MICR 304 Immunology & Serology

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

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

3. Key Players in Immunology

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. Polar cationicresidues electrostatic interaction with negatively charged surface of microbial target Apolar residues embedding into lipid membrane of microbe Sequestration of Polar and Apolar Amino Acid Residues 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 Mature Pro • 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: • 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] Pro (-) Mature (+) Pre

17. 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

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 • respiratory • gastrointestinal • urogenital HBD2 http://www.cathdb.info/bsm/data/v3_1_0/molscript/1fqqA00.850.jpg

20. Cathelicidins http://www.expasy.org/cgi-bin/view-pdb?pdb=1pg1 • 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 http://modbase.compbio.ucsf.edu

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. 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: A Cationic Hydrophobic Hydrolase 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. 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 • 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. T-Ly AMPs as Chemoattractants 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 http://z.about.com/d/pregnancy/1/0/y/Y/3/newbornvernix.jpg

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. Janeway’s Immunobiology (2008), 7th edition Slonczewski (2009) Lippincott’s Biochemistry (1994), 2nd edition 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 References