Download Santosh Kumar Tiwari

Design of synthetic-hybrid bacteriocins from
enterocin E50-52 and pediocin PA-1
for therapeutic applications
Santosh Kumar Tiwari, PhD
Assistant Professor
Department of Genetics
Maharshi Dayanand University
Rohtak-124001, Haryana
Email: [email protected]
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 antibioticresistant 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
BACTIBASE dataset (version 2, July 2009)
small peptides
antimicrobial activity
Diversity
BACTIBASE dataset (version 2, July 2009)
Total bacteriocins :177
Gram-positive bacteria : 156 (113 from lactic acid bacteria)
Gram-negative bacteria : 18
Archaea domain : 3
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
The residues in red have positive net charge, blue are hydrophobic. Dha, dehydroalanine;
Dhb, dehydrobutyrine; Lan, lanthionine; Mla, methyllanthionine; S, thioether bridge
NATURE REVIEWS | MICROBIOLOGY VOLUME 4 | JULY 2006 | 531
Mode of action of bacteriocins
Design and Synthesis of Hybrid Bacteriocins
Pediocin PA-1
TTKNYGNGVCNSVNWCQCGNVWASCNLATGCAAWLCKLA
Enterocin E50-52
Design and Synthesis of Hybrid Bacteriocins
Methods for detection of antimicrobial activity
Producer
strain
Indicator
strain
Agar Well Diffusion Assay (AWDA)
Spot assay plate method
100
% inhibition of growth of indicator strain
120
% growth of indicator strain
100
80
60
40
20
0
80
60
40
20
0
50
Control
Treated
25
12.5 6.25 3.12 1.56 0.78 0.39 0.19 0.095
Amount of bacteriocin (l)
AU/ml OR MIC
Percentage inhibition of indicator strain
Minimum Inhibitory Concentration (MIC)
0.4
0.4
Enterocin E50-52
(B
)
Pediocin PA-1
0.3
Growth (A595)
Growth (A595)
0.3
0.2
0.1
0.2
0.1
0.0
0.0
0.00
1.56
3.12
6.25
12.5
25
50
100
200
0.00
1.56
3.12
Concentration ( M)
12.5
25
50
100
200
Concentration ( M)
0.4
0.4
EP(C
)
PE(D
)
0.3
Growth (A595)
0.3
Growth (A595)
6.25
0.2
0.2
0.1
0.1
0.0
0.0
0.00
1.56
3.12
6.25
12.5
25
Concentration ( M)
50
100
200
0.00
1.56
3.12
6.25
12.5
25
Concentration ( M)
50
100
200
Comparison of MIC of WT and hybrid bacteriocins
ATP Efflux
35x10-12
10x10-12
8.5x10-12
30x10-12
9x10-12
Control
8.0x10
25x10-12
20x10-12
15x10-12
10x10-12
Internal ATP conc (M)
Internal ATP conc ( M)
Internal ATP conc ( M)
-12
7.5x10-12
7.0x10
PA1
-12
E50
EP
6.5x10
PE
8x10-12
7x10-12
6x10-12
5x10-12
-12
5x10-12
4x10-12
3x10-12
6.0x10-12
0
0
15
30
45
Time (min)
Micrococcus luteus ATCC 10420
0
15
30
45
Time (min)
Salmonella enteritidis 20E1090
0
15
30
TIme (min)
E. coli O157:H7
45
DiSC3(5)
Probe
150
0.1
52.1
104.1
156.1
208.1
260.1
312.1
364.1
416.1
468.1
520.1
572.1
624.1
676.1
728.1
780.1
832.1
884.1
936.1
988.1
1040.1
1092.1
1144.1
1196.1
1248.1
1300.1
1352.1
1404.1
1456.1
1508.1
1560.1
1612.1
Fluorescence (au)
Dissipation of membrane potential
Micrococcus luteus ATCC 10420
250
200
Cells
E50-52
50
Bacteriocin
Time (s)
EP
PE
100
Nigericin
Valinomycin
Glucose
0
Dissipation of membrane potential
E. coli untreated
Fluorescence (au)
(au)
1000
950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
0.1
E. coli treated with
EDTA only
E. coli treated with
lysozyme and EDTA
Vancomycin
Glucose
Nigericin E50-52
DiSC3(5)
0.0
100
200
300
400
500
600
700
800
900
Time (s)
1000
1100
1200
1300
1400
1500
Inhibition pattern of target bacteria by wildtype and
hybrid bacteriocins
0.8
0.8
0.8
Control
PA1
0.6
0.6
E50
0.6
0.4
A595
A595
A595
EP
0.4
0.4
PE
0.2
0.2
0.2
0.0
0.0
0
2
4
6
8
10
12
14
TIme (h)
Micrococcus luteus ATCC 10420
16
0.0
0
2
4
6
8
10
12
14
TIme (h)
Salmonella enteritidis 20E1090
16
0
2
4
6
8
10
TIme (h)
E. coli O157:H7
12
14
16
Tiwari et al. 2015. Applied and Environmental Microbiology 81: 1661-1667.
Bacteriocins in our laboratory
UGC
CSIR
ICMR
Bacteriocins
Pediocin LB44
Wisellicin LM85
DST
Mode of Action
IUSSTF
DBT
Research Group
MSc Dissertation
Naveen
PhD completed
Aabha
PhD completed
Ramanjeet
PhD Student
Vijay
PhD Student
1.
Aabha
2.
Komal
3.
Gitika
4.
Anu
5.
Gita
6.
Parul
7.
Nidhi
8.
Karishma
9.
Monica
10. Nandita,
Manoj
Project Fellow
11. Jyoti
12. Ritu
13. Bhawana
Poonam
Project Fellow
14. Sonia
15. Pooja
Acknowledgements
Prof. Sheela Srivastav
Department of Genetics
University of Delhi South Campus, New Delhi
Prof. Michael L. Chikindas
School of Environmental and Biological Sciences
Rutgers State University, New Jersey
ICMR
CSIR
Felicitated for Indo-US Research Fellow by IUSSTF, New Delhi.