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

2. Urbanization?

3. 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.

4. 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

5. 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

6. 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

7. Bacterial Cell Wall

8. 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:

9. 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:

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

11. 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

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

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

14. Hydrolases as antibiotic targets
MMBR Mar 1998

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

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

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

18. 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

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

20. Phenotype of amidase mutants
Cell Division
Cell Separation
Amidase Mutants

21. What is the role of amidase in Caulobacter cells?

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

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

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

25. What happens when amidase activity is removed from the cell

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

27. Amidase depletion causes cell division defects in Caulobacter

28. Effect of over-activity of amidase on Caulobacter cells

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

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

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

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

33. 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

34. Role of environment in antibiotic resistance

35. Ecology of Antibiotics

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

38. Spread of antibiotic resistance

39. Antibiotics are communication molecules of bacteria
Humans communicate with poetry, bacteria….

40. 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

41. Urbanization?

42. 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)

43. School of Natural Sciences
Environmental and Natural Resources Management
Jyoti Sharma:
Life Sciences
Shailja Singh:
Seema Sherawat :
Anindita Chakrabarty:
Richa Priyadarshini:

44. School of Natural Sciences
Dept. of Chemistry
Subhabrata Sen: Gouriprasanna Roy:
Bimlesh Lochab:
Parthapratim Munshi: