Design of synthetic-hybrid bacteriocins from enterocin E50-52 and pediocin PA-1 for therapeutic applications Santosh Kumar Tiwari, PhD Assistant Professor.

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: [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 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.