CECROPINS

Family of Inducible antimicrobial oligopeptides ; small proteins of about 35

amino acid residues active against both Gram-positive and Gram-negative bacteria

THE ROLE OF ANTIMICROBIAL PEPTIDES

These peptides play an important role in

the offense and defense systems of different organisms .

Are an evolutionarily conserved component of the innate immune response.

COMMON CHARACTERISTICS

- Amino acid compositionAmino acid composition- AmphipathicityAmphipathicity

- Cationic chargeCationic charge - SizeSize

- their synthesis is of type ribosomaltheir synthesis is of type ribosomal

All these characteristics allow them to attach to and insert into membrane

bilayers to form pores .

Why study Cecropins?

The importance of the study of these antimicrobial peptides is enhanced by the phenomena of resistance to conventional antibiotics (which occur with increasing frequency), due to improper use of the

same. Against these peptides, in fact, until now there have been no phenomena of

resistance.

CECROPIN

Inducible antimicrobial oligopeptide

discovered for the first time in the hemolymph of Lepidoptera

The Family of CECROPINS

• They are present in insects, amphibians and mammals.

• These peptides are long from 31 to 39 a.a.

• Domain N-term : basic and hydrophilic

• Domanin C-term : hydrophobic

• The D- enantiomers retain antibacterial activity.

CECROPINS

Linear peptides that are structured to

α -helix in hydrophobic ambient. They assume

random structure in solution.

CECROPINS

• They perform host defense functions and often act in synergy, providing rapid non-specific defence against invading micro-organisms .

• Most of these natural broad-spectrum peptide antibiotics have limited cytotoxicity to human cells and are being developed as therapeutics against pathogens resistant to classical antibiotics.

The butterfly Hyalophora

Cecropia produces 3 different

cecropine, called type A, B and D

Cecropins A, B and D :

- Are linear peptides- Are basic peptides - Consist of 35-37 residues that

show 62-65% homology in the primary sequence

CECROPIN- A : DATA

Formula :

C184H313N53O46

KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH2

CECROPIN- A• Isolated from the silkworm Hyalophora

cecropia .

• Composed of 37 amino acids.

• Kills bacteria by dissipating transmembrane electrochemical ion-gradients.

• Cecropin A induces changes in gene expression profiles in Escherichia coli exposed to sub lethal doses, apart from any lethal effects caused by membrane perturbation.

CECROPIN- B : DATA

KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL-NH2

Linear and cationic

CECROPIN- B• Isolated from the giant silkmoth, Hyalophora

cecropia. • It enhances the activities of betalactams in

experimental rat models of Gram-negative septic shock.

• Cecropin B has been shown to reduce significantly the lethality of Escherichia coli and plasma endotoxin levels in a rat model of septic shock .

• Can affect host cell to promote wound repair.

Cecropin –D :

- It appears in the hemolymph at much later times after bacterial infection than Cecropin A or Cecropin B .

- It possesses potent antibacterial activities against a panel of Gram positive and negative bacteria without haemolytic activity against human red blood cells.

- These peptides are more active against Bacillus megaterium than Cecropin A.

• Some time later, many other Cecropine were isolated from organisms other than Hyalophora cecropia .

• Cecropins isolated from other insects have been given various names… bactericidin, lepidopteran, sarcotoxin, etc. All of these peptides are structurally related.

Cecropin- AD :• The peptide is a hybrid analog consisting of

cecropin A-(1-11) and D-(12-37).

• The hybrid protein is 5-55 times as active as cecropin D against some bacteria and slightly more active than cecropin A. It is 6 times more active than cecropin A against Bacillus subtilis Bs11.

• It has reported that Cecropin AD promotes cell death by apoptosis in Nasopharyngeal carcinoma cells.

Cecropin P1 :

- It has been isolated originally as anti Escherichia coli factor from pig intestines .

