Super-res microscopy method reveals cause of chronic infections

Researchers at the University of California, Berkeley, using fluorescent labeling and employing super-resolution microscopy, discovered how many bacterial diseases attack, including cholera, lung infections in cystic fibrosis patients, and chronic sinusitis.

The imaging method enabled the researchers to examine the structure of bacterial biofilms, as well as identify genetic targets for potential drugs that could break up the bacterial community and expose the bugs to the killing power of antibiotics.

“In their natural habitat, 99.9 percent of all bacteria live as a community and attach to surfaces as biofilms; according to the National Institutes of Health, 80 percent of all infections in humans are related to biofilms,” explains lead researcher Veysel Berk, a postdoctoral fellow in the Department of Physics and the California Institute for Quantitative Biosciences (QB3) at UC Berkeley.

Combining super-resolution microscopy with Berk's fluorescent labeling method, which allows continuous labeling of growing and dividing cells in culture, biologists will be able to record stop-motion video of “how bacteria build their castles,” he says.

3D reconstruction of the bacterial biofilm made by cholera bacteria
3D reconstruction of the bacterial biofilm made by cholera bacteria. Bacterial cells (blue) attach to surfaces with a glue-like protein (green) and cement themselves together with another protein (gray). The bacterial clusters then cover themselves with a protective shell (red) made of proteins and sugar molecules. (Image courtesy of Veysel Berk)

“This work has led to new insights into the development of these complex structures and will no doubt pave the way to new approaches to fighting infectious disease and also bacteriological applications in environmental and industrial settings,” says Steven Chu, a former UC Berkeley professor of physics and of molecular and cell biology and former director of the Lawrence Berkeley National Laboratory (LBNL).

To study a biofilm formed by cholera bacteria (Vibrio cholerae), Berk built his own super-resolution microscope in the basement of UC Berkeley’s Stanley Hall based on a 2007 design by coauthor Xiaowei Zhuang, Chu’s former post-doctoral student who is now a professor at Harvard University (Cambridge, MA). To actually see these cells as they divided to form “castles,” Berk devised a new technique called continuous immunostaining that allowed him to track four separate target molecules by means of four separate fluorescent dyes.

Berk discovered that, over a six-hour period, a single bacterium laid down a glue to attach itself to a surface, then divided into daughter cells, making certain to cement each daughter to itself before splitting in two. The daughters continued to divide until they formed a cluster, at which point the bacteria secreted a protein that encased the cluster like the shell of a building. The clusters are separated by microchannels that may allow nutrients in and waste out, he explains.

“If we can find a drug to get rid of the glue protein, we can move the building as a whole. Or if we can get rid of the cement protein, we can dissolve everything and collapse the building, providing antibiotic access,” says Berk. “These can be targets for site-specific, antibiotic medicines in the future.”

He suspected that super-resolution microscopy--which obtains 20 nm resolution--could reveal the unknown structure of biofilms by highlighting only part of the image at a time using photo-switchable probes and compiling thousands of images into a single snapshot, which takes only a few minutes to compile.

Then, Berk and his team had to figure out how to label the cells with fluorescent dyes to continuously monitor their growth and division. So, he suspected that a critically balanced concentration of fluorescent stain – low enough to prevent background, but high enough to have efficient staining – would work just as well and eliminate the need to flush out excess dye for fear it would create a background glow.

“The classical approach is first staining, then destaining, then taking only a single snapshot,” explains Berk. “We found a way to do staining and keep all the fluorescent probes inside the solution while we do the imaging, so we can continuously monitor everything, starting from a single cell all the way to a mature biofilm. Instead of one snapshot, we are recording a whole movie.”

The team's findings appear in the July 13 issue of Science; for more information, please visit http://www.sciencemag.org/content/337/6091/236.abstract.

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