We’re Outta Here
Novel imaging technique tracks individual bacterial cells as they leave their biofilm community
Heidi Opdyke
- Associate Dean of Marketing and Communications, MCS
- Email opdyke@andrew.cmu.edu
- Phone 412-268-9982
An innovative imaging technique developed at Carnegie Mellon University reveals, for the first time, single bacterial cells leaving their biofilm community. Watching the bacteria in real-time at high resolution affords unprecedented views that advance the understanding of how single cells in biofilms move and how biofilms disperse. The findings, published in PLOS Biology, provide fundamental insights into the mechanisms underlying how pathogens in biofilms spread.
Most bacteria spend much of their lives in multicellular communities called biofilms. Living in the biofilm allows bacteria to collectively acquire nutrients and resist threats, including antibiotics and chlorination. By some estimates, up to 70 percent of human bacterial infections are caused by biofilm-forming bacteria.
Although attached to surfaces, biofilms are not static. Many types, like those formed by Vibrio cholerae, undergo repeated rounds of biofilm formation and disassembly, allowing the newly free bacteria to roam.
“Being able to transition in and out of the biofilms is critical for bacteria to be able to spread between niches. It could be between some environmental locations or, more relevantly, it could be between hosts or infection sites,” said Drew Bridges, assistant professor in the Department of Biological Sciences.
Biofilm disassembly and dispersal play a key role in disease spread, but studying these processes with microscopy and related imaging techniques has been impossible. Until now.
“No one had been able to image biofilm dispersal with the sort of resolution that we were able to achieve,” Bridges said. “And it is because of FAP labeling technology.”
FAPs, short for fluorogen activating proteins, emit fluorescent light only when bound to a fluorogen, an otherwise non-fluorescent dye. They emit light in a region of the visible spectrum that is not commonly utilized — the far-red region. Far-red light is typically less toxic to living organisms and better for imaging through tissues.
FAPs are an ideal workaround for a common problem scientists face when trying to image biofilms. Traditional fluorescent proteins require oxygen to emit light. But in biofilms, the bacteria are so densely packed that oxygen becomes scarce, preventing the dyes from lighting up. Bridges said it was a challenge to do good microscopy without having probes that worked in biofilms.
“It was a problem that I figured we would just have to work around. And then, when I got to Carnegie Mellon, I learned about FAPs. And they’re the perfect alternative because their mechanism is very different from how other fluorescent proteins work. They're not sensitive to oxygen limitation,” Bridges said.
FAPs were developed at Carnegie Mellon in 2008. Since then, CMU researchers and collaborators have published more than 150 papers developing FAP technology for diverse biological applications. This study marks the first time FAPs have been used to image biofilms.
Working closely with project scientist and FAP expert Robert van de Weerd, the Bridges lab incorporated FAPs into the genome of the Vibrio cholerae bacteria. The scientists added malachite green-derived fluorogens to the growing bacterial colony, which bound to the FAPs and emitted far-red fluorescence. Using spinning-disc confocal microscopy, the team followed cells in V. cholerae biofilms as they moved, disassembled and dispersed.
The real-time, single-cell imaging revealed that the bacteria start dispersing from the edges, which wasn’t necessarily surprising. What did interest Bridges was seeing that a sub-population of cells, about 20-25%, stays behind and never leave. He’s investigating further to determine whether their staying is based on simply being trapped or if there’s something else going on.
The imaging also revealed the development of localized dynamic regions, or dispersal “hot spots,” where cells exhibited large outward displacements. They also observed that some cells in the biofilm’s periphery didn’t leave but instead compressed toward the biofilm core. Bridges’ hypothesis is that cells themselves are a major mechanical component in the biofilm, and, as they start to leave, the overall structure collapses.
Overall, Bridges said the findings suggest a model in which certain areas of biofilms become more fluid-like, enabling localized outward motion of cells even from the interior. At the same time, the more rigid cell groups undergo compression to fill newly unoccupied space. The Bridges lab is investigating how these localized differences in mechanical properties are established during biofilm development and dispersal. They also plan to apply the FAP labeling technology to other notorious biofilm formers.
This work was supported by NIH grant R00AI158939, a Shurl and Kay Curci Foundation grant, a Kaufman Foundation New Investigator Research Grant, a Damon Runyon Cancer Research Foundation Dale F. Frey Award for Breakthrough Scientists and startup funds from Carnegie Mellon University.
