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Kinetics of antimicrobial peptide activity
measured on individual bacterial cells using highspeed atomic force microscopy
Georg E. Fantner, Roberto J. Barbero, David S. Gray and Angela M. Belcher
Department of Materials Science and Engineering, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA, Department of
Biological Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, USA.
NATURE NANOTECHNOLOGY / 14 MARCH 2010
清華大學分子醫學研究所
張晃猷教授實驗室
許方瑜
2010.03.25
Emerging limitations of AFM when it comes to
biological samples
• Cells are too fragile to withstand the forces
administered from the cantilever.
• Some biological events progress more
swiftly than the cantilever.
• Scan size must be optimized to obtain
representative and informative images.
High-speed AFM
• Micro-fabricated cantilever
- a thousand times lighter than
conventional one
- integrated tips
• Advantages:
- increased imaging speed in tapping
mode
- keep the forces applied on cells low
Higher resonance frequency of small cantilever
for high-speed AFM
Antimicrobial peptides (AMPs)
• Bactericidal mechanism of
AMPs:
(www.dermagen.se/Technology/Action.aspx)
- membrane disruption
- targeting to intracellular components to
inhibit cell wall, nucleic acid or protein
synthesis
- naturally produced by many species,
such as insects, mammals,
amphibians…etc
• CM15
CM15 shows a-helical structure.
(PDB ID: 2JMY)
- synthetic peptide
- hybrid of cecropin A and bee venom
peptide mellitin
- contains 15 amino acid sequence:
KWKLFKKIGAVLKVL
1
3
2
Nature Reviews Microbiology 3, 238-250 (March 2005)
Cell disruption by CM15 monitored in real-time
• Variations of surface states were observed among bacterium 1 and 2.
Bacterium 2 resists CM15 until t = 78 s.
Simultaneous imaging of a population of E. coli treated
with CM15
• At about 12 min, some bacteria still remained unaffected. After about 30
minutes of treatment, all the bacteria cell surface became corrugated.
Morphological changes correlates with the viability state
of bacterial cells
• Bacteria were stained with LIVE/DEAD dye to differentiate live and
dead cells.
Bulk measurement of CM15 antimicrobial activity
Early-stage kinetics measured by high-speed AFM
Summary
• High-speed AFM with small cantilever allows
monitoring biological events in real-time possible
without artifacts from prolonged and complicated
sample processing.
• Simultaneous observation from a single to a
population of bacteria provides convincing
results.
Thank you for your attention.