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: 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 BACTIBASE dataset (version 2, July 2009) Ribosomally synthesized small peptides antimicrobial activity

Diversity Total bacteriocins :177 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 Spot assay plate method Producer strain Indicator strain Agar Well Diffusion Assay (AWDA) Percentage inhibition of indicator strain AU/ml OR MIC

Minimum Inhibitory Concentration (MIC) Pediocin PA-1 (D) (B) (C) PE Enterocin E50-52 EP

Comparison of MIC of WT and hybrid bacteriocins

ATP Efflux Micrococcus luteus ATCC 10420 Control PA1 E50 EP PE Micrococcus luteus ATCC 10420 Salmonella enteritidis 20E1090 E. coli O157:H7

Dissipation of membrane potential Micrococcus luteus ATCC 10420 Valinomycin Bacteriocin Nigericin Cells Glucose Time (s) DiSC3(5) Probe Fluorescence (au)

Dissipation of membrane potential E. coli untreated DiSC3(5) Glucose Nigericin Vancomycin E. coli treated with lysozyme and EDTA EDTA only Time (s) Fluorescence (au) (au) E50-52 0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 850 800 750 700 650 600 550 450 400 350 300 250 150 200 100 50 0.1 500 950 1000 900

Inhibition pattern of target bacteria by wildtype and hybrid bacteriocins Control PA1 E50 EP PE Micrococcus luteus ATCC 10420 Salmonella enteritidis 20E1090 E. coli O157:H7

Tiwari et al. 2015. Applied and Environmental Microbiology 81: 1661-1667.

Bacteriocins in our laboratory UGC CSIR Plantaricin LD1 Enterocin LD3 Halocin HA1, 3 Bacteriocins Pediocin LB44 Wisellicin LM85 DST ICMR Mode of Action Hybrid bacteriocins HTP screening Halocin HA3 DBT IUSSTF

Research Group MSc Dissertation Aabha Komal Gitika Anu Gita Parul Nidhi Karishma Monica Nandita, Jyoti Ritu Bhawana Sonia Pooja Naveen PhD completed Aabha PhD completed Ramanjeet PhD Student Vijay PhD Student Manoj Project Fellow Poonam Project Fellow

Acknowledgements ICMR CSIR 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.