- This peptide is not of mammalian origin, but is produced by the pig intestinal parasitic nematode, Ascaris suum, and human Ascaris lumbricoides

- Is bactericidal against a wide range of micro-organisms:

Aeromonas hydrophila, Bacillus subtilis , Escherichia coli; Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pneumocystis carinii, Saccharomyces cerevisiae .

CECROPIN- XJ:

• Is extremely heat-stable and tolerates extreme acidic, basic, and high salt environments.

• Has the ability to kill ampicillin- resistant Staphylococcus aureus and Staphylococcus enterica .

• It is also resistant to 24 hours digestion by artificial gastric juice.

Function of Cecropin

• The site of action of the antimicrobial activity is the cell membrane.

• Cecropin- A folds into final structure while superficially adsorbed to a membrane surface. Interactions with deeper hydrophobic regions of the bilayer appear to be unnecessary for folding. Its conformation is predominantly α-helical with some β structure.

To investigate the nature of its membrane activity, the interactions of cecropin with

synthetic lipid vesicles, bacteria and planar lipid membranes were investigated using

various techniques .

Action of Cecropins

• Just in vesicles, the action of cecropin A is concentration dependent, forming :

- ion channels at low peptide to lipid ratios

- pores ,large enough to pass probe molecules, at higher peptide to lipid ratios.

Action of Cecropin- A in vescicles.

• Cecropin A is equally effective on anionic and neutral vesicles, even though anionic vesicles bound more peptide.

• Cholesterol doesn’t prevent dissipation of ion gradients by low concentrations of peptide, but inhibits release of encapsulated probe by high concentrations of peptide.

The bactericidal action of Cecropins is correlated with the permeabilization of the membrane, that is , infact, always followed

by irreversible cytolysis and death of the bacteria.

Action of Cecropins

Many of these peptides directly act by altering the membrane of target cells: the interaction of these molecules with some

chiral receptor membrane seems excluded, since analogues of these

peptides consisting of all enantiomers D are active as the natural ones.

Positioning on the cytoplasmic membranePositioning on the cytoplasmic membrane

The longitudinal axis of the helical structure, and the transverse axes of any β

structure, are preferentially oriented parallel to the

membrane surface. These findings suggest a

mechanism of action against vesicles that involves

cooperative action of two or more peptides to produce a

transmembrane defect.

Toroidal Model

The toroidal pore is formed when the alpha helices of Cecropins intercalate in the lipid

bilayer

The peptide binds to the membrane and when it reaches the critical value pep/lip 1:20 changes its orientation

Barrel Stave or Toroidal Pore?

Barrel Stave Toroidal

All the Cecropins cause the formation of a toroidal pore in the phospholipid bilayer

One exception: Cecropin- P1

• Isolated from the intestine of a pig .• Is not very active against Gram-negative.• It has been suggested a mechanism of type

"detergent" :

The amphipatic monomers of α- helix are arranged on the membrane surface and then rotate by inserting the hydrophobic residues in the lipid bilayer, and destroying the packing.

Action of Cecropin- P1

Factors for the selectivity of Cecropins

• The different composition of the The different composition of the membranesmembranes

- Bacterial membranes : are rich in anionic phospholipids such as phosphatidylserine and phosphatidylglycerol; this then determines an electrostatic interaction of the peptide positively charged with the membrane itself, which is the basis of the subsequent effect of disturbance of the bilayer.

Factors for the selectivity of Cecropins

- Eukaryotic cells: for example, the haemocytes are characterized by a high number of zwitterionic phospholipids, such as phosphatidylcholine, sphingomyelin and phosphatidylethanolamine; are also rich in cholesterol, absent in bacteria, which appears to inhibit the action of these peptides conferring a certain resistance to the membranes.

Factors for the selectivity of Cecropins

• The value of membrane potentialThe value of membrane potential : : A potential more negative in the cell, typical

of the bacterial cells (100-150 mV), facilitates the interaction of Cecropins with the lipid layer.

Interaction with :

Gram +Gram +

The peptide is probably attracted by the acids teicoici and teicuronici

and other anionic groups which are located

externally to the layer of peptidoglycan.

