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					MICR 304 Immunology & Serology
Lecture 2 Antimicrobial Peptides Chapters 2.3 and 2.4

Immunology
• Recognition of self and non-self
– Antigens

• Elimination of non-self
– Exogenous targets
 Microbes  Allergens  Foreign material

– Endogenous targets
 Tumors

Key Players in Immunology
Innate Adaptive
Lymphocytes (B-Ly, T-Ly)

Cells

Phagocytes Epithelial Cells NK Cells

Defense Proteins

Complement Antimicrobial (Poly)Peptides

Antibodies

Antimicrobial (Poly)Peptides are Widespread in Nature

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

Antimicrobial Peptides
• • • • Wide spread in nature Gene-encoded Small (< 100 amino acids) Cationic

– positive net charge at physiological pH – Arginine and/or lysine rich

Amphiphilic

• Hydrophobic • Various structures
– alpha-helical – beta-sheet – circular

Typical Structures of Antimicrobial Peptides
a-helix

b-sheet

From Lippincott’s Biochemistry

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

Membrane Targeted Action of Antimicrobial Peptides
+++

• Amphiphilic
– Cationic – Hydrophobic
+++

• Microbial killing through membrane permeabilization

Protegrin-Treated N. gonorrhoeae
Scanning EM

Qu et al., 1996

Control

PG-1 treated

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

Active Learning Exercise
• How could bacteria become resistant to AMPs?

Reported Antimicrobial Activity of Antimicrobial Peptides
• Bacteria (gram+, gram, Chlamydia, Mycobacteria) • Fungi (Aspergillus, Candida, Cryptococcus) • Virus (enveloped: e.g.Herpes, HIV)

• Protozoa (Cryptosporidia, Giardia, Plasmodium)

Posttranslational Processing of Antimicrobial Peptides
 Specific functions?

Pre

Pro

Mature

• Many AMPs are initially produced as pre-propeptides • 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

Selected Antimicrobial Peptides
• Defensins:
Mammals, Insects, Plants

• Protegrins:
Pigs

• Magainins:
Frogs

Defensins
• Structure:
– 6 cysteines with 3 disulfide linkages – alpha- and beta-defensins with distinct cysteine connectivity (DEFA and DEFB)
– Preproprotein

• Synthesis:

Pre

Pro (-)

Mature (+)

• Expression • Activity

– Maturation dependent, constitutive or inducible – Broad spectrum antimicrobial activity at mM concentrations [mg/ml]

Structure of Defensins
• Antiparallel b-sheet
– Sometimes combined with a-helix

• Form dimers in solution

http://www.expasy.org/cgi-bin/get-pdb.pl?1DFN

Human a-Defensins (DEFA)
• Human Neutrophil peptides HNP1-4 (DEFA1-4)
– Stored in secondary granules as mature peptide
Neutrophil (TEM)

• 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
HD5-Immunostain

Human Beta-Defensins (DEFB): Epithelial Defensins HBD1-4
• HBD1 (DEFB1): constitutively produced
– – – – – – – – urogenital gastrointestinal mammary glands respiratory skin respiratory gastrointestinal urogenital
HBD2

• HBD2 (DEFB2): inducible

http://www.cathdb.info/bsm/data/v3_1_0/molscript/1fqqA00.850.jpg

Cathelicidins
• Heterogeneous group of antimicrobial peptides • Share a common propiece: cathelin • Antimicrobial activity generated with removal of propiece
– Protegrins in pigs – LL 37 in humans (derived from hCAP18)
LL37
http://modbase.compbio.ucsf.edu http://www.expasy.org/cgi-bin/view-pdb?pdb=1pg1

PG1

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

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 grampositive bacteria • Species specific activity against gram-negative bacteria by action like antimicrobial peptides

Lysozyme
PG
CM OM (LPS) PG CM

Gram+

Gram-

Potential Role of Lysozyme as Down Regulator in Inflammation
• PG is recognized via TLR-2 • Triggers inflammatory response • If degraded by lysozyme, less proinflammatory signals will be generated
Ganz et al., 2002

A: Parent strain C57/bl6 B: Parent strain 129Sv C: -/- lyso knock out mouse

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

Transgenic Mice Expressing HD5 Withstand Oral Infection with S. typhimurium
Ileum 6 h p.i.

12 h after p.i.
Ileum 24 h p.i.

Salzman et al ., 2003

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

AMP Beyond Killing
• LPS- binding
– down regulation of inflammation

• Chemotactic properties
– recruit naïve T-cells – mast cells

• Wound healing
– stimulation of angiogenesis – Increased collagen production

• Lectin function • Anticancer activity
– lactoferrin derivates

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

AMPs as Chemoattractants
T-Ly

HBD2

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

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

References
• Janeway et al. (2008) Immunobiology, 7th edition • Prescott et al (2002) Microbiology, 5th edition • Lippincott’s Biochemistry (1994), 2nd edition

• • • • • • • • •

Baht et al., 2007 Boman 2003 Bowdish et al., 2006 Duerr and Peschel 2002 Elsbach 2003 Ganz et al 2002 Porter et al 1997 Qu et al 1996 Salzman et al 2003


				
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