Antimicrobial Peptides

Question 1

Polymyxins:

Backgroud
Spectrum of activity

Answer 1

family of 5 compounds (A-E) produced by Bacillus polymyxa and related bacteria. Only polymyxins B + E (colistin) are used therapeutically. They are cyclic polypeptides with a long hydrophobic tail (lipopeptide)
Act like a cationic detergent by binding to cell membrane and causing leakage of cytoplasm by increasing solubility of cell membrane bilayers. Not entirely selective, so colistin does exhibit toxicity.

Most G-ves including P. aeruginosa
Mainly used topically; B for local eye & ear infections, B & E for local skin infections.
Given orally to decontaminate those with profound neutropenia.
Colistin is inhaled or injected, being increasingly used IV, side-effects include nephrotoxicity.

Question 2

Antimicrobial peptides in nature:

Found where?
Importance?
Examples?

Answer 2

Found in lymph of insects, granules of neutrophils and frog skin shown to contain antibacterial peptides.
Are important components of natural defences of most living organisms against microbial infection
Defencins, Bacteriocins, Mutacins, clavacins

Question 3

2 cationic peptide common features:

Answer 3

net positive charge of at least +2

Have a hydrophilic and hydrophobic face (amphipathic)

Question 4

Activity in vitro

Answer 4

MICs in a range that is competitive with those of other potent AA against resistant microorganisms.
Most cationic peptide don’t induce resistant mutants
At concs close to MIC, peptides kill bacteria more quickly than conventional AA
Burholderia cepacia is a natrually resistant bacterium

Question 5

MOA of Antimicrobial peptides

Answer 5

All peptides interact with the membranes.
These are divided into the classes membrane disruptive and non-membrane disruptive.
The cross gram -ve OM via self-promoted pathway, they then interact with divalent cation binding sites on lipopolysaccharide where they competitively displace Mg2+ and Ca2+. OM is disrupted and peptide translocates the membrane through destabilised areas which promotes uptake of peptides.

Question 6

Interaction of AMP with bacterial CM

Answer 6

AMP associate with outer monolayer of CM and interact electrostatically, and then insert into lipid bilayer.
Membrane-disruptive peptides MOA is unclear but results in rapid depolarization of bacterial cell and rapid death (as quick as 5 mins)

Question 7

characteristics of bacteria that favour the formation of channels (3)

Answer 7

large transmembrane potentials
large number of -vely charged lipids
lack of cationic lipids and chlesterol

peptides are selective to bacteria

Question 8

1. ) Membrane disruptive peptides

2. ) Non-membrane disruptive peptides

Answer 8

Membrane-disruptive peptides MOA is unclear but results in rapid depolarization of bacterial cell and rapid death (as quick as 5 mins)

Thought to act on cytoplasmic targets
translocate across CM without causing large pertubations; any membrane disruption is transient
Once in cytoplasm, thought to interact with DNA, RNA and/or cellular proteins and inhibit synthesis.
Slower acting than membrane-acting ones.

Question 9

4 hypotheses for action of how peptides cause cell death

Answer 9

Cell leakage/lysis
Channel formation
Carpet membrane dissolution
Attacking internal targets

Question 10

Other roles of AMPs

Answer 10

Some antibiotics promote endotoxaemia, and some cationic peptides neutralise LPS and prevent this.
Some AMPs stimulate immune system
Some suppress inflammatory response

Question 11

Host Defence Peptides

Answer 11

Examples: defensis & cathelicidins
important part of their activity is their binding to LPS
They neutralise endotoxin and can reduce proinflammatory response, preventing cytokine storm and organ damage.
HDPs may also prevent activation of macrophages by LPS, they may also upregulate production of chemokines. they could also promote angiogenesis and wound healing

Question 12

Possible roles of cationic peptides in innate immunity (6)

Answer 12

Promote wound repair
neutralise endotoxins
stimulate mast cells to release histamine
stimulate chemotaxis
Inhibit proteases to limit tissue injury
kill bacteria

Question 13

In vivo activity of peptides

Answer 13

not much data out there
reports of efficacy of compounds vs S. aureus, MRSA & P. aeruginosa in animal models
Various peptides for topical treatment is under trial
none yet for systemic use (under trials)

Question 14

Developing New AMPs

Answer 14

Study of natural AMPS- purification of large quantities not usually possible. Screenin of short peptides for AA activity.
Computer aided designs also employed

Question 15

Synthetic cyclic peptides

Answer 15

Cyclic peptides composed of an even number of altering D- and L- amino acids.
They selectively target and self-assemble in bacterial membranes. They form multimeric, hollow tubular structures called peptide nanotubes. Rapid AA activity through increased membrane permeability

Question 16

Beta-peptides

Answer 16

rationally designed sequences of B-amino acids
Synthetic peptidomimetics of natural AMPs
designed to avoid limitations of naturally existing (and other) peptides. They have improved biostability and less likely to be destroyed by proteases.

Question 17

Daptomycin

Background
MOA 3 steps
Spectrum of activity

Answer 17

A semisynthetic cyclic polypetide
binds to bacterial membranes and causes rapid depolarization of membrane potential. Less toxic than many peptides.
Binding & insertion to membrane
Oligimerization & channel formation
depolarisation and ion efflux leads to inhibition of DNA, RNA and protein synthesis (cell death)
active vs G+ve cocci only
licensed for complicated and soft tissue infections and endocarditis

Question 18

Nisin

Answer 18

Lantibiotic produced by strains of Lactococcus lactis
food preservative
bactericidal vs G+ves
cell membrane permeabiliser
targets lipid II (a precursor for cell wall biosynthesis)
used for almost 50 years, but little resistance reported
bioengineered Nisin derivative effective vs L. monocytogenes in mice