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Chemistry ChemIntra

Anastasia Armitage

FtsZ localisation as a marker for identifying the action of novel antibacterials

microscopy image of escherichia coli cells

National Institute of Allergy and Infectious Diseases

What is FtsZ?

FtsZ is a protein that plays an essential role in cell division in most bacteria. It is involved in the formation of the Z ring, which constricts to split the bacterial cell into two.

Changes in the expression and/or localisation of FtsZ could show how bacterial cell division is being affected, and so could help determine the mode of action of antibacterials.

Project aim: to study how FtsZ can be used as a marker to elucidate the action of antibacterials

Methodology

Antibiotics to test:

PROTEIN SYNTHESIS INHIBITORS

CELL WALL SYNTHESIS INHIBITORS

NUCLEIC ACID SYNTHESIS INHIBITORS

Apramycin

Ampicillin

Ciprofloxacin

Gentamicin

Cefotaxime

Rifampicin

Kanamycin

Flucloxacillin

  

Spectinomycin

Vancomycin

  

Tetracycline

    

Chloramphenicol

    

Streptomycin

    

Viable cell counts:

We carried out time course viable cell counts to optimise the incubation, taking samples out at 0, 15, 30, and 60 minutes. An antibiotic from each class was tested, along with a control sample. Our results show that there is no significant cell death observed after 1 hour, so we decided on this incubation time.

graph showing viable cell counts for control, rifampicin, ampicillin, and chloramphenicol-treated samples at different incubation times

Treatment + microscopy

  • An overnight culture of Escherichia coli K12 FtsZ-mNeon, which produces a chromosomally-encoded FtsZ-GFP fusion protein, was diluted 1:20 in LB and grown to mid-exponential phase
  • Bacteria were incubated with sub-MIC concentrations of 5 antibiotics (+ control) for 1 hour
  • Added sample to microscope slides with agar pads
  • Samples were imaged with confocal microscopy to observe the number of Z rings and cell length
Initial results:

confocal microscopy image of e. coli k12 ftsz-mneon cells with no treatment

confocal microscopy image of e. coli k12 ftsz-mneon cells treated with rifampicin, cefotaxime and kanamycin

Initial results show that treatment with antibiotics causes cells to elongate and form multiple Z rings, compared with the control cells which remained short with a single Z ring.

Next steps

  • Repeat the experiment for the rest of the antibiotics
  • Use a stain to make the bacterial cell membrane appear red under the microscope
  • Test a new antibiotic with an unknown mode of action

    image of a cell stained with FM 4-64

    Zupan et al., 2013, PNAS

Email: Anastasiya.Armitage@warwick.ac.uk

LinkedIn:

https://www.linkedin.com/in/anastasiya-armitage-043b29202/ 

Project supervisors:

Dr. Antonia Sagona

Dr. Jessica Lewis