US researchers have developed a new powerful microscopy technique and used it to show proteins killing bacteria in real time, thus revealing the deadly workings of antimicrobial peptides (AMPs), naturally occurring proteins that scientists are pursuing as a new approach to treating bacterial infections.
Led by Massachusetts Institute of Technology (MIT) Professor Angela Belcher, the researchers modified an existing technology called high-speed atomic force microscopy (AFM) to allow them to see the onset of bacterial cell death induced by AMPs in real time, on a cell by cell basis. They have written about their work in a paper published in the 14 March online edition of Nature Nanotechnology.
For the last twenty years, scientists have been looking for a way of treating bacterial infections by getting naturally occuring AMPs to kill them. Most AMPs kill bacteria by punching holes in their cell membranes, thus destroying the delicate equilibrium they maintain between themselves and their environment. Other AMPs destroy bacteria by getting inside them and interfering with their internal cellular machinery.
There has been a lot of interest in developing AMP-based drugs to replace antibiotics, but as yet none have been approved for market.
Belcher, who is MIT’s Germeshausen Professor of Materials Science and Engineering and Biological Engineering and a member of the MIT Koch Institute for Integrative Cancer Research, told the press that the new type of high-speed atomic force microscopy (AFM) could help perfect the technique of using AMP to kill bacteria, and also aid better understanding of how cells respond to viral infections and other drugs.
One area that could be vital is to understand how bacteria become resistant to AMPs (until a few years ago scientists thought they couldn’t, but recent research has revealed they can).
Paul Hansma, a physics professor at the University of California at Santa Barbara (UCSB), has been working on AFM for 20 years. He was not involved in the study and suggested the new technique could be used to study cell death in mammals, for instance to see what happens when nerve cells die in Alzheimer’s patients.
“This paper is a highly significant advance in the state-of-the-art imaging of cellular processes,” said Hansma.
Lead author Dr Georg Fantner, a postdoctoral associate in Belcher’s lab who had been working on high-speed AFM at UCSB, brought his experience to MIT. While he and other scientists had developed new high-speed AFM techniques, they hadn’t optimized them to study living cells: this then became the new focus of the MIT team.
Invented in 1986, AFM is a type of scanning probe microscopy (SPM), a set of related technologies for imaging and measuring surfaces down to the level of molecules and groups of atoms. Electron microscopy also works at this scale, but it requires a vacuum so you can’t use it with living samples.
At the centre of AFM is a mechanical technology that “feels” the surface to be examined with an extremely sharp tip (3 to 50 nanometers radius of curvature) that is mounted on a flexible cantilever so the tip can follow the contours of the surface.
As the tip moves across the surface of the object being investigated, it undergoes different forces of interaction with the surface which affect the movement of the cantilever. These tiny movements are fed to selective sensors and can be used as the basis for seeing the shape and investigating other properties of the surface.
However, conventional AFM technology takes several minutes to produce one image, making it unsuitable for looking at a series of events in succession.
For this study, the MIT researchers used a cantilever that was about 1,000 times smaller than the one used in coventional AFM. This enabled them to increase the imaging speed without harming the bacteria. Another factor that helped keep the bacteria alive was that they performed the measurements in a liquid.
By using the new AFM set up the MIT team was able to take images every 13 seconds for several minutes while they looked at bacteria treated with an AMP called CM15.
They wrote that:
“The increased time resolution (13 s per image) allows the characterization of the initial stages of the action of the antimicrobial peptide CM15 on individual Escherichia coli cells with nanometre resolution.”
They found that AMP-induced cell death appeared to occur in two stages: a short incubation period followed by “a more rapid execution phase”.
What surprised them was that the the incubation phase took anything from 13 to 80 seconds to complete.
Co-author Roberto Barbero, a graduate student in the MIT team, told the press that:
“Not all of the cells started dying at the exact same time, even though they were genetically identical and were exposed to the peptide at the same time.”
An Erwin-Schrodinger Fellowship, the National Institutes of Health, Army Research Office and Austrian Research Promotion Agency provided funds for the study.
“Kinetics of antimicrobial peptide activity measured on individual bacterial cells using high-speed atomic force microscopy.”
Georg E. Fantner, Roberto J. Barbero, David S. Gray & Angela M. Belcher.
DOI:10.1038/nnano.2010.29
Source: MIT, SPM website.
Written by: Catharine Paddock, PhD