<figure><img src="image_1.png" /></figure>

HAL
open science

# Research project avian defensins as alternative to antibiotics

Anne-Christine Lalmanach

<figure><img src="image_2.png" /></figure>

## To cite this version:

Anne-Christine Lalmanach. Research project avian defensins as alternative to antibiotics. 1ères ren-
contres de recherche en santé animale, Syndicat de l'Industrie du Médicament et Réactif Vétérinaires
(SIMV). FRA., Dec 2014, Lille, France. hal-01601510

HAL Id: hal-01601510

https://hal.science/hal-01601510

Submitted on 5 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_3.png" /></figure>

Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License
Research project
avian defensins as alternative to
antibiotics

Anne-Christine Lalmanach

UMR 1282 Infectiologie et Santé Publique,

Pathogeny of avian colibacillosis group

INRA, Centre de Recherche de Tours,

France
Defensins

* Molecular arsenal of innate immunity.

* Cationic peptides, β-sheet structure, 6 cysteins highly conserved (3 S-S bonds).

* Three families: α (only in mammals),
  β -> throughout evolution,
  θ (only in primates).
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

# Defensins: biological activity

## Antimicrobial activity: bacterial membrane disruptors

Direct killing

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Internal targets

<figure><img src="image_8.png" /></figure>

Bilayer disruption

<figure><img src="image_9.png" /></figure>

Specific binding to
lipid receptors,
e.g. lipid II

<figure><img src="image_10.png" /></figure>

Disruption of
membrane-
bound machineries

<figure><img src="image_11.png" /></figure>

HDP

## Immuno-modulatory activity

Immune modulation

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

Monocyte

<figure><img src="image_14.png" /></figure>

Granulocyte

<figure><img src="image_15.png" /></figure>

Lymphocyte

Recruitment and activation
of immune cells

<figure><img src="image_16.png" /></figure>

Enhanced pathogen
killing and clearance

<figure><img src="image_17.png" /></figure>

Controlled
inflammation
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

# Birds defensins

* Avian β-defensins (AvBDs), 14 members (for review: van Dijk et al, Vet Immunol Immunopathol, 2008)

* Transcriptomic analysis in tissues :

<figure><img src="image_20.png" /><figcaption>Male chickens, 3 weeks old (broilers), from Lynn et al, Immunogenetics, 2004</figcaption></figure>
# Cells producing AvBDs in vivo :

**heterophils granulocytes** (Harwig et al, FEBS Lett, 1994; Evans et al, J Leukoc Biol, 1994)

<figure><img src="image_21.png" /></figure>

Intact cell mass spectrometry
(ICM-MS): peptidic map
(Derache et al, Antimicrob Agents
Chemother, 2009)

<figure><img src="image_22.png" /></figure>

**epithelial cells**

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

Overexpression in intestinal EC from chicken resistant to *Salmonella*
(Derache et al, Dev
Comp Immunol, 2009)

A-C Lalmanach _ Rencontres Recherche -Industriels SIMV
BioFIT Lille, 2-3 Décembre 2014
<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

# Preparation of AvBDs (1)

* Recombinant avian β-defensins (AvBD)

Various system used: E coli (Origami strains), Pichia pastoris, Insect cells...
Unsuccessfully

* Synthetic peptide

Delicate chemistry, high cost.

* Structural features

AvBD2 natural purified or synthetic peptide, 3D structure solved by NMR

<figure><img src="image_27.png" /><figcaption>AvBD2</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>L1
K31
V20</figcaption></figure>

<figure><img src="image_29.png" /><figcaption>AvBD2 K31A</figcaption></figure>
<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

# Preparation of AvBDs (2)

* Avian β-defensins (AvBD), extraction and purification from chicken bone marrow (Derache C et al, 2009, Antimicrob Agents Chemother)

BM collected from 10 ROSS PM3 chickens (femur and tibia)

<figure><img src="image_32.png" /></figure>

Peptides extraction with 10% acetic acid

<figure><img src="image_33.png" /></figure>

Separation by size-exclusion chromatography (Biogel P10, Biorad)

<figure><img src="image_34.png" /></figure>

Purification by RP-HPLC (C18)

<figure><img src="image_35.png" /></figure>

Peak analysis by MALDI-TOF mass spectrometry

Estimated yields :

AvBD7 : 1.5 mg,

AvBD2 : 1.9 mg.

