antimicrobial activity, including immunomodulation and cell migration. Their mechanisms of action generally involve interactions with and disruption of microbial membranes through electrostatic and hydrophobic interactions.

1. Antimicrobial peptides-readings
●
Text : Immunobiology 7th Edition by
Janeway and Travers et al. 2008.
●
Principals of Mucosal Immunology. Philip
Smith, Thomas T MacDonald, Richard S.
Blumberg. Eds. Garland Science 2012.

2. What are antimicrobial peptides?
●
Antimicrobial peptides- defined as polypeptide
antimicrobial substances
●
More than 100 of these peptides identified in
fungi, insects, amphibians and humans.
●
Between 12-50 amino acids;half the residues
hydrophobic
●
Generally cationic and amphipathic molecules
●
A role in innate defense mechanism of many
species

3. Classification of antimicrobial
peptides
●
Major classes include
●
(a) beta-sheets with S-S bonds
●
(b) linear alpha-helices
●
(c) exended coils
●
(d) loop structures
●
Predominant class is the linear, amphipathic,
α-helical peptide

4. What are the functions of
antimicrobial peptides??
●
Antimicrobial peptides participate in the
innate immune system
●
Can protect host from invasive microbial
infections
●
New evidence views antimicrobial peptides
as multifunctional molecules that link innate
immune response to adaptive immune
system
●

5. What are the functions of
antimicrobial peptides??
●
Mediate of cross-talk between 2 wings of
immune sytem. How?
●
Achieved by cytokine and chemokine
production(immunomodulation)
●
Also by facilitating immune and inflammatory
cell migration
●
●

6. What are the functions of
antimicrobial peptides??
●
α and β-defensins, and cathelicidin extend
neutrophil lifespan(by suppression of
neutrophil apoptosis)
●
Induce secretion of histamine and
prostaglandins from mast cells
●
Induce cytokine release from T cells

7. Where do we find antimicrobial
peptides?
●
In cells- neutrophils, Paneth cells, ????
●
In body fluids-complement proteins
●
In body secretions such as saliva and tears
●
In intestinal mucus layers-peptides bound to
mucins detected in rectal mucus
●
Β-defensin of Epithelial cells of trachea-LPS
dependent production via rel/NFKappaB
●

8. Where do we find antimicrobial
peptides?
●
α-Defensins in neutrophils and Paneth cells
●
β-defensins from epithelia of many organs
including skin
●
Cathelicidins in secretory granules of
neutrophils and in NK cells, T cells, B cells,
mast cells and epithelial cells
●
In insect venom, frog skin,?????
●

9. How about D analogs of these
peptides?
●
Can be effective as well
●
In nature, ___ sugars and ___ amino acids
are found?
●
Be familiar with D and L and R and S
designations in organic chemistry.

10. Antimicrobial peptides
●
Magainin 1-from frog
●
GIGKFLHSAGKFGKAFVGEIMKS
●
Broad activity vs bacteria, protozoa, yeast,
fungi, viruses(HSV-1 and HSV-2)
●
Cytotoxic against some cancer cells

11. Antimicrobial peptides
●
Aurein-net charge 1
●
GLFDIIKKIAESI
●
The antibiotic and anti-cancer active aurein
peptides from the australian bell frogs Litoria
aurea and Litoria raniformis; the solution
structure of aurein 1.2.
●
Rozek T, Wegener KL, Bowie JH, Olver IN,
Carver JA, Wallace JC, Tyler MJ.
●
Eur J Biochem. 2000 Sep;267(17):5330-41.
●

12. Antimicrobial peptides-porcine
●
Protegrin-porcine leukocytes-cathelicidin family
●
RGGRLCYCRRRFCVCVGR - NH2 (disulfide
bridge:6 - 15 and 8 - 13)
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H - Arg - Gly - Gly - Arg - Leu - Cys - Tyr - Cys -
Arg - Arg - Arg - Phe - Cys - Val - Cys - Val - Gly -
Arg - NH2 (disulfide bridge:6 - 15 and 8 – 13)
●
Active vs Escherichia coli, Listeria
monocytogenes, candida albicans, and enveloped
viruses

