Antimicrobial Peptides: Defensins and antimicrobial peptides

Question 1

Major subdivisions of the immune system

Answer 1

• The innate or non-specific immune system
• The adaptive or specific immune sytem

Question 2

Innate immunity

Answer 2

• Natural, none-adaptive, nonspecific.
• Phagocytes: Monocytes, macrophages, PMN neutrophils
• Natural Killers (NK) cells
• Complement system
• Exterior defenses: Skin, Stomach acifdity, Mucus, Cilia, Microflora, Lysozyme in tears, Flushing of urinary tract.

Question 3

Summary on Innate Immunity

Anatomical barriers

Answer 3

• Epithelial surfaces, especially our skin, form a ohysical barrier impermeable to most infectious agents.
• Mucus, tears and saliva containing (lysozyme, phospholipase and defensins) can prevent infection.
• The normal flora can prevent the colonization of pathogenic bacteria by antimicrobial or by competing with pathogenic bacteria.

Question 4

Summary on Innate Immunity

Humoral barriers and inflammation

Role for locally secreted small peptides

Answer 4

• Inflammation is one of the first responses of our immune system, stimulated by chemical factors released by injured cells called chemokines,
• Lactoferrin and transferin - Bind iron, essential for bacteria, limiting bacterial growth
• Lysozyme - Breaks down the cell wall of bacteria.
• Interferons - Limit virus replication in cells
• Interleukin 1 - Induces fever and the production of acute phase proteins.

Question 5

Summary on Innate Immunity

Complemet system

Answer 5

A group of small serum proteins (opsoins) that bind antigens and thus enhance phagocytosis.

Question 6

Summary on Innate Immunity

Phagocytes

Answer 6

• The neutrophils, also called granulocytes, most efficient phagocytic cells, contain toxic substances that kill or inhbit growth of bacteris and fungi.
• Macrophages, phagocyte intracellular pathogens, regulate inflammation adn tissue healing.
• Natural killer cells that destroy compormised cells (infected or tumorous).
• Other cells, mast cells, eosinophils.

Question 7

Innate vs adpative Immne Response

Answer 7

Innate immune response protect the host during the early phase of an infection.
The innate response greatly influences the formation of the subsequent adaptive response.

Question 8

Determinants recognized by the innate immune response

Answer 8

• PAMPs - Pathogen associated molecular patterns
• PRRs - Pattern recognition receptors

Question 9

PAMPs
Pattern Associated Molecular Patterns

Answer 9

• Non-specific (not antigen specific) receptor recognition.
• Part of innate antimicrobial defense.

Question 10

PRRs
Pattern Recognition Receptors

Answer 10

• Toll-like receptors on phagocytic cells
• The nucleotide-binding oligomerization domain (NODs) - like receptors (NLRs)
• The retinoic acid-inducible gene (RIG) - I - like receptors (RLRs)
• Membrane C-type lectin receptors (CLRs)
• DNA receptors (cytosolic sensors for DNA)

Question 11

PRRs
Pattern Recognition Receptors

Answer 11

PRRs recognize conserved microbe (pathogen) - associated molecular patterns (MAMPs/PAMPs), pathogen - specific virlence factors and danger - associated molecular patterns (DAMPs; tissue injury)

Question 12

Innate Immunity from Invertebrates to vertebrates

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Answer 12

Focus on AMPs

Question 13

Antimicrobial Peptides
(AMPs)

Answer 13

Human host defense AMPs and proteins are key components of innate immunity, they play a critocal role in warding off invading microbial pathogens.
These peptides vary from 10 to 150 amino acids with a net charge between -3 and + 20, and a hydrophobic content below 60%.

Question 14

Main features of AMPs

Answer 14

• Small, cationic peptides with potent antomicrobial activity.
• Most widely used host defense molecule in nature.
• Produced and secreted by epithelial lined surfaces of tissues that routinely encounter microbes or cell types involved in host defense - skin, lungs, GI tract, urinary tract, etc.
• Natural antibiotics
• Disrup bacterial cell membrane and exert additional antimicrobial and hysiological functions.

Question 15

Prototypic Groups of Antimicrobial Peptides

Answer 15

• Defensins
• Melittin
• Cathelicidins
• Other AMP

Question 16

Classification of AMPs from eukaryote organisms

Answer 16

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Question 17

Multiple functions of AMPs in host defense

Answer 17

Question 18

AMPs selectively bind to microbial membranes

Answer 18

Question 19

Models of AMPs antimicrobial membrane disruption

Answer 19

Question 20

Classification of AMPs

Structure

Answer 20

• Alpha-helix
• Beta-sheet
• Both alpha-helix and beta-sheet
• Linear extension

Question 21

Defensins

Definition

Answer 21

Small (18-45 amino acids) cysteine-rich cationic proteins characterzied by many Beta-sheets and a framework of disulfide-linked cysteines. They are divided into three sub-classes as alpha, beta, and omega according to their genomic organization, cysteine spacing and intramolecular disulfide bonds.
Widely expressed across various life kingdoms, including animals (vertebrate and invertebrate), plnats, and fungi.

