Protein engineering: promising approach for the...

Protein engineering: promising approach for the development of new antimicrobials

Ismail Fliss & Riadh Hammami

STELA conference, May13-14 2013


Team at INAF: identification, caracterisation and study of the structure/fonction relationship of bactériocins produced by lactic acid and probiotic bacteria.

Team at CHUQ research centre, laboratory of medical chemistry: synthesis of bioactive peptides.

FQRNT-Team project


Large scale synthesis of new, more stable antimicrobial molecules with larger spectrum of antimicrobial activity from bacteriocin peptide sequences.

Microcine J25

General objective


Peptides or polypeptides naturally produced by several bacterial strains of food and environmental origin.

Antimicrobial activity (bactericidal or bacteriostatic) against microorganisms phylogenetically related to the producing strain.

Bacteriocins


Widespread phenomenon (Lactococci and Lactobacilli);

Broad spectrum of antimicrobial activity;

Acid and heat resistance;

150 Gram (+) and 25 Gram (-) bacteriocins: BACTIBASE

Bacteriocins


Nisin is the only bacteriocin approved in more than 50 countries. Carnobacterium maltaromaticum CB1: antimicrobial agent for preservation of meat products (Health Canada, food additive) C. divergens M35: bioconservation agent for smoked sea products (Health Canada, new foods section) Pediococcus acidilactici UL5 (pediocin producer): probiotic strain for veterinary use (Health Canada, Natural health products)

This image cannot currently be displayed.

Legal status of bacteriocins


Food : natural food additives (Nisin)

Pharmaceutical: skin infections, antibiotic resistant bacteria

Veterinary: Treatment and prevention of bovine mastitis (WIPE OUT® Immucell)

Applications

http://goatsupplies.netfirms.com/ProductImages/wipeout.jpg


Advantages and limits

Limits Advantages

Food grade substances: completely

degraded in the digestive tract

High biological activity: 103 to 106

times higher than other antimicrobials,

including antibiotics;

High thermal stability: compatible with

various heat treatments

Narrow spectrum of inhibition activity

Instability in biological matrices

(proteases, extreme pH, salt).

Low production yields (10%): difficulty

for large-scale production, high costs,

etc.


Microbiological approach: selection of highly productive strains (L. lactis diacetylatis UL719, P. acidilactici UL5), optimization of fermentation processes (continuous fermentation with immobilized cells) and purification (immunoaffinity). Molecular biology approach: cloning and production of recombinant bacteriocins Protein engineering approach: peptide synthesis (knowledge of the structural characteristics and relationships with biological activity)

Improving yield


Microcin J25

Main Objective

Elucidate the relationship between the primary and secondary structures of bacteriocins and their biological function. The ultimate goal is to exploit the knowledge generated for the large-scale synthesis of new, more stable and biologically active antimicrobial molecules with a broad spectrum of inhibition activity.


Microcin J25


Microcin J25

21 AA Unique structure : Lasso 3 β-sheets


Microcin J25


Structure-function relationship of microcin J25

Several studies using molecular biology and chemical synthesis have attempted to produce microcin J25 or active derivatives but without success. Lack of knowledge about the relationship between the particular Lasso structure of microcin and its biological activity: - Role of the N-terminal 1-8 loop - The role of the C-terminal tail (with two segments: 9-17 and 18-21) - The presence of some amide bridges (cyclisation G1-E8) - The presence of some specific AA.


GGAGH VPEYF VGIGT PISFYG

Is overall conformation a prerequisite for Microcin biological activity?

J25

C1

C2

C3 GGAGH VPCYF VGIGT PICFYG

CGAGH VPCYF VGIGT PISFYG

CGAGH VPEYF VGIGT PICFYG C8 C18

C1 G1

C8


Tridimensional structure of microcin-derived peptides

J25 C1 C2 C3


Is the whole sequence of microcin required for its biological activity?

GGAGH VPEYF VGIGT PISFY G

--YF VGIGT PISFY G

-CYF VGIGT PICFY G

KCYF VGIGT PICFY G

J25

9-21

8-21

7-21


J25 7-21



GGAGH VPEYF VGIGT PISFY G

GWKGK WKCYF VGIGT PICFY G

J25

WK_7-21

Does the overall net charge influence Microcin biological activity?

