Design of synthetic-hybrid bacteriocins from enterocin E50-52 and pediocin PA-1 for therapeutic applications Santosh Kumar Tiwari, PhD Assistant Professor.
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Design of synthetic-hybrid bacteriocins from enterocin E50-52 and pediocin PA-1
for therapeutic applications
Santosh Kumar Tiwari, PhDAssistant ProfessorDepartment of GeneticsMaharshi Dayanand UniversityRohtak-124001, HaryanaEmail: santoshgenetics@gmail.com
6th World Congress on Biotechnology, October 05-07, 2015, New Delhi
Antibiotics Vs Resistance
World Health Organization (WHO) foreseen: Almost one billion people will be infected with Mycobacterium tuberculosis
between the years 2000 and 2020.
About 35 million humans will die till 2020 as a result of tuberculosis in antibiotic-resistant form .
Over 70% of bacterial pathogens that cause fatal infections are likely to be resistant to at least one of the drugs (Infectious Disease Society of America, IDSA).
Several preventive measures have been taken to avoid the microbial resistance development, but still there is an urgent need for new antimicrobial agents and new strategies to overcome problematic resistant pathogens.
Antimicrobial peptides (AMPs), particularly bacteriocins produced by bacteria, may be an important contributor in this context as they often have a relatively narrow killing spectrum (Nes et al. 2007).
Bacteriocins
Ribosomally synthesized small peptides antimicrobial activity
BACTIBASE dataset (version 2, July 2009)
Diversity
Total bacteriocins :177Gram-positive bacteria : 156 (113 from lactic acid bacteria)
Gram-negative bacteria : 18 Archaea domain : 3
BACTIBASE dataset (version 2, July 2009)
Therapeutic potential of bacteriocins Thuricin CD isolated from Bacillus thuringiensis DPC6431, specifically
eliminates Clostridium difficile without disrupting the beneficial microbial community (Rea et al. 2010).
Nisin, mersacidin and lacticin 3147 can eradicate infections caused by Streptococcus pneumoniae, MRSA in mice, tooth diseases in dogs and bovine mastitis in dairy cows (Òkuda et al. 2013).
Microcin J25 has been shown to drastically reduce Salmonella infection in a mouse model (Lopez et al. 2007).
Fermenticin HV6b and nisin ZP inhibit wide range of pathogens, spermicidal and anticancerous activity as reported to induce apoptosis in cancerous cells (Kaur et al. 2013; Kamrajan et al. 2015).
Nisin: A bacteriocin produced by Lactococcus lactis
NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | JULY 2006 | 531
The residues in red have positive net charge, blue are hydrophobic. Dha, dehydroalanine; Dhb, dehydrobutyrine; Lan, lanthionine; Mla, methyllanthionine; S, thioether bridge
Mode of action of bacteriocins
Pediocin PA-1
Design and Synthesis of Hybrid Bacteriocins
TTKNYGNGVCNSVNWCQCGNVWASCNLATGCAAWLCKLA
Enterocin E50-52
Design and Synthesis of Hybrid Bacteriocins
Methods for detection of antimicrobial activity
Amount of bacteriocin (l)
50 25 12.5 6.25 3.12 1.56 0.78 0.39 0.19 0.095
% in
hibi
tion
of g
row
th o
f in
dica
tor
stra
in
0
20
40
60
80
100
AU/ml OR MIC
Control Treated
% g
row
th o
f in
dica
tor
stra
in
0
20
40
60
80
100
120
Percentage inhibition of indicator strain
Spot assay plate method
Producer strain
Indicator strain
Agar Well Diffusion Assay (AWDA)
Concentration (M)
Gro
wth
(A
595)
0.0
0.1
0.2
0.3
0.4
200100502512.56.253.121.560.00
Pediocin PA-1
(D)
(B)
(C)
Concentration (M)
Gro
wth
(A
595)
0.0
0.1
0.2
0.3
0.4
200100502512.56.253.121.560.00
Concentration (M)
Gro
wth
(A
595)
0.0
0.1
0.2
0.3
0.4
200100502512.56.253.121.560.00
Concentration (M)
Gro
wth
(A
595)
0.0
0.1
0.2
0.3
0.4
200100502512.56.253.121.560.00
PE
Enterocin E50-52
EP
Minimum Inhibitory Concentration (MIC)
Comparison of MIC of WT and hybrid bacteriocins
TIme (min)
0 15 30 45
Inte
rnal
AT
P c
on
c (
M)
3x10-12
4x10-12
5x10-12
6x10-12
7x10-12
8x10-12
9x10-12
10x10-12
Time (min)
0 15 30 45
Inte
rnal
AT
P c
on
c (
M)
6.0x10-12
6.5x10-12
7.0x10-12
7.5x10-12
8.0x10-12
8.5x10-12
ATP Efflux
Time (min)
0 15 30 45
Inte
rnal
ATP
con
c (
M)
0
5x10-12
10x10-12
15x10-12
20x10-12
25x10-12
30x10-12
35x10-12
Micrococcus luteus ATCC 10420 Salmonella enteritidis 20E1090 E. coli O157:H7
PE
PA1
EPE50
Control
Dissipation of membrane potential
ValinomycinBacteriocinNigericin
Cells
Glucose
Time (s)
DiSC3(5)Probe
Fluo
resc
ence
(au)
Micrococcus luteus ATCC 10420
E. coli untreated
DiSC3(5)
Glucose Nigericin Vancomycin
E. coli treated with lysozyme and EDTA
E. coli treated with EDTA only
Time (s)
Flu
ores
cen
ce (
au)
(au
)
E50-52
0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
850
800750700650600550
450400
350300250
150200
10050
0.1
500
9501000
900
Dissipation of membrane potential
TIme (h)
0 2 4 6 8 10 12 14 16
A59
5
0.0
0.2
0.4
0.6
0.8
TIme (h)
0 2 4 6 8 10 12 14 16
A59
5
0.0
0.2
0.4
0.6
0.8
TIme (h)
0 2 4 6 8 10 12 14 16
A59
5
0.0
0.2
0.4
0.6
0.8
Micrococcus luteus ATCC 10420 Salmonella enteritidis 20E1090 E. coli O157:H7
PE
PA1
EP
E50
Control
Inhibition pattern of target bacteria by wildtype and hybrid bacteriocins
Tiwari et al. 2015. Applied and Environmental Microbiology 81: 1661-1667.
Bacteriocins in our laboratory
Bacteriocins
CSIR
Plantaricin LD
1
Enterocin LD
3
Pediocin LB44Wisellicin LM85
HTP screening
Hybrid bacte
riocin
s
Halocin HA1, 3
IUSSTF DBT
DST
UGC
Halocin HA3
Mode of ActionICMR
Research Group
NaveenPhD completed
AabhaPhD completed
VijayPhD Student
ManojProject Fellow
MSc Dissertation
1.Aabha
2.Komal
3.Gitika
4.Anu
5.Gita
6.Parul
7.Nidhi
8.Karishma
9.Monica
10.Nandita,
11.Jyoti
12.Ritu
13.Bhawana
14.Sonia
15.Pooja
RamanjeetPhD Student
PoonamProject Fellow
Acknowledgements
Prof. Sheela SrivastavDepartment of GeneticsUniversity of Delhi South Campus, New Delhi
Prof. Michael L. ChikindasSchool of Environmental and Biological SciencesRutgers State University, New Jersey
CSIRICMR
Felicitated for Indo-US Research Fellow by IUSSTF, New Delhi.
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