13. Indolicidin-cow neutrophils
●
Indolicidin-cathelicidin family
●ILPWKWPWWPWRR – NH2
●
Ile - Leu - Pro - Trp - Lys - Trp - Pro - Trp -
Trp - Pro - Trp - Arg - Arg -NH2
●
What is the charge on the above peptide?
●
Isolated from cow neutrophils
●
IN vitro activity vs gram +ve/gram -ve,
protozoa,fungi and HIV

14. Defensins, histatins and
cathelicidins are 3 important
peptides in humans●
small, cationic and amphipathic.
●
Exhibit broad-spectrum activity against
Gram-positive and Gram-negative bacteria,
yeasts, fungi and enveloped viruses
●

15. Defensins
●
alpha-defensins and beta-defensins
●
Cationic, non-glycosylated peptides with six
cysteine residue
●
Cysteines form three intramolecular disulfide
bridges
●
We triple-stranded beta-sheet

16. Histatins-saliva
●
small, cationic, histidine-rich peptides
●
random coil conformation in aqueous
solvents
●
alpha-helices in non-aqueous solvents

17. Cathelicidins
●
random coil conformation in a hydrophilic
environment
●
Alpha helical in hydrophobic medium

18. Antimicrobial peptides-from cells in
human gut
●
Lysozyme and Defensins (bactericidal proteins)
●
Paneth cells (PCs)-terminally differentiated,
specialized secretory cells located at the base
of the crypts of Lieberkühn in the small
intestine.
●
Paneth cells located adjacent to stem cells-role
in defendepithelial cell renewal??
●
MyD88-dependent toll-like receptor (TLR) and
other receptors sense bacteria
●

19. Paneth cell secrete antimicrobials
to regulate gut microbiome
●
PCs secrete granules containing
defensins/cryptdins(mice) and lysozyme-into
the lumen
●
Homeostatic relationship between host and
microbes(
●
Maintain gastrointestinal barrier; regulate gut
microbiome
●
Paneth cell dysfuction in intestinal
inflammation (Crohn's disease)

20. Paneth cells located below
intestinal stem cell- intestinal
glands●
Histology

21. Paneth cells
●
Paneth cells

22. PalG1from cow rumen vs
Vancomycin-resistant Enterococci
(VRE)●
Enterococcus faecalis- human enterococcal
infections
●
Being charecterized
●
palG1 has potential in the treatment of
enterococcal infections

23. Antimicrobial peptides from saliva
●
Major secretion-submandibular glands,
sublingual glands and parotid glands(these
are paired glands).
●
Also secreted by many minor salivary
glands(in lamina propria and oral mucosa)
●
Connective tissue(lamina propria)
+epithelia=mucosa
●
Minor salivary glands-lingual, buccal and
labial glands, palatine glands and
glossopalatine folds

24. Lysozyme and lactoferrin from both
major and minor salivary glands
●
Salivary lysozyme-hydrolyzes beta(1-4)
bond between N-acetylmuramic acid and N-
acetylglucosamine in the peptidoglycan layer
of the bacterial cell wall.
●
Perturbed cell wall integrity-bacteria more
suceptible to lysis due to the hypoosmotic
conditions in saliva
●
Lactoferrin-cationic peptide binds iron thus
hindering bacterial growth

25. Antimicrobial peptides from saliva
●
Perioxidase slows down acid production and
growth of many oral microbes and fungi.
●
Lysozyme promotes lysis of bacteria in
conjunction with other antibacterial systems.
●
Histatins from human saliva

26. Functions of saliva-focus on
antimicrobial peptides
●
Peptides

27. Antimicrobial peptides from saliva
●
In body fluids

28. Antimicrobial peptides-complement
●
Complement system made of nearly 30
proteins circulating in blood plasma.
●
Most are inactive; cleaved by a protease and
converted into a protease(active).
●
Components of the system serve as the
substrate of a prior component and then as
an enzyme to activate a subsequent
component.
●

