Antibiotics Resistance: Lessons learned from Bacterial Cell Division Richa Priyadarshini Assistant Professor Dept. of Life Sciences School of Natural Sciences Shiv Nadar University

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On September 18 this year the Obama Administration announced the White House National Strategy for Combating Antibiotic Resistant Bacteria (CARB). The strategy outlines bold steps to slow the public health threat of antibiotic resistant bacteria, including efforts to stimulate innovative research.

Study of Antibiotic Resistance Two Approaches towards studying antibiotic resistance: Study of bacterial cell wall biogenesis using model organism (E. coli and Caulobacter) Use on metagenomics

It's not in the open we feel comforted but in the shadows It's not in the open we feel comforted but in the shadows. … We can't feel at home with the infinite sky above and around us. Space must be cut off, shaped, defined, for us to inhabit. From cradle to coffin, it's enclosure that defines us. —Robert Morgan

Why study bacterial cell shape? Varma et al. J. Bac. 2004 Priyadarshini et al. J. Bac. 2006 Kruse et al. Mol. Micro. 2004 Cell wall provided shape to the bacterial cell

Bacterial Cell Wall

Targets of cell wall-active antibiotics. S. aureus peptidoglycan synthesis and targets of cell wall-active antibiotics. The inhibition of enzymatic reactions is indicated by blocked arrows; the inhibition of cell wall synthesis by the binding of antibiotics to peptidoglycan precursors is indicated by half-moon symbols; pentaglycine bridge cleavage by lysostaphin and membrane disruption/depolarization by daptomycin are indicated by arrows. (Adapted from reference 31 with the permission of the publisher.)‏ McCallum N et al. Antimicrob. Agents Chemother. 2011;55:1391-1402

Mechanism Leading to Antibiotic Resistance Genetic mutations lead to: changes in binding proteins ribosomes membrane structure inactivating enzymes Fig. 1: The main genetic mechanisms leading to antibiotic resistance are genetic mutation (single point mutations or major deletions or rearrangements), expression of a latent resistance gene and acquisition of genes or DNA segments with resistance determinants. Some of the genes are inherited, some emerge through random mutations in bacterial DNA and some are imported from other bacteria. These genetic changes code for changes in binding proteins (a), ribosomes (b), membrane structure (c) or inactivating enzymes (d). Adapted with permission fromScientific American (1998;March:46-53). Photo: Christine Kenney Conly J CMAJ 2002;167:885-891

A population-based antibiotic-resistance mechanism in bacteria. HH Lee et al. Nature 467, 82-85 (2010) doi:10.1038/nature09354

Strategies for control of antibiotic resistance Minimize the use of antibiotic improving infection control developing new antibiotics Global policy for tracking the emergence of antibiotic resistance

Cell wall modifying enzymes (Penicillin Binding Proteins) 1 PBP 2/ PBP 3 PBP 1A/ 1B/1C Holtje et al. MMBR Mar 1998

Cell wall modifying enzymes (Hydrolases) D,D-carboxypeptidae PBP5 & PBP6 Amidase 1 Endopeptidase PBP4 &PBP7 Holtje et al. MMBR Mar 1998

Hydrolases as antibiotic targets MMBR Mar 1998

Hydrolases as antibiotic targets Penicillin (Beta-Lactams) Vancomycin MMBR Mar 1998

How is the activity of cell wall hydrolases regulated? Penicillin Vancomycin Bacitracin MMBR Mar 1998

How is the activity of cell wall hydrolases regulated? Penicillin Vancomycin Bacitracin fosfomycin Cycloserine MMBR Mar 1998

Caulobacter crescentus: elegant model system Dimorphic Asymmetric cell division Genome is sequenced and genetic tools are available Easy to obtain synchronized cell population Courtesy Yves Brun

Cell wall modifying enzymes (Hydrolases) 1 Amidase E. coli has 3 amidases: AmiA, AmiB and AmiC

Phenotype of amidase mutants Cell Division Cell Separation Amidase Mutants

What is the role of amidase in Caulobacter cells?

Localization of amidase in Caulobacter Caulobacter has only one annotated amidase Ami-mCherry

Cell envelope and divisome components Typas et al. Microbiol. Mol. Biol. Rev. 2006

Amidase localizes to mid-cell after FtsN 0min 15min 30min 45min 60min 75min 90min FtsN-YFP Ami-mCherry Merge

What happens when amidase activity is removed from the cell

Xylose inducible promoter system Off No Xylose Xylose ON -Xylose promoter -Gene of interest

Amidase depletion causes cell division defects in Caulobacter

Effect of over-activity of amidase on Caulobacter cells

Overexpression of amidase causes cell chaining and filamentation Time after induction 6hr 7hr 8hr 10hr 0.2% glu 0.3% xyl Amidase overexpression

Overexpression of amidase causes cell chaining Amidase overexpression Time lapse at 30°C with xylose

Balance between cell wall degradation and synthesis is disturbed in amidase overexpression strains Wild Type Amidase overexpression PBP3 Amidase Cell wall

Overexpression of amidase in absence of PBP3 causes lysis PBP3 depletion (Temperature sensitive mutant) and amidase overexpression. Amidase tagged with mCherry

Summary Amidase is essential for viability in Caulobacter Amidase depletion and overexpression causes cell division defects and lysis of cells Amidase overexpression disturbs the delicate balance between synthesis and hydrolysis of the septal peptidoglycan

Role of environment in antibiotic resistance

Ecology of Antibiotics

Bacteria found in soil contain antibiotic resistance genes D'Costa et al., Science. 311 (5759): 374-377

Spread of antibiotic resistance

Antibiotics are communication molecules of bacteria Humans communicate with poetry, bacteria…. www.sciencedaily.com

Metagenomic Approaches to Combat antibiotic resistance metagenomic tools to understand the microbial composition of certain unique environments identify the presence of antibiotic resistance genes and pathways identify pathways and genes responsible for production of novel antimicrobial compounds

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Acknowledgements Lab members: Ph.D. Students- Deepika Chauhan and Amrita Dubey Vinita Tomar (Lab Assistant) Dept. Life Sciences Dr. Rupamanjari Ghosh (Director SONS) Shiv Nadar University Duke University Collaborators Christine Jacobs-Wagner (Yale University)

School of Natural Sciences Environmental and Natural Resources Management Jyoti Sharma: http://snu.edu.in/naturalsciences/jyoti_kumar_sharma_profile.aspx Life Sciences Shailja Singh: http://snu.edu.in/naturalsciences/Shailja_Singh_profile.aspx Seema Sherawat : http://snu.edu.in/naturalsciences/Seema_Sehrawat_profile.aspx Anindita Chakrabarty: http://snu.edu.in/naturalsciences/anindita_chakrabarty.aspx Richa Priyadarshini: http://snu.edu.in/naturalsciences/Richa_Priyadarshini_profile.aspx

School of Natural Sciences Dept. of Chemistry Subhabrata Sen: http://snu.edu.in/naturalsciences/Subhabrata_Sen_profile.aspx Gouriprasanna Roy: http://snu.edu.in/naturalsciences/gouriprasanna_roy_profile.aspx Bimlesh Lochab: http://snu.edu.in/naturalsciences/Bimlesh_Lochab_profile.aspx Parthapratim Munshi:   http://snu.edu.in/naturalsciences/parthapratim_munshi_profile.aspx