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Bioengineering Lantibiotics for
Therapeutic success
Dr Des Fielddes.field@ucc.ie
QuebecNot Quebec
Food Veterinary Medicine
Health & Pharma
Food Safety and Shelf life extension Anti-mastitis treatment
Potential for use as Therapeutics
Bacterially produced, small, heat-stable, gene-encoded
peptides that are active against other bacteria and to which
the producer has a specific immunity mechanism.
-More than100 lantibiotics identified
wide variation in structures
wide variety of post-translational
modifications
Ile SerIle Thr Ser Leu Cys Thr Pro Gly Cys
OH
OH OH
OH
IleIle Leu Cys Pro Gly CysDhaDhb Dha Dhb
SHSH Lanthionine
Ile Dhb Ala
Ile
Dha
Leu
Ala Abu
Pro Gly
Ala K
S
S
Lanthionine-containing antibiotics
β-methyl-
lanthionine
Class I bacteriocins
Undergo post-translational modification (by modification enzymes)
Dairy products: processed cheese and cheese spreads; direct acidified cheeses;pasteurised dairy desserts;fresh and recombined milk; •Liquid egg •Dressings and sauces • Not very effective against Listeriamonocytogenes
Membranephospholipids
Wallpeptidoglycan
Breukink lab; Sahl lab
Lipid II
1.
2.
• Multiple activities
• Two domains separated by a ‘hinge’
Transport/
Processing
Alan
Structural
B C
J
D
Lanthionine
synthesis
Further
modifications
M
in out
T P
Processing
Gene-encoded biosynthesis
Potential for bioengineering through manipulation of the
structural peptide
Generate vast population of variant derivatives
A
‘Unlike the mammalian immune system, the
bacterium has no mechanism for generating a
diverse population of (lantibiotics)’.
‘……can be viewed as a kind of primitive antibody,
even having constant and variable regions.’
Liu & Hansen J Biol Chem 1992 267:25078
‘the extent to which (lantibiotics) can be
structurally altered without destroying their
character is unknown’
‘Unlike the mammalian immune system, the
bacterium has no mechanism for generating a
diverse population of (lantibiotics)’.
Natural variants
However, natural variants could be
regarded as diverse populations
Nisin A
Nisin Z
Nisin Q
Nisin U
Nisin U2
Nisin F
Nisin P
Nisin H
Cotter, P.D., C. Hill and R.P. Ross. 2005. Curr. Prot. Pept. Sci. 6:61.
57 derivatives
• nisin – was only example >8 derivatives described in literature
• mutants have been very useful for fundamental analysis
• unfortunately there was little success in enhancing activity
b
a
Complete Alanine Scanning of Lacticin 3147
‘the extent to which (lantibiotics)
can be structurally altered without
destroying their character is
known!!!’
‘CAN be viewed as a kind of primitive antibody,
even having constant and variable regions.’
Cotter et al 2006 Mol Microbiol
Development of systems for Lantibiotic Mutagenesis
A B T C I P R K F E G
Random Mutagenesis Saturation Mutagenesis Directed Mutagenesis
Q
Bioengineering Nisin
X X X
Random
NZ9800
pPTPL
NZ9800
pPTPL-nisA
NZ9800
pPTPL-nisAK22T
NZ9700 NZ9800 NZ9800
nisA-K22S
A. B.
NZ9800
pPTPL
NZ9800
pPTPL-nisA
NZ9800
pPTPL-nisAK22T
NZ9700 NZ9800 NZ9800
nisA-K22S
A. B.
Nisin Mutant
High-throughput Robotics
systems
Bioengineering Nisin
Random Banks
Single X Banks
Double X Banks
NisF/Z/Q S29X
Triple X Banks
8,000variants
3,456 variants
6,432 variants
432 variants
19,200 variants
In total: 37,520 variants…and counting!