<figure><img src="image_36.png" /></figure>

M/Z: 3916
AvBD2
# Antibacterial activity of AvBDs

## Minimal inhibitory concentration, MIC
measured by radial diffusion assay

MIC in µM
95% confidence interval

<figure><img src="image_37.png" /></figure>

<table><thead><tr><th>Bacterial strains</th><th>L-AvBD2</th><th>D-AvBD2</th><th>L-AvBD2-Acm</th><th>AvBD2-K31A</th><th>AvBD2-K31A-Acm</th></tr></thead><tbody><tr><td><strong>Gram +</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>B. cereus</em></td><td><strong>0.31</strong><br>0.19-0.47</td><td><strong>0.34</strong><br>0.19-0.53</td><td><strong>3.47<sup>a</sup></strong><br>2.12-4.87</td><td><strong>0.75<sup>b</sup></strong><br>0.56-0.97</td><td><strong>&gt;58.26</strong></td></tr><tr><td><em>L. monocytogenes</em></td><td><strong>0.20</strong><br>0.09-0.36</td><td><strong>0.20</strong><br>0.11-0.34</td><td><strong>3.21<sup>a</sup></strong><br>0.98-6.13</td><td><strong>1.55<sup>c</sup></strong><br>1.05-2.14</td><td><strong>23,3-58,26</strong></td></tr><tr><td><em>S. aureus</em></td><td><strong>0.58</strong><br>0.05-1.79</td><td><strong>1.37</strong><br>0.35-3.03</td><td><strong>&gt;114.99</strong></td><td><strong>6.56<sup>c</sup></strong><br>0-32.5</td><td><strong>&gt;116.52</strong></td></tr><tr><td><strong>Gram -</strong></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><em>E. coli</em></td><td><strong>1.09</strong><br>0.68-1.57</td><td><strong>1.05</strong><br>0.73-1.40</td><td><strong>1.23</strong><br>0.32-2.49</td><td><strong>2.07<sup>d</sup></strong><br>0.11-5.20</td><td><strong>23,3-58,26</strong></td></tr><tr><td><em>S. Enteritidis</em></td><td><strong>0.48</strong><br>0.20-0.86</td><td><strong>0.52</strong><br>0.26-0.87</td><td><strong>1.31<sup>c</sup></strong><br>0.64-2.13</td><td><strong>2.55<sup>c</sup></strong><br>0.77-4.64</td><td><strong>&gt;58.26</strong></td></tr><tr><td><em>S. Typhimurium</em></td><td><strong>3.67</strong><br>1.73-5.99</td><td><strong>1.59</strong><br>1.83-5.96</td><td><strong>5.18<sup>a</sup></strong><br>0-6.06</td><td><strong>7.08<sup>c</sup></strong><br>0.57-10.23</td><td><strong>&gt;58.26</strong></td></tr></tbody></table>

<footnote><sup>a</sup>, P=0.0002, <sup>b</sup>, P=0.0003, <sup>c</sup>, P<0.0001, <sup>d</sup>, P=0.016.</footnote>
<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

# Resistance of AvBDs to proteolysis

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

Separation of reaction products
by RP-HPLC
and M/S analysis
+ antimicrobial assay

- Conservation of integrity and of activity of AvBD2 and 7 after reaction with trypsin, chymotrypsin, elastase, cathepsin D and H.

- Partial susceptibility of AvBD2 to cathepsin B and S.

- Total susceptibility of AvBD2 to cathepsin L and K.

- Total resistance of AvBD7 to 9 proteases tested (conservation of antimicrobial activity)
# Therapeutic use of AvBDs

<figure><img src="image_42.png" /></figure>

Investigate the following issues :

<figure><img src="image_43.png" /></figure>

Activity of AvBDs on pertinent bacterial isolates (field strains, clinical isolates, MDR strains...): in progress.

<figure><img src="image_44.png" /></figure>

Synergistic antimicrobial effect between AvBDs: in progress.

<figure><img src="image_45.png" /></figure>

Chemotactic and stimulatory activity towards host cells (leukocytes, epithelial cells): in progress.

<figure><img src="image_46.png" /></figure>

When administered in vivo, are AvBDs protective/curative against pathogenic bacteria?
<figure><img src="image_47.png" /></figure>

<figure><img src="image_48.png" /></figure>

# Therapeutic use of AvBDs

<figure><img src="image_49.png" /></figure>

* Are AvBDs protective/curative in vivo against pathogenic bacteria?

-> *In vivo* administration of AvBDs in animal infectious model (letal salmonellosis in mice, Maiti et al, Antimic Agents Chemother. 2014; Benincasa et al, BMC Microbiol. 2010).

<figure><img src="image_50.png" /></figure>

Five Balb/c mice
per group

Inoculation of *Salmonella typhimurium* (10² to 10³ CFU, i.p.)

<figure><img src="image_51.png" /></figure>

Administration of AvB2 or AvBD7
(200 µg/mice, i.p.) or H2O

Monitoring of
animal survival.
Measurement of
organ colonisation

<figure><img src="image_52.png" /></figure>
<figure><img src="image_53.png" /></figure>

# Therapeutic use of AvBDs

## Remaining issues to be examined

* Acceptability of therapeutic molecules from animal products origin

* If acceptable, economic interest with added value to byproducts (bones, carcasses).

* If not acceptable, strategies of production of AvBD2 and AvBD7: recombinant, synthetic
Contributors

INRA ISP, PAC group

G. Bailleul

N. Lallier

A. Trotereau

N. Chanteloup

R. Guabiraba-Brito

C. Schouler

A-C. Lalmanach

<figure><img src="image_54.png" /></figure>

INRA
SCIENCE & IMPACT

<figure><img src="image_55.png" /></figure>

UNIVERSITÉ
FRANÇOIS - RABELAIS
TOURS

INRA Mass spectrometry group

G. Harichaux

A.P. Teixeira-Gomes

V. Labas

INRA Animal facilities (PFIE)

E. Guitton and coll.

<figure><img src="image_56.png" /></figure>

INSERM U1100 CEPR,
University of Tours

F. Lecaille

G. Lalmanach

<figure><img src="image_57.png" /></figure>

CNRS, UPR 4301 CBM,
Orléans

A. Delmas

C. Landon