Antimicrobial Peptides An ancient immune system
Antimicrobial peptides:What are they? • Small proteins of cationic charge involved in host innate immune defense
Antimicrobial peptides:Where are they? • In everything from Amoebas to Humans (incl. Bacteria), • Abundant in vertebrates in: • External Mucosa • eyes, mouth, genitourinary, skin, lung, trachea • Circulating cells • Neutrophils, platelets • Intestinal tract (duodenum) • in humans; Paneth cells are the source
The principal defense molecules secreted by Paneth cells are alpha-defensins, also known as cryptdins. Paneth cells are stimulated to secrete defensins when exposed to bacteria (both Gram positive and negative types) or bacterial products (lipopolysaccharide, muramyl dipeptide and lipid A). In addition to defensins, Paneth cells secrete lysozyme and phospholipase A2, both of which have clear antimicrobial activity. This battery of secretory molecules gives Paneth cells a potent arsenal against a broad spectrum of agents, including bacteria, fungi and even some enveloped viruses (gastrointestinal barrier).
Ouelette, Infect and Immun 1999, 67: 6643 Immunohistochemistryshowing Cryptidin 4 in Paneth cell secretory granules (A,B). C and D are the controls to the experiment to show specificity (peimmune serum/preadsorption).
Antimicrobial peptides:What do they do? • Antifungal (Candida, Cryptococcus, Aspergillus) • Antiparasitic (Malaria, Leishmania, Trypanosoma) • Antibacterial • Gram positive • Gram negative • Antiviral (Herpesvirus, influenza virus, HIV) ! They are chemotactic.
Antimicrobial peptides:Why are they picky? • Bacteria have : • more negative charge • recall: peptides are cationic • no sterols, little phosphatidylcholine
Antimicrobial peptides:How are they made? • NFB-type genetic element seems universal • like acute phase response in humans • The propiece is cleaved upon delivery of molecule to phagosome (host protection?) signal propiece mature
Yount et al., J. Biol. Chem. 274, 26249-26258, 1999 Immunohistochemical staining of BNBD-12. Cytospin preparations of peripheral blood leukocytes (A and B) and bone marrow cells (C and D) were incubated with anti-BNBD-12 IgG (A and C) or preimmune IgG (B and D). A, peripheral leukocytes. All cells shown are neutrophils except the three eosinophils (E). B, negative control staining of leukocytes using preimmune IgG as the first antibody. C, BNBD-12 immunoreactivity in neutrophilic precursors. A spectrum of cells at different stages of maturation is shown. MB, myeloblast; PM, promyelocyte; MC, myelocyte; MMC, metamyelocyte; NB; neutrophilic band. D, Staining of bone marrow cells using preimmune IgG as the first antibody.
Antimicrobial peptides are found stored in granules but can also be de novo synthesised upon stimulation with e.g. E.coli/LPS, triggering through Toll-like Receptors.
Antimicrobial peptides:How do they work? Acute inflammation: • Killing of bacteria, release of bacterial inflammatory mediators (LPS, LTA), regulation of macrophage response; • adherence of PMN and chemotaxis, stimulation of mast cells to release histamine. • fibroblast growth and adherence, apoptosis of viral- or bacteria- infected host cells, inhibition of fibrin clot lysis (thereby limiting bacterial spread), inhibition of proteases (thus limiting tissue injury). • Chronic inflammation: • Recruitment of T cells, enhancement of monocyte chemotaxis, • regulation of macrophage response.
Antimicrobial peptidesHow important are they really? • Defensins: Wilson et al., Science 1999, 286:113 Mice with genetically engineered deficiency of a metalloproteinase (needed for extracellular cleavage and activation of a-defensins) show increased susceptibility to bacterial infection • Cathelicidins: Bals et al., Infect Immun 1999, 67:6084 In a mouse model of septicemia, LL-37 binds to and neutralises LPS and protects against endotoxic shock. • Histatins: Nishikata et al., Biochem Biophys Res Commun. 1991;174:625 role in protection from periodontal disease by inhibiting protease of B. gingivalis
SUMMARY • Antimicrobial peptides are found in our mucosa and neutrophil granules and act on phospholipids in cell membranes via their hydrophobic and positively-charged domains. • Defensin molecules insert into membranes, where they interact with one another to form pores that disrupt membrane function, leading to cell killing. • Due to the higher concentration of negatively-charged phospholipids in bacterial membranes than vertebrate membranes, defensins preferentially bind to and disrupt bacterial cells. • Clinical Application • Antimicrobial peptides are tested in clinical trials for treatment of • Acne, oral candidiasis. • !!Bacteria have evolved countermeasures to the action of antimicrobial peptides (changes in wall composition, transporter systems).
Reading recommendation Robert Bals: Epithelial antimicrobial peptides in host defense against infection. Review. Respir Res 2000, 1:141-150