Anti microbial peptides.pdf

9/12/2022 Siddu Lakshmi Prasanna, Sr. M.Sc (Agri.) 1

University of Agricultural Sciences, GKVK,
Bengaluru
Department of Plant Pathology
Seminar- 2
Siddu Lakshmi Prasanna
PALB 7314
Sr. M.Sc (Agri.)
Title: Antimicrobial compounds - Role in
Plant disease management
9/12/2022 2
Siddu Lakshmi Prasanna, Sr. M.Sc
(Agri.)

Flow of seminar
Introduction
Types of antimicrobial
compounds
Classification
Mode of action
Case studies
Conclusion

Fungicides and its residues in environment:

An antimicrobial compound which is derived from microbial source that kills
microorganisms or stops their growth.
• Includes Antifungal
Antiviral
Antibacterial
Antinematicidal etc.,
Introduction
What is an antimicrobial compound?
Why?????
✓ No harmful residues present in the environment.
✓ Prevents antibiotic resistance by the microorganisms due to complex nature.
✓ Specific in killing microorganisms

Types of antimicrobial compounds:
➢ Secondary metabolites - which are not required for normal growth and development
of the organism.
➢ Produced by plants, fungi, bacteria etc.,

Classification of bacterial secondary metabolites

Antimicrobial peptides (AMP’s)
Eukaryotic AMP’s Prokaryotic AMP’s/Bacteriocins
▪ Less specific
▪ Broad spectrum
▪ Requires high (micro molar)
concentration
▪ More specific
▪ Narrow target spectrum
▪ High potency and specificity
▪ Requires comparatively less (pico and
nano molar) concentration
▪ Located on mobile genetic elements,
conjugative plasmids or transposons
Source of antimicrobial peptides:
(Prashantkumar et al., 2018)

9/12/2022
Siddu Lakshmi Prasanna, Sr. M.Sc (Agri.)
9
Prokaryotic AMP’s
Gram negative bacteria
Microcins Colicins
Small Large
Class I
• < 5 KDA
• > Post translatio-
nal modifications
Ex: Microcin C7,
J25
Class II
• 5-20 KDA
• Post translational
modifications are less
Ex: Microcin E492
Gram Positive bacteria
Class I Class II
Lantibiotics non lantibiotics
• 19-38 AA
• Post translational
modifications occur
• Great structural variation
Ex: Nisin, Subtilin, Lacticin
• 25-60 AA
• Non modified
antimicrobial
peptides
• Binds to specific
receptors

✓ Bacteriocins were
first classified by
Klaenhammer
(1993)
Bacteriocins
Class I lantibiotics –
modified amino acid
lanthionine and small
peptides <5 kDa
Class II cystibiotics –
one or more disulfide
bonds , <10 kDa, heat
stable, and membrane-
active
Class III thiolbiotics-
active –SH group, <30
kDa, and heat-labile
Class IV complex
proteins- containing
one or more lipids or
carbohydrate moieties
Klaenhammer
(1993)
Classification of Bacteriocins

✓ Bacteriocins are classified according to chemical structure, heat stability, molecular
mass, enzymatic sensitivity, presence of modified amino acids, and mode of action
(Motta et al., 2008).
✓ Abriouel et al. (2011) classified Bacillus bacteriocins into 3 categories:
Class I (the post-
translationally modified
peptides)
Class II (the
nonmodified peptides)
Class III (the large
proteins).

Lantibiotics are among the best-characterized AMPs.
• These small microbial peptide antibiotics possess a variety of unusual amino acid
residues, genetic determinants, and biosynthesis mechanisms.
• During maturation, the premature peptides undergo post-translational modifications
through the introduction of unusual thioether amino acids, such as lanthionine and methyl
lanthione, together with the proteolytic removal of leader peptides.
Serine Dehydration 2,3-didehydroalanine + cysteine Lanthionine
(Lan)
Dehydration
2,3-didehydrobutyrine + cysteine
Threonine Methyl
lanthionine
(MeLan)

• Mature lantibiotics typically contain one or more unusual dehydro residues that do not
participate in lanthionine bridges and may thus be useful components in the design of
novel biomolecules.
• Ex: Arias and colleagues (2013) identified amylolysin, a putative lantibiotic that was
isolated from the B. amyloliquefaciens GA1 strain.
A putative lantibiotic gene cluster containing a structural gene (am/A) and genes
responsible for modification (am/M), transport (am/T), regulation (am/KR), and
immunity (am/FE) has been identified through genome characterization.

