1. MICR 304 Immunology & Serology
Lecture 2
Antimicrobial Peptides
Chapter 1.1 – 1.17

2. Immunology Recognition of self and non-self Elimination of non-self
Antigens
Elimination of non-self
Exogenous targets
Microbes
Allergens
Foreign material
Endogenous targets
Tumors

3. Key Players in Immunology
Innate
Adaptive
Cells
Phagocytes
Epithelial Cells NK Cells
Lymphocytes
(B-Ly, T-Ly)
Effector molecules
Complement
Antimicrobial (Poly)Peptides
Antimicrobial lipids?
Antibodies

4. Antimicrobial (Poly)Peptides are Widespread in Nature

5. Antimicrobial (Poly)Peptides in Mammalian First Line Defense
On body surfaces (skin, mucosa)
In phagocytes (neutrophils, macrophages)
In body fluids
(Tomas Ganz)

6. Antimicrobial Peptides
Wide spread in nature
Gene-encoded
Small (< 100 amino acids)
Cationic
positive net charge at physiological pH
Arginine and/or lysine rich
Hydrophobic
Various structures
alpha-helical
beta-sheet
circular
Amphiphilic

7. Typical Structures of Antimicrobial Peptides
a-helix
b-sheet
From Lippincott’s
Biochemistry

8. Sequestration of Polar and Apolar Amino Acid Residues
Polar cationic residues
electrostatic interaction with negatively charged surface of microbial target
Apolar residues
embedding into lipid membrane of microbe
The a-Helical Wheel

9. Membrane Targeted Action of Antimicrobial Peptides
+ + +
Amphiphilic
Cationic
Hydrophobic
Microbial killing through membrane permeabilization
+ + +
+ + +

10. Protegrin-Treated N. gonorrhoeae
Scanning EM
Qu et al., 1996
Control PG-1 treated

11. Factors Affecting Antimicrobial Activity
Salt pH
Divalent cations
Nutrients
Under physiological conditions often high concentrations required!

12. Active Learning Exercise
How could bacteria become resistant to AMPs?

13. Reported Antimicrobial Activity of Antimicrobial Peptides
Bacteria (gram+, gram-, Chlamydia, Mycobacteria)
Fungi (Aspergillus, Candida, Cryptococcus)
Viruses (enveloped: e.g.Herpes, HIV)
Protozoa (Cryptosporidia, Giardia, Plasmodium)

14. Posttranslational Processing of Antimicrobial Peptides
 Specific functions?
Pre
Pro
Mature
Many AMPs are initially produced as pre-pro-peptides
Pre-piece targets to ER and is cleaved off upon entering ER
Some peptides are fully processed and stored as mature peptide (e.g. HNPs in PMN)
Some peptides are stored as propeptides and cleavage occurs upon delivery
Bactenecins: in bovine PMNs
Human defensin 5: in Paneth cells

15. Selected Antimicrobial Peptides
Defensins:
Mammals, Insects, Plants
Cathelicidins:
Mammals
Magainins:
Frogs

16. Defensins Structure: Synthesis: Expression Activity
6 cysteines with 3 disulfide linkages
alpha- and beta-defensins with distinct cysteine connectivity (DEFA and DEFB)
Synthesis:
Preproprotein
Expression
Maturation dependent, constitutive or inducible
Activity
Broad spectrum antimicrobial activity at mM concentrations [mg/ml]
Pre
Pro (-)
Mature (+)

17. Structure of Defensins
Antiparallel b-sheet
Sometimes combined with a-helix
Form dimers in solution

18. Human a-Defensins (DEFA)
Human Neutrophil peptides HNP1-4 (DEFA1-4)
Stored in secondary granules of neutrophils as mature peptide
Also found in other immune cells
Human Defensin HD5 and HD6 (DEFA5,6)
HD5 stored as precursor in granules of Paneth cells in small intestine
Inducible in epithelial cells of urogenital tract
Neutrophil (TEM)
HD5-Immunostain

19. Human Beta-Defensins (DEFB): Epithelial Defensins HBD1-4
HBD1 (DEFB1): constitutively produced
urogenital
gastrointestinal
mammary glands
respiratory
HBD2 (DEFB2): inducible
skin
HBD2

