Antibiotics Resistance: Lessons learned from Bacterial Cell

Division

Richa Priyadarshini

Assistant Professor

Dept. of Life Sciences

School of Natural Sciences

Shiv Nadar University

Urbanization?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 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

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?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

Bacterial Cell WallBacterial Cell Wall

Targets of cell wall-active antibiotics .

McCallum N et al. Antimicrob. Agents Chemother. 2011;55:1391-1402

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

HH Lee et al. Nature 467, 82-85 (2010) doi:10.1038/nature09354

A population-based antibiotic-resistance mechanism in bacteria.

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

Holtje et al. MMBR Mar 1998

1

Cell wall modifying enzymes (Penicillin Binding Proteins)

PBP 2/ PBP 3

PBP 1A/ 1B/1C

Holtje et al. MMBR Mar 1998

1

Cell wall modifying enzymes (Hydrolases)

Amidase

Endopeptidase PBP4 &PBP7

D,D-carboxypeptidae PBP5 & PBP6

Hydrolases as antibiotic targetsHydrolases as antibiotic targets

MMBR Mar 1998

MMBR Mar 1998

Penicillin (Beta-Lactams) Vancomycin

Hydrolases as antibiotic targets

MMBR Mar 1998

Penicillin Vancomycin

Bacitracin

How is the activity of cell wall How is the activity of cell wall hydrolases regulated? hydrolases regulated?

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?

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

1

Amidase

E. coli has 3 amidases: AmiA, AmiB and AmiC

Cell wall modifying enzymes (Hydrolases)

Phenotype of amidase mutantsPhenotype of amidase mutants

Cell Division

Amidase Mutants

Cell Separation

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

Localization of amidase in Localization of amidase in CaulobacterCaulobacter

Ami-mCherryCaulobacter has only one annotated amidase

Cell envelope and divisome componentsCell envelope and divisome components

Typas et al. Microbiol. Mol. Biol. Rev. 2006

Divisome

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

What happens when amidase What happens when amidase activity is removed from the cellactivity is removed from the cell

Xylose inducible promoter system

No Xylose

Xylose

Off

ON-Xylose promoter

-Gene of interest

Amidase depletion causes cell division Amidase depletion causes cell division defects in defects in CaulobacterCaulobacter

Effect of over-activity of amidase on Effect of over-activity of amidase on Caulobacter cellsCaulobacter cells

0.2% glu

6hr 7hr 8hr 10hr

0.3% xyl

Overexpression of amidase causes cell chaining and filamentation

Amidase overexpression

Time after induction

Overexpression of amidase causes Overexpression of amidase causes cell chaining cell chaining

Amidase overexpressionTime lapse at 30°C with xylose

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

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

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

Role of environment in Role of environment in antibiotic resistanceantibiotic resistance

Ecology of AntibioticsEcology of Antibiotics

Bacteria found in soil Bacteria found in soil contain antibiotic contain antibiotic resistance genesresistance genes

D'Costa et al., Science. 311 (5759): 374-377

Spread of antibiotic resistanceSpread of antibiotic resistance

Antibiotics are communication Antibiotics are communication molecules of bacteriamolecules of bacteria

www.sciencedaily.com

Humans communicate with poetry, bacteria….

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

Urbanization?Urbanization?

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)

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

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