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Antibiotics Resistance: Lessons
learned from Bacterial Cell
Division
Richa Priyadarshini
Assistant Professor
Dept. of Life Sciences
School of Natural Sciences
Shiv Nadar University
Urbanization?
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:
1. Study of bacterial cell wall biogenesis
using model organism (E. coli and
Caulobacter)
2. Use on metagenomics
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.
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
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
1
Amidase
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
Ami-mCherry
Caulobacter has only one
annotated amidase
Cell envelope and divisome components
Divisome
Typas et al. Microbiol. Mol.
Biol. Rev. 2006
Amidase localizes to mid-cell after FtsN
0min
FtsN-YFP
Ami-mCherry
Merge
15min
30min
45min
60min
75min
90min
What happens when amidase
activity is removed from the cell
Xylose inducible promoter system
No Xylose
Off
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
Urbanization?
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