Question 22

Defensins

Distribution

Answer 22

In plants and fungi, they are produced by a wide variety of tissues. In animals they are produced by cells of the innate immune system and epithelial cells.
An organism usually produces many differents defensins, some of which are stored inside the cells, and others are secreted into the extracellular medium.

Question 23

Defensins

Function

Answer 23

They are host defense peptides, with members displaying either direct antimicrobial activity, immune signaling activities, or both.
They are variously active against bacteria, fungi and many enveloped and non-enveloped viruses.

Question 24

Defensins

Structure and diversity

Answer 24

Question 25

Classification of Human Defensins

Answer 25

Question 26

Human Defensins | Expression

Answer 26

* Ubiquitous in the animal kingdom, in human are expressed abundantly in granules of profesional phagocytes, alpha-defensins are produced by neutrophils, macrophages and instestinal Paneth cells, while beta-defensins are mainly produced by most leukocytes and epithelial cells. * Defensins response can be induced by insult or injury to tissues/cells (bacteria, LPS, etc.)

Question 27

Human Defensins | Expression

Answer 27

The following tissues and systmes are protected by defensins: * Skin -HBD-1 is constitutively expressed * Mucosal and epithelial surfaces (oral cavity) * Lining of the gastrointestinal tract (GI) * Lining of the genitourinary tract (UT) * Lining of the respiratory tract (RT)

Question 28

Human Defensins | Immunomodulatory and Antimicrobial Functions

Answer 28

Question 29

Cathelicidins | Definition

Answer 29

Family of multifunctional AMPs found exclusively in vertebrates so far (>30), including mammals, birds, reptiles, amphibians and fishes.

Question 30

Cathelicidins | Distribution

Answer 30

* Cathelicidins are small, cationic, antimicrobial peptides found in several species, including famr animals (cattle, horses, pigs, sheep, goats, chickens, rabbits and in some species of fish). * In humans, the CAMP gene encodes the peptide precursos hCAP-18 whihc is cleaved inot the active forms LL-37 and FALL-39 and both are primarily stored in the lysosomes of macrophages and neutrophils.

Question 31

Cathelicidins | Function

Answer 31

* The mechanism triggering cathelicidin action involves the disintegration (damaging and puncturing) of cell membranes of organism toward which the peptide is active. * Cathelicidin rapidly destroys the lipoprotein membranes of microbes enveloped in phagosomes after fusion with lysosomes in macrophages. * These peptides show a broad spectrum of antimicrobial activity against bacteria, enveloped virsues and fungi.

Question 32

Cathelicidin LL-37 strucuture

Answer 32

Question 33

Cathelicidin antimicrobial killing mechanisms in Gram-bacteria

Answer 33

Question 34

The pleiotorpic propeties of cathelicidin LL-37 in relation to the different cells and tissues

Answer 34

Question 35

Human LL37 can cativate the inflammasome

Answer 35

Question 36

Mechanisms of microbial resistnace to AMPs

Answer 36

Question 37

Differential bacterila resistance mechanisms to AMPs

Answer 37

Question 38

AMPs as mediators of human host defense

Answer 38

Question 39

AMPs contribute to intestinal tract homeostasis and innate immune defense

Answer 39

Question 40

AMPs contribute to innate immune defense at the respitatory tract

Answer 40

Question 41

AMPs at the innate urothelial defense

Answer 41

Question 42

AMPs in thre Urinary Tract

Answer 42

* **Defensis:** *Defb1-/-* mice exhbit increased rates of spotaneous bacteriuria. DEFA1 CNV in UTI patients. * **Cathelicidin:** Increased renal UPEC burden in *Camp-/-* mice after experimental UTI. * **Lipocalin 2 (NGAL):** Increased renal UPEC burden in *Lcn2-/-* mice after experimental UTI. * **Ribonucleases:** RNase 4, 6 and 7 have antimicrobial activity in the urinary tract.

Question 43

AMPS: Potential for therapeutics in UTI

Answer 43

**The Good: ** * Bactericidal at micromolar concentrations * Microbial resistance is limited * Synergy with conventional antibiotics * Immunomodulation **The Challenge:** * Cost of production and delivery * Safety/tolerability

Question 44

Dysregulation of AMPs is associated with inflammatory and autoimmune diseases | Clinical Significance

Answer 44

* The alpha defensin peptides are increased in chronic inflammatory conditions * Alpha defensin are increased in several cancers, inclduign colorectal cancer. * A reduction of ileal alpha defensins may predispose to Crohn's disease * Patients with rosacea have elevated levels of cathelicidins * LL-37 is thought to play a role in psoriasis pathogenesis (along with other anti-microbial peptides).

Question 45

Summary

Answer 45

* AMPs are small cationic proteins, extensively expressed along multicellular organisms and with potent capcity of killing pathogens. * AMPs also boost specific innate immune responses and exert selective immunomodulatory effects on the host. * Upon exposure to danger, AMPs create an overall balance by inhibiting microbial growth, attenuating exacerbted inflammatory resposnses. * Overall, AMPs functions as natural antibiotics and immune regulators. * AMPs are promising potential therapeutics, useful in the control of human disease.