Gram –Gram –

Initially the peptide interacts with the

polyanionic molecules of lipopolysaccharide of the outer membrane and is

then able to make it permeable or to be

received inside.

Future applications in medicine of Cecropins.

Cecropins inhibit bladder tumor cell Cecropins inhibit bladder tumor cell

viability and proliferationviability and proliferation

Background: bladder cancer’s therapy

Intravesical chemotherapy and BCG-immunotherapy are both associated with

significant side effects. Toxicities vary from mild cystitic symptoms to severe sepsis and have a negative impact on patient

compliance. Therefore, the identification of new potent intravesical agents is highly

desirable to reduce toxicity and to improve long-term outcome .

Methods

The antiproliferative and cytotoxic potential of the Cecropins A and B was quantified by assays in four bladder cancer cell lines as well as in murine and human fibroblast cell

lines.

Scanning electron microscopy (SEM) was performed to visualize the morphological changes induced by Cecropin A and B in

bladder tumor cells and benign fibroblasts.

Results

1. Cecropin A and B inhibit bladder cancer cell proliferation and viability in a dose-dependent fashion.

2. Benign fibroblasts were significantly less or not at all susceptible to Cecropin A and B.

3. Both Cecropins induced an increase in LDH release from bladder tumor cells whereas benign fibroblasts were not affected.

4. Transfection of human bladder cancer cells with Cecropin genes reduces their tumorigenicity in nude mouse models too.

ConclusionsConclusions

Cecropin A and B exert selective cytotoxic and antiproliferative efficacy in bladder

cancer cells while sparing targets of benign murine or human fibroblast origin. Both peptides may offer novel therapeutic

strategies for the treatment of bladder cancer with limited cytotoxic effects on

benign cells.

The hibrid peptide Cecropina- Melittin

exerts a leishmaniasicidal action.

Leishmaniasis

Disease caused by protozoan parasites that belong to the genus Leishmania

and is transmitted by the bite of certain

species of sand fly.

• The hibrid Cecropin- Melittin most active against Leishmaniasis is CA(1-8)M(1-18)

• A 4 μM the proliferation of promastigotes is inhibited up to 95% .

• The action of the peptide in Leishmaniasis is very rapid. At a concentration of 2.5 pM, the 90% of the final effect is reached in the first 5 minutes of incubation.

Factors of interactionFactors of interaction

• Temperature : the maximum inhibition of cell viability is reached from 20 °C.

• Effect of divalent cations : The lipophosphoglican ( LPG ) is one of major components of promastigotes’ membrane, and it binds Ca2 + cations. If we treat the cells with EDTA (Ethylenediaminetetraacetic acid ) , which sequesters these cations, you can observe a strong awareness of promastigotes to the peptide .

Alterations causedAlterations caused

• Damage of the flagellar pocket , that after the treatment is devoid of microtubules .

• Increase of the permeability of mebrane: it causes the accumulation of proline that affects both the pH gradient and the membrane potential , the two components involved in the maintenance ofenergetic capacity by translocation of protons.

Other Recent Studies:

• It has been demonstrated a specific tumoricidal activity of both Cecropin A and B against mammalian leukemia, lymphoma and colon carcinoma cell lines as well as small cell lung cancer and gastric cancer cells . In vivo, Cecropin B improves survival of mice bearing ascitic colon adenocarcinomas .

Cecropins are especially promising candidates for anticancer therapy in humans because they demonstrate

several unique features:

1. Their selectivity for malignant cells and their potentially pronounced lytic activity against high-grade tumor cells allow for an optimal therapy in vivo with low therapeutic concentrations and limited side effects .

2. The anti-tumoral effect of Cecropins has been hypothesized to be unaffected by the multidrug resistance (MDR) phenotype observed in many cancer types.

3. Classic chemotherapeutic agents such as mitomycin which are widely used for intravesical instillation are highly unstable in urine. In contrast, Cecropins are largely resistent against serum and urine proteolysis because of their specific biochemical structures, rendering them ideal candidates for intravesical tumor therapy .