29. Antimicrobial peptides-complement
●
Sequential activation produces an expanding
cascade of activity (like the blood clotting
system
●
Complement proteins provide an effector
mechanim to enhance the specific antibody-
antigen interactions
●
antibodies "finger" the target, complement
destroys it
●
Produced by liver and by monocytes, these
proteins punch holes in bacteria

30. Complement pathways
●
Complement

31. Ponericins from venon of ants
●
Predatory ant Pachycondyla goeldii

32. Cecropins from cecropin moth
●
31 - 37 AA residues Gram+/Gram- bacteria.
●
Lyse
membrane

33. Plant sources-Neem leaf extracts
●
Shown to be effective against Enterococcus
faecalis
●
E faecalis is a major microbe recovered from
root canal when endodontic treatment has
failed

34. Proline-rich Bactenicins against
gram-ves
●
Bac-7 can enter bacterial cells without
damaging membranes
●
Uses SbmA- a bacterial proton-drive
transporter to enter bacterial cells
●
10-residue proline-rich peptide with two
arginine residues enters best
●
Sadler et al. Biochemistry, 2002, 41 (48), pp
14150–14157

35. PAF-26 is a de novo designed
hexapeptide antifungal
●
PAF-26- a de novo-designed hexapeptide
●
2 motifs-N-terminal cationic region and a C-
terminal hydrophobic region.
●
PAF26 -dynamic antifungal mode-of-action
that involves electrostatic interaction with cell
membrane, internalisation, and cell killing
●
Mode of fungal killing still unclear
●

36. Current research on PAF-26
●
Attached a fluorescent tag
●
analysed by live cell imaging
●
in the fungal model Neurospora crassa
●
the human fungal pathogen Aspergillus
fumigatus

37. Newer antimicrobial peptides-shark
squalamine
Squalamine-from liver tissue of deep water
shark
amino sterol cationic peptide analog-anti-
obesity and anti angiogenesis

38. Newer peptide antimicrobials
●
An antimicrobial peptide library screen
against Candida albicans
●
600 9-mer peptides tested
●
A 9-mer peptide analaog RLWLAIGRG-NH2
of insect defensin Protoetiamycin being
tested for antibacterial activity

39. Sarcosine is N-methylglycine
●
An intermediate in glycine synthesis and
degradation
●
Zn and Sn complexes (Organotin) or tin
carboxylates
●
Antibacterial
●
Antifungal

40. ●
Scheme

41. Sarcosine complexes more active
against gram negatives
●
Bidentate carboxyl ion can coordinate Zn
●
Complexes with Sn and Zn are more lipid
soluble
●
Complexes can enter cell walls more easily
●
More studies are needed
●

42. Antimicrobial peptides from
Lactococcus bacteria
●
Nisin- 34 aa peptide
●
Other peptides from Lactococcus
●
Signal peptide of L lactis was fused to codon
sequence of antimicrobials
●
Codon sequence cloned under nisin-
inducible promoter and bacteria transformed
into recombinant strain
●
Vozing et al. ACS Synth. Biol., 2013, 2 (11),
pp 643–650

43. Antimicrobial from Neem plant
●
Tripeptide N-Ala-Phe-Cys-C has been
isolated
●
MW=321.5 Da
●
●

44. Recombinant L lactis produces
antimicrobials active vs gram -ves
●
A3APO and Alyteserin showed activity
against gram -ve E coli and Salmonella
●
A3APO and Alyteserin currently being
investigated against gram negatives

45. Release of antimicrobial peptides
●
Paneth cells release antimicrobial peptides
upon stimulation by the proinflammatory
cytokine, interferon gamma(IFN-γ)
●
Neutrophils-
●

46. Antimicrobial peptides-release of
defensins by neutrophils
●
94aa-75aa-29-30aa

47. Structure of antimicrobial peptides
●
Most are cationic and amphipathic
●
Presence of multiple lysine and arginine
residues
●
Simplest ones are alpha-helical or beta-
hairpin
●
Alpha helix and hairpins are structural motifs