-1

+3


J25 WK-7-21



Synthesized peptides

Peptide Sequence link Mass (Da) Net charge

MccJ25* GGAGHVPEYFVGIGTPISFYG 1-8 2 145.0 -1 C1 CGAGHVPCYFVGIGTPISFYG 1-8 2 145.0 0 C2 CGAGHVPEYFVGIGTPICFYG 1-18 2 145.0 -1 C3 GGAGHVPCYFVGIGTPICFYG 8-18 2 145.0 0

1-8L GGAGHVPE------------- - 723.0 -1 1-10C GGAGHVPEYF----------- 1-8 1033.0 -1

NC1 GGAGHVPCYF-GKG---CFYG 8-18 1717.8 +1 NC2 GGAGHVPCYF-KKK---CFYG 8-18 1861.0 +3 NC3 GGAGHVPCYF-WKW---CFYG 8-18 1976.8 +1

9-21L --------YFVGIGTPISFYG-NH2 - 1420.0 0 9-21C --------YFVGIGTPISFYG 9-18 1420.0 0 8-21C -------CYFVGIGTPICFYG 8-18 1540.0 0 7-21C ------KCYFVGIGTPICFYG 8-18 1668.0 +1

WK_7-21 GWKGKWKCYFVGIGTPICFYG 8-18 2410.0 +3


Minimal inhibitory concentration (MHI) of the peptides

Strains MIC (µM)

MccJ25* NC1 NC2 NC3 C1 C3 8-21C 7-21C WK_7_21

Salmonella enterica subsp. enterica ATCC 14028 6.5 - - - 125-250 - - - -

Salmonella enterica subsp. enterica ATCC 8387 0.1 - - 31.3 1.0 15.6-31.3 1.0 7.8-15.6 7.8

Salmonella enterica subsp. enterica ATCC 29628 6.5 - 250 - - - - - -

Salmonella enterica subsp. enterica ATCC 8400 0.8 - - - 62.5-125 - - 125-250 62.5

Salmonella enterica subsp. enterica ATCC 9607 1.6 - - - 250 - - - -

Salmonella enterica subsp. enterica ATCC 9700 0.4 - - - 250 - - - -

Escherichia coli ATCC 11229 0.2 - - - 62.5 - 125-250 - 250

Escherichia coli ATCC 25922 3.3 - - - 250.0 - - 250 - Escherichia coli ATCC 15144 - - - - - - - - -

Escherichia coli O157 H7 ATCC 35150 - - - - - - - - -

Escherichia coli MC4100 ATCC 35695 6.5 - - - 31.3-62.5 - - 125 -

Escherichia coli DH5a 6.5 - - - - - - - - Escherichia coli LR 05 - - - - >250 - - >250 >250

Listeria ivanovii HPB28 - - - - >250 - - >250 250


0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

3.00 30.00 300.00

OD

595n

m

Concentration (µM)

7-21C-2S C1-2S C2-2S C3-2S-1 C3-2S-2 1-8L 8-21C-2S 9-21C-2S 9-21L 1-10C

Inhibition activity against E. coli


Inhibition activity against S. enterica ATCC 8387

0

20

40

60

80

100

MccJ25 C1 C3 NC3 8-21C 7-21C WK_7_21

Inhi

bitio

n (%

)

0.5 µM 7.8 µM


Growth inhibition of Listeria ivanovii by active peptides at 250µM

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 4 8 12 16 20 24

Opt

ical

den

sity

(595

nm)

Time (Hours)

Control C1

7-21C WK_7-21


Electrostatic Potential (Face A)

Microcin J25 WK_7-21


Microcin J25 WK_7-21

Electrostatic Potential (Face B)


Secondary structure

Circular dichroïsm experiments :

The secondary structure of active peptides is very

similar to that of native microcin J25 (β sheets +

coils)


CONCLUSIONS

R1: yes, the lasso structure is important but not prerequisite for the biological activity.

R2: No, for example the ring plays an important role in the antimicrobial activity of microcin (C1) but its deletion doesn’t totally abolish the inhibitory activity (7-21)

R3: yes, the substitution of some amino acids to increase the overall charge seems to enhance the activity of microcin and extend its spectrum of action toward Gram+ (WK-7-21)

PERSPECTIVES

The new molecules can be synthesized on a large scale with high yields.

Opens the door to applications of these molecules in several areas (food, medical and veterinary) where the search for these new molecules is becoming more urgent.

Acknowledgements

François Bédard

Eric Biron

Pedro Alvares

Muriel Subirade

Fond québécois de recherche sur la nature et les technologies (FQRNT-Équipe)

Destruction of membrane potential

Nucleolytic and related activities

Inhibition of protein synthesis

Out

In

Leakage of cellular content

(ions, ATP)

insertion and pore formation

Nisin

Lipid II

Pediocin

Man-PTS

Electrostatic interactions

Translocation into the cytoplasm (e.g. colicins)

Bacteriocin

OR

Target recognition

Mem

bran

e

e.g. Sakacin

Receptor

or

+ + +

- - - -

-

Attraction électrostatique

Perturbation de la

membrane Insertion bioénergétique

et interaction

Peptides antimicrobiens: Mode d’action général

+ +

D’une manière générale le mode d’action se situe au niveau de la membrane :

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