antibiotics resistance: lessons learned from bacterial cell division richa priyadarshini assistant...
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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
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Urbanization?Urbanization?
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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.
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Study of Antibiotic ResistanceStudy 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
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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
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Why study bacterial cell shape?Why study bacterial cell shape?
Kruse et al. Mol. Micro. 2004Priyadarshini et al. J. Bac. 2006Varma et al. J. Bac. 2004
Cell wall provided shape to the bacterial cell
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Bacterial Cell WallBacterial Cell Wall
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Targets of cell wall-active antibiotics .
McCallum N et al. Antimicrob. Agents Chemother. 2011;55:1391-1402
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Conly J CMAJ 2002;167:885-891
Mechanism Leading to Antibiotic Resistance
Genetic mutations lead to: changes in binding proteins ribosomes membrane structure inactivating enzymes
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HH Lee et al. Nature 467, 82-85 (2010) doi:10.1038/nature09354
A population-based antibiotic-resistance mechanism in bacteria.
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Strategies for control of antibiotic Strategies for control of antibiotic resistanceresistance
Minimize the use of antibiotic Minimize the use of antibiotic
improving infection control
developing new antibiotics
Global policy for tracking the emergence of antibiotic resistance
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Holtje et al. MMBR Mar 1998
1
Cell wall modifying enzymes (Penicillin Binding Proteins)
PBP 2/ PBP 3
PBP 1A/ 1B/1C
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Holtje et al. MMBR Mar 1998
1
Cell wall modifying enzymes (Hydrolases)
Amidase
Endopeptidase PBP4 &PBP7
D,D-carboxypeptidae PBP5 & PBP6
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Hydrolases as antibiotic targetsHydrolases as antibiotic targets
MMBR Mar 1998
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MMBR Mar 1998
Penicillin (Beta-Lactams) Vancomycin
Hydrolases as antibiotic targets
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MMBR Mar 1998
Penicillin Vancomycin
Bacitracin
How is the activity of cell wall How is the activity of cell wall hydrolases regulated? hydrolases regulated?
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MMBR Mar 1998
Penicillin Vancomycin
Bacitracinfosfomycin Cycloserine
How is the activity of cell wall How is the activity of cell wall hydrolases regulated? hydrolases regulated?
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Caulobacter crescentusCaulobacter crescentus: elegant : elegant model systemmodel system
• Dimorphic
•Asymmetric cell division
•Genome is sequenced and genetic tools are available
•Easy to obtain synchronized cell population
Courtesy Yves Brun
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1
Amidase
E. coli has 3 amidases: AmiA, AmiB and AmiC
Cell wall modifying enzymes (Hydrolases)
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Phenotype of amidase mutantsPhenotype of amidase mutants
Cell Division
Amidase Mutants
Cell Separation
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What is the role of amidase in What is the role of amidase in Caulobacter cells?Caulobacter cells?
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Localization of amidase in Localization of amidase in CaulobacterCaulobacter
Ami-mCherryCaulobacter has only one annotated amidase
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Cell envelope and divisome componentsCell envelope and divisome components
Typas et al. Microbiol. Mol. Biol. Rev. 2006
Divisome
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Amidase localizes to mid-cell after FtsNAmidase localizes to mid-cell after FtsN
FtsN-YFP
Ami-mCherry
Merge
0min 15min 30min 45min 60min 75min 90min
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What happens when amidase What happens when amidase activity is removed from the cellactivity is removed from the cell
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Xylose inducible promoter system
No Xylose
Xylose
Off
ON-Xylose promoter
-Gene of interest
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Amidase depletion causes cell division Amidase depletion causes cell division defects in defects in CaulobacterCaulobacter
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Effect of over-activity of amidase on Effect of over-activity of amidase on Caulobacter cellsCaulobacter cells
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0.2% glu
6hr 7hr 8hr 10hr
0.3% xyl
Overexpression of amidase causes cell chaining and filamentation
Amidase overexpression
Time after induction
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Overexpression of amidase causes Overexpression of amidase causes cell chaining cell chaining
Amidase overexpressionTime lapse at 30°C with xylose
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Balance between cell wall degradation and synthesis is Balance between cell wall degradation and synthesis is disturbed in amidase overexpression strainsdisturbed in amidase overexpression strains
PBP3
Amidase
Cell wall
Wild Type
Amidase overexpression
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Overexpression of amidase in absence of Overexpression of amidase in absence of PBP3 causes lysis PBP3 causes lysis
PBP3 depletion (Temperature sensitive mutant) and amidase overexpression. Amidase tagged with mCherry
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SummarySummary
Amidase is essential for viability in Amidase is essential for viability in Caulobacter Caulobacter
Amidase depletion and overexpression causes Amidase depletion and overexpression causes cell division defects and lysis of cellscell division defects and lysis of cells
Amidase overexpression disturbs the delicate Amidase overexpression disturbs the delicate balance between synthesis and hydrolysis of the balance between synthesis and hydrolysis of the septal peptidoglycanseptal peptidoglycan
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Role of environment in Role of environment in antibiotic resistanceantibiotic resistance
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Ecology of AntibioticsEcology of Antibiotics
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Bacteria found in soil Bacteria found in soil contain antibiotic contain antibiotic resistance genesresistance genes
D'Costa et al., Science. 311 (5759): 374-377
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Spread of antibiotic resistanceSpread of antibiotic resistance
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Antibiotics are communication Antibiotics are communication molecules of bacteriamolecules of bacteria
www.sciencedaily.com
Humans communicate with poetry, bacteria….
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Metagenomic Approaches to Metagenomic Approaches to Combat antibiotic resistanceCombat 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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Urbanization?Urbanization?
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AcknowledgementsAcknowledgements
Lab members:Lab members:
Ph.D. Students- Deepika Chauhan and Amrita DubeyPh.D. Students- Deepika Chauhan and Amrita Dubey
Vinita Tomar (Lab Assistant)Vinita Tomar (Lab Assistant)
Dept. Life SciencesDept. Life Sciences
Dr. Rupamanjari Ghosh (Director SONS)Dr. Rupamanjari Ghosh (Director SONS)
Shiv Nadar UniversityShiv Nadar University
Duke UniversityDuke University
CollaboratorsCollaborators
Christine Jacobs-Wagner (Yale University)Christine Jacobs-Wagner (Yale University)
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School of Natural SciencesSchool of Natural Sciences
Environmental and Natural Resources ManagementEnvironmental and Natural Resources Management
Jyoti Sharma:http://snu.edu.in/naturalsciences/jyoti_kumar_sharma_profile.aspx
Life SciencesLife Sciences
Shailja Singh: Shailja Singh: http://snu.edu.in/naturalsciences/Shailja_Singh_profile.aspxhttp://snu.edu.in/naturalsciences/Shailja_Singh_profile.aspx
Seema Sherawat : Seema Sherawat : http://snu.edu.in/naturalsciences/Seema_Sehrawat_profile.aspxhttp://snu.edu.in/naturalsciences/Seema_Sehrawat_profile.aspx
Anindita Chakrabarty: http://snu.edu.in/naturalsciences/anindita_chakrabarty.aspx
Richa Priyadarshini:Richa Priyadarshini:http://snu.edu.in/naturalsciences/Richa_Priyadarshini_profile.aspxhttp://snu.edu.in/naturalsciences/Richa_Priyadarshini_profile.aspx
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School of Natural SciencesSchool of Natural Sciences
Dept. of ChemistryDept. of ChemistrySubhabrata 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