48. Primary and secondary structures
of petides-review
●
Alpha helix, beta sheets and random coils
are examples of secondary structures of
proteins
●

49. Alpha helices held by H-bonds
within a strand
●
Intra-chain hydrogen bonds stabilize the helix
●
Some proteins-keratin and collagen-almost
entirely alpha helical in structure.
●
Most globular proteins contain both alpha helical
and beta pleated sheat regions in addition to
regions without alpha helical or beta pleated
sheats???
●

50. How does an alpha helix form?
●
An alpha helix is formed by making a rope
coil in a left handed direction
●
In proteins, the rope is represented by the N-
C-C-N-C-C-N .... backbone of the polypeptide
chain.

51. Alpha helix
●
Alpha

52. Beta sheets held by H-bonds
between strands
●
Parallel

53. Anti-parallel Beta sheets are more
stable
●
Anti

54. A beta-hairpin
●
Image

55. Intermolecular hydrogen bonding in
beta-sheats
●
Compared with alpha helices, beta sheats
held by intermolecular hydrogen bonds

56. H-bonds in silk-a beta sheat
●
The hydrogen on the amide of one protein chain
is hydrogen bonded to the amide oxygen of the
neighboring protein chain.
●
The pleated sheet effect arises form the fact that
the amide structure is planar while the "bends"
occur at the carbon containing the side chain.
●
Can you follow the N-C-C-N-C-C backbone on
next slide??

57. Beta sheets-silk
●
Gly-Ala

58. Secondary structre of silk arises from a
basic primary structure
●
The basic primary structure is: -gly-ala-gly-
ala-gly-ala-.
●
glycine and alanine=75-80% of the amino
acids
●
another 10-15% is serine and the final 10 %
contain bulky side chains such as in tyr, arg,
val, asp, and glu.

59. Effect of amino acids on secondary
structures
●
Charged amino acid side chains destabilize
the alpha helical or beta-pleated sheat
structures.
●
amino acids containing hydrophobic side
chains are compatible with the formation of
alpha helices and beta pleated sheats.

60. Mechanism of action of antimicrobial
peptides●
Exact mechanism still unknown
●
cationic, hydrophobic peptides of the prototypic
sequence KKAAAXAAAAAXAAWAAXAAAKKKK-
amide
●
Peptide–lipid interactions lead to membrane
permeation
●

61. Mechanism of action
●
Carpet model-lysine interacts with anionic
phospholipid head groups
●
Hydophobic region then interacts with the
lipid bilayer disrupting bacterial membrane
●
Hydrophobicity of core residues determines
ability of peptide to insert into the membrane
●
Next slide

62. Amino acid sequence Core segment hydrophobicity Molecular mass (Da)
KKAAAFAAAAAFAAWAAFAAAKKKK-NH2 1.18 2480
KKKKKKAAAFAAAAAFAAWAAFAAA-NH2 1.18 2480
KKAAAAFAAFAAWFAAFAAAAKKKK-NH2 1.43 2,556
KKAFAAAAAFAAWAAFAKKKK-NH2 1.45 2,196
KKKAAAFAAWAAFAKKK-NH2 1 47 1,835
RRRAAFAAWAAFAARRR-NH2 1.47 2,003
KKKKKKAAFAAWAAFAA-NH2 1.47 1,835
kkkkkkaafaawaafaa-NH2( 1.47 1,835
RRRRRRAAFAAWAAFAA-NH2 1.47 2,003
KKKKKKAAAAFWAAAAF-NH2 1.47 1,835
KKKKKKAAFAAFAAFAA-NH2 1.49 1,796
KKKKKKAAWAAWAAWAA-NH2 1.45 1,913

63. Circular dichroism(CD) is a useful
tool in peptide drug discovery
●
Light absorption spectroscopy that helps
determine secondary strucure of polypeptides
●
measures difference in absorbance of right-
and left-circularly polarized light
●
http://www.cryst.bbk.ac.uk/PPS2/course/secti
on8/ss-960531_21.html