L. lactisHP
S. aureus RF122
S. aureus SA113
S. aureus
NCDO1499
S. agalactiae ATCC
L. mono
MRSA
VISA
hVISA
VRE
E. faecium
E. faecalis
B. cereus
S. pyogenes
S. mutans
S. dysgalactiae
S. mitis
E. durans
S.uberis
E. casseliflavus
I Dhb Ala
IDha
L
Ala Abu
P G
Ala N
AAbu
Ala
HG
Abu
A
L
G
Ala
M
M K Abu Ala Dha KS I H VK
A
BC D
E
5
10
15
20
2530
SS
S S
S
I4V S29A/OthersM21V
Food Veterinary Medicine
Health & Pharma
Food Safety and Shelf life extension Anti-mastitis treatment
Potential for use as Therapeutics
Veterinary Medicine
Health & Pharma
Anti-mastitis treatment
Potential for use as Therapeutics
350,000 Pounds of Hot Dogs, Corn Dogs Recalled Amid Fears of Listeria ContaminationPosted by CNN Wire
Hot dog meat trial
(Lux-labelled Listeria)
Field et al 2008 Mol Microbio[
Field et al 2010 Microbiol Biotechnol
Enhanced anti-listeria activityM21V WT
Listeria monocytogenes
Model Food studies with Nisin V (M21V) fermentate in
combination with essential oils e.g. cinnamaldehyde,
carvacrol
Field et al., 2015 AEM
In food, Nisin is used as a fermentate containing 2.5% nisin (e.g. nisaplin® )
Chocolate Milk Commercial Soup
AORI Ts
A B T C I P R K F E G
A B T C I P R K F E GV
Mutate nisA to nisV
1 nt change
A B T C I P R K F E G
V B T C I P R K F E G
50%
50%
VORI Ts
Cotter et al., 2002 AEMCotter et al., 2006 Mol Microbiol
L. monocytogenes LO28
Nisin A Nisin V
Ruling by EU/EPA in 2011 that Nisin V is excluded from Directive2009/41/EC
Not regulated as a GMM!
Food
Health & Pharma
Food Safety and Shelf life extension
Potential for use as Therapeutics
Veterinary MedicineAnti-mastitis treatmentFood Safety and Shelf life extension
Health & Pharma
Food
Potential for use as Therapeutics
Antibiofilm activity /Synergy with antibiotics
Methicillin-resistant S. pseudintermedius (MRSP):opportunistic animal pathogen responsible for skin, soft tissue, and surgical site infections.Multi-drug resistant, ability to form Biofilms
Biofilms of S. pseudintermedius DSM 21284
Wt I4V
Field et al 2015 PLoS ONE
Field et al 2016 Frontiers in Micro
Peptide S. aureus 54072
Mg/L
S. aureus 20777
Mg/L
S. aureus NCDO 1499
Mg/L
S.aureusRF122
Mg/L
L. lactisMG1363
Mg/L
L. lactisHP
Mg/L
B. LongumUCC
Mg/L
L. acidophilusUCC
Mg/L
Nisin A 1 8 0.25 2 0.06 <0.125 0.06 0.03
Variant 1 0.25 0.5 0.06 0.25 0.06 0.125 0.06 0.06
Variant 2 4 0.25 1 2 0.5 0.5 0.12 0.12
Variant 3 0.25 0.125 0.03 1 0.125 0.125 0.24 0.12
The BAD The GOOD
Nisin versus Bovine Mastitis pathogens
Veterinary MedicineAnti-mastitis treatment
Food
Health & Pharma
Food Safety and Shelf life extension
Potential for use as Therapeutics
Veterinary MedicineAnti-mastitis treatment
FoodFood Safety and Shelf life extension
The Outer membrane (OM) of the Gram
negative cell wall which acts as a barrier for
the cell, restricting the access of the peptides
to the cytoplasmic membrane
Enhanced derivatives against Gram Negatives
5 10 15 20 25
0.2
0.6
1.0
1.4 Untreated
Nisin S29A 15M
Colistin 0.75g/ml
Nisin S29A 15M + Col
0.75g/ml
Time (Hrs)
OD
60
0n
m
P. aeruginosa PA-01
Field et al., 2012 PLoS ONE
Field et al .,2016 Frontiers in Micro
But…..some bacteria have evolved nisin resistance mechanisms!!!!!
• Nisin Resistance Protein (NSR) degrades nisin by cleaving the peptide bond
between MeLan28 in ring E and Ser29
• NSR protein provides a 20-fold increased resistance towards nisin
The Nisin Resistance Protein (NSR)
632 Da
2720 Da
+
Khosa et al 2016 Frontiers in Micro
NSR homologues are found in many pathogenic and non-pathogenic strains including:
-E. faecium-S. epidermidis-Strep. sanguinis-Strep. dysgalactiae-S. agalactiae-Corynebacterium casei-C. ammoniagenes
More widespread than we thought!!!
Ser29X derivatives
Molecular modelling provides strong evidence of a rigidity and inflexibility in the region around the nisincleave point for the S29P derivative compared to the wild type Nisin A.
37,520
340000000000000000000
Cork Bacteriocin Group
University College Cork
Prof Colin HillProf Paul RossDr Des FieldDr Ciorsdan CampionKevin EganJenna EllisAngeliki AngelopolouFanny CharlotInes BaghouPhiliswa Mbandlwa
Teagasc
Dr Paul CotterDr Sheila MorganDr Mary ReaPaula O’ConnorHarsh MathurElaine LawtonCleriane Andre
The Irish Government under the National Development Plan
Funding
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