Mode of action (MOA) of AMP’s
• Therapeutic agents
• Mainly there are two classes:
1. Direct killing- 2. Immunomodulation
Membrane targeting Non- membrane
targeting
A. Receptor mediated
(Nano molar) - Ex: Nisin
Mesentericin
B. Non- receptor mediated
(Micro molar)
components of membrane
Transmembrane Transmembrane
pore models non pore models
1.Barrel stave model
2.Toroidal pore model
1. Carpet model
2. Detergent like model
• Have intracellular targets
• Inhibit cell wall synthesis
• Interact with various
precursor molecules required
for cellwall synthesis
✓ Activate immune cells
✓ AMP’s produced by many
immune cells such as
neutrophils, macrophages
etc.,
✓ Immune responses are
activated by activation,
attraction, differentiation
process etc.,

Mode of action (MOA) of AMP’s
(Prashant kumar et al., 2018)

Non receptor mediated membrane targeting

Transmembrane models

Objective: Biocontrol activity of lipopeptides against phytopathogens and
modulation of lipopeptide production in the presence of phytopathogens
(Cawoy et al., 2015)

Materials and methods:
Bacillus strains S499 and GA1.
Isolates QST713/QST2808.
B. amyloliquifaciens Strain FZB42 and its mutants used.
Fungal cultures:
Cladosporium cucumerinum,
B. cinerea,
F. oxysporum and
Pythium aphanidermatum used for antagonism assays.
UPLC-MS analysis
Samples analysed by reverse phase coupled with a single quadrupole MS.
Method used is, based on acetonitrile gradients, that allowed the simultaneous detection
of all three LP families (Iturin, Fengycin, Surfactin).
MALDI-TOF analysis for LP production and antagonism on natural root exudates

Relative proportion of LP
production in supernatants
and analysed by UPLC - MS
analysis
B. s- Bacillus subtilis
B. a- Bacillus amyloliquifaciens
B. p- Bacillus pumilus

Antagonism potential of Bacillus strains with pathogens

Correlations between LP concentration and intensity of antagonism

Intensity of the antagonism by B.
amyloliquefaciens strain FZB42 and its
lipopeptide mutants against pathogens

Lipopeptide production influencing by pathogen

MALDI-MS imaging shows the involvement of LPs in B. a. 98s
antagonism against F. oxysporum

Objective: Characterization of two rhizosphere-associated Bacillus velezensis
isolates (Y6 and F7) possess strong antagonistic activity against Ralstonia
solanacearum and Fusarium oxysporum f.sp. cubense under both laboratory
and greenhouse conditions.
(Cao et al., 2018)

Bacillus sps. isolated from the rhizosphere soil of tomato plants in Yuejin Farm, Guangzhou,
China
Pathogens:
Ralstonia solanacearum
Fusarium oxysporum f. sp. cubense
In vitro evaluation
Plate confrontation assay
Spot-on-lawn assay
Identification and quantification of LPs by UPLC–MS. Both methanol and acetonitrile/water
extracts were analyzed by reverse phase Ultra-Performance Liquid Chromatography coupled
with a triple quadrupole MS.
RNA extraction and qPCR analysis to identify the lipopeptide expression genes.

Isolation and identification of two Bacillus strains with significant
antibacterial activity.

Identification of the lipopeptide compounds secreted by Y6 and F7.

Quantification of lipopeptide compounds produced by Y6 and F7

LP production is strongly
stimulated in the presence of
R. solanacearum.

The presence of R. solanacearum affects expression of LP biosynthesis genes.
Expression of the LP biosynthesis genes (srfAB, ituC and fenD for synthesis of
surfactin, iturin, and fengycin, respectively) was monitored in the two isolates in
the presence or absence of the co-culturing pathogen Ralstonia solanacearum.

LP production is significantly stimulated during interaction with R. solanacearum (RS).

Iturin and fengycin play a redundant role in antagonism against R. solanacearum.
The antagonistic activity of Y6 (WT) and its derived mutants including
srfAA , ituA , fenC and ituA fenC against the pathogen R. solanacearum was
tested using a spot-on-lawn assay.

Iturin is the primary factor responsible for antifungal activity against Fusarium
oxysporum f.sp. cubense in vitro.

Iturin is the primary factor responsible for antifungal activity against Fusarium
oxysporum f.sp. cubense in vivo.

(Khan et al., 2008 )
Objective: To evaluate the nematicidal potential of P. polymyxa strain
GBR-1 against Meloidogyne incognita under in vitro and green house
conditions.

• Paenibacillus polymyxa GBR-1 strain
• Cell free filtrate
• Bacterial suspension
➢ Root knot nematode - Meloidogyne incognita

Effect of bacterial culture filtrate on egg hatch and juvenile
mortality of M. incognita in vitro assay

Effect of application of various concentrations of bacterial culture filtrate
extract and bacterial suspension on root galling

extract and bacterial suspension on final population

extract and bacterial suspension on fresh root and shoot weight

Effect of application of various concentrations of bacterial suspension on
root galling, multiplication and tomato plant growth

Potential applications and beneficial roles of AMPs of the Bacillus species
➢ Rapid growth rate of the organism, which result in short fermentation cycles
➢ High capacity for protein secretion into the extracellular medium
➢ In whole genome of Bacillus sps. contains secondary metabolite clusters,
antimicrobial genes which helps in using as potential bio-control agents.
➢ Bacillus-derived peptides have shown antibacterial, antifungal, antiviral,
antitumor, antiamebocytic, and antimycoplasmic activities (Yilmaz et al.,
2006); Chen et al., 2008).

Anti microbial peptides.pdf

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