64. CD spectra of the F17-6K peptide in an aqueous buffer (Aq) containing 20 mM Tris-HCl and 20
mM NaCl, pH 7.4, and in the same buffer with the addition of 20 mM SDS. The spectrum of all-d
F17-6K in SDS is presented for comparison. [θ], ellipticity (measured in degrees × square
centimeter per decimole).
Stark M et al. Antimicrob. Agents Chemother. 2002;46:3585-
3590

65. CD spectra of poly-L-lysine

66. Mechanism of action against
bacteria
●
Gram +ve
cell wallcell wall
membranemembrane

67. Mechanism of action against
bacteria
●
Gram negative
●
OMOM
cell wallcell wall
Membrane
LPS

68. Mechanisms
●
Mech.

69. Mechanisms
●
Mech.

70. Antimicrobial peptides differ from
peptide antibiotics of bacteria
●
These peptides differ from most (but not all)
peptide antibiotics of bacteria
●
Also differ from peptide antibiotics from fungi
●
Fungi synthesize peptide antibiotics by
specialized metabolic pathways

71. How do we measure the activity of
antimicrobial peptides???
●
Therapeutic potential of antimicrobial
peptides is compared using Minimum
inhibitory concentration assays
●
MIC is lowest concentration at which the
drug prevents growth of inoculum of 100000
cells/ml of exponentially growing bacteria
●

72. Drawbacks of MIC testing for
efficacy of antimicrobial peptides
●
Lacks info on actual killing of bacterial cells
●
Can't differentiate between bactericidal and
bacteriostatic activities
●
Some use cell viability as a more reliable
measure of bactericidal activity

73. Safety profile of antimicrobial
peptides
●
There are some toxicity concerns
●

74. How we deliver antimicrobial
peptides??
●
Nanodized drug delivery systems being
studied
●
Chitosan-based nanoparticles can be used

75. Novel strategies to deliver
antimicrobial peptides
●
Insert gene encoding peptide into
bacteriophage
●
Maybe effective vs MRSA
●
UCLA doctoral thesis with Timothy Lu,
Michael Coris at Boston University

76. Useful websites
●
http://users.rcn.com/jkimball.ma.ultranet/BiologyPa
●
http://www.biog1445.org/demo/01/fibrous_v_glob.h
●
(http://www.bbcm.univ.trieste.it/ tossi/pag1.ht∼
m).

77. References and Resources●
http://aps.unmc.edu/AP/database/antiB.ph
●
Nagakoa et al. Modulation of Neutrophil
Apoptosis by Antimicrobial Peptides(2012).
ISRN Microbiology.
http://dx.doi.org/10.5402/2012/345791
●
http://cariology.wikifoundry.com/page/Formation+of
●
Stark et al(2002). Cationic Hydrophobic
Peptides with Antimicrobial Activity.
Antimicrobial agents and
Chemotherapy.46(11). 3580-3585
●

78. References and Resources
●
Charles L. Bevins & Nita H. Salzman(2011)
Paneth cells, antimicrobial peptides and
maintenance of intestinal homeostasis.
Nature Reviews Microbiology 9, 356-368
●
François Niyonsaba et al. (2010)
Antimicrobial Peptides Human β-Defensins
and Cathelicidin LL-37 Induce the Secretion
of a Pruritogenic Cytokine IL-31 by Human
Mast Cells. J Immun 184(7) 3526-3534.

79. References and Resources
●
Rozek et al.(2000). The antibiotic and
anticancer active aurein peptides from the
australian bell frogs Litoria aurea and Litoria
raniformis the solution structure of aurein
1.2.Eur J Biochem.Sep;267(17):5330-41.
●
Mor et al(1991). Isolation, amino acid
sequence, and synthesis of dermaseptin, a
novel antimicrobial peptide of amphibian skin.
30(36):8824-8830.