20. Cathelicidins Heterogeneous group of antimicrobial peptides
Share a common propiece: cathelin
Antimicrobial activity generated with removal of propiece
LL 37 in humans (derived from hCAP18)
Up-regulated by Vit D3
Protegrins in pigs
PG1
LL37

21. Antimicrobial (Poly)Peptides
Larger Proteins with other functions that contain domains acting like antimicrobial peptides
Lysozyme (peptidoglycan hydrolase)
Lactoferrin (iron binding)
Secretory Leukocyte Protease Inhibitor
Hemoglobin derivatives

22. Lysozyme: A Cationic Hydrophobic Hydrolase
The first described protein of innate defense (Alexander Fleming, 1927)
Predominant peptide in all body secretions (up to mg/ml!)
Peptidoglycan hydrolase
Good activity against gram-positive bacteria
Species specific activity against gram-negative bacteria by action like antimicrobial peptides
Lysozyme
OM (LPS) PG PG CM CM
Gram Gram-

23. Potential Role of Lysozyme as Down Regulator in Inflammation
PG is recognized via TLR-2
Triggers inflammatory response
If degraded by lysozyme, less pro-inflammatory signals will be generated
Ganz et al., 2002
A: Parent strain C57/bl6
B: Parent strain 129Sv
C: -/- lyso knock out mouse

24. Do Antimicrobial Peptides Play an Important Role in vivo?
S. typhi: typhoid fever in humans, systemic disease
S. typhimurium: typhoid fever in mice, enteritis in humans
Mouse small intestine defensins (cryptdins): inactive against S. typhimurium
Human small intestine defensins (HD5): active against S. typhimurium
Transgenic mice expressing human defensin HD5 should be protected against S. typhimurium

25. Transgenic Mice Expressing HD5 With-stand Oral Infection with S
Transgenic Mice Expressing HD5 With-stand Oral Infection with S. typhimurium

26. Each Species has Multiple Types of AMP
> 800 sequences for AMPs known
Each species expresses ~ 15 – 20 different peptides

27. AMP Beyond Killing LPS- binding Chemotactic properties Wound healing
down regulation of inflammation
Chemotactic properties
recruit naïve T-cells
mast cells
Wound healing
stimulation of angiogenesis
Increased collagen production
Lectin function
Toxin and virus aggregation
Anticancer activity
lactoferrin derivates

28. LPS Binding by AMP
Dual function of selected antimicrobial peptides: direct bactericidal activity and LPS-binding
LPS triggers release of proinflammatory cytokines
LPS alone can lead to symptoms of septic shock
Binding of LPS down regulates inflammation

29. AMPs as Chemoattractants
T-Ly
HBD2

30. APMs as Lectins Wang et al., 2003
Retrocyclin is a theta (circular) defensin
Recognizes and binds carbohydrate-containing surface molecules
Binds to gp120 of HIV and prevents HIV binding to CD4
Protect cells from HIV-1 infection

31. Antimicrobial Lipids Emerging arm of innate defense
Free fatty acids in vernix caseosa act synergistically with LL37 (Tollin et al., 2005)
Host derived lipids contribute to the inherent antimicrobial activity mucosal secretions alone and in conjunction with antimicrobial lipids (Do et al., 2008)
Cholesteryl esters

32. Today’s Take Home Message
Antimicrobial peptides are natural amphipathic peptide antibiotics
Cationic charge required for electrostatic attraction
Hydrophobicity required for target membrane insertion
Antimicrobial spectrum varies
AMPs are multifunctional
Each species seems to have multiple types of antimicrobial peptides
Antimicrobial lipids represent an emerging arm of innate immunity

33. References Baht et al., 2007 Boman 2003
Bowdish et al., 2006
Do et al., 2008
Duerr and Peschel 2002
Elsbach 2003
Ganz et al., 2002
Porter et al., 1997
Qu et al., 1996
Salzman et al., 2003
Tollin et al., 2005
Janeway’s Immunobiology (2008), 7th edition
Slonczewski (2009)
Lippincott’s Biochemistry (1994), 2nd edition