Researchers at Pitt have produced the most detailed image to date of a bacteriophage–phage for short–that has allowed them to see for the first time the structural makeup of the part of the phage that directly attaches to its target Mycobacterium cell.
"Now you've got like a spec sheet for going in and designing phages so that they'll bind to different kinds of cells," said Graham Hatfull , the Eberly Family Professor of Biotechnology in the Kenneth P. Dietrich School of Arts and Sciences.
That's important because of what a phage, which is a kind of virus, does after it binds to a bacterial cell: it pierces a hole in the cell membrane and injects its own DNA turning the bacteria into a phage factory.
With antibiotic resistance on the rise, in some cases phages are the only option to fight off bacterial infections. They are, however, picky assassins; a particular kind of phage will typically attack just one strain of bacteria. The ability to engineer a phage to seek out and destroy a specific bacteria could be a medical game-changer.
The research was published in the journal Cell.
There are about 1031 phages on the planet–about one trillion for every grain of sand–and they have been evolving for billions of years. Despite all the variation this long history has given rise to, they almost all share similar components: a capsid, a tail tube, and a tail tip.
A phage capsid sits like a head atop the narrow tail tube. Researchers have been able to capture high-resolution imagery of phage capsids for a while now for a couple of reasons.
"First, it's gigantic and easy to find," said Krista Freeman , a research associate in Hatfull's lab. But the capsid has another advantage when it comes to cryo-electron microscopy, or cryo-EM, one of two imaging techniques used in this work. The capsid is comprised of 60 symmetrical parts that can be averaged together to boost the signal. Imaging a phage using cryo-EM actually entails stitching together a lot of images from different angles. Because of the symmetry, relatively few images are needed to assemble enough information to piece together an entire capsid.
The rest of the phage's body is quite small by comparison, and less symmetric.
"So you have to be more careful," Freeman said. "You have to find more particles, and do more hunting, and do more manual manipulations. It's much less automated than getting the big structure."
Phages are bundles of tangled, intertwined proteins that loop around and through the structure. They are less like a globe, with its information painted on the surface, and more like a sculpture of a flower–constructed out of Slinkies.In practice, this means it requires taking tens of thousands of images of phages, all oriented in different ways, to piece together a complete image.
With this massive amount of data, and a massive amount of computing power, Freeman was able to recreate the tail tube and, perhaps most tantalizingly, the tip of the tail, which binds to the bacteria. Currently, researchers do not know why a particular phage attacks a particular strain of bacteria. "The tip of the tail, that's the part that's recognizing the bacteria cell," Hatfull said. "We're especially interested in it for this reason."
Their high-definition images have allowed them to see structures that had previously only been resolved to a fuzzy grayscale, indicative of the density of electrons.
"Now you can show every molecular component in this thing," Freeman said. "And it's just breathtaking." It's also revealing structural information that researchers can explore to better understand the point of contact between a phage and its bacterial target.
"It was astonishing to find out how complex the tail tip structure is," she said. "It's an intricate assembly of 10 distinct proteins. There are countless intimate interactions between these that enable the overall structure to do the complicated job of finding, binding to, and infecting the bacterial host."
The images are made dynamic thanks to work done by Raphael Park and his colleagues at Scripps Research who used another kind of imaging, cryo-electron tomography (cryo-ET), which images phages bound to the bacterial cell, highlighting the entire phage and where it attaches to the bacterial cell surface.
In some images, the phage's DNA is plainly visible inside the capsid, in others, it has made its way through the cell wall of the bacteria. Between these two steps, "there are some subtle differences," in the structure of the phage Hatfull said. These may point to the mechanism by which the DNA is triggered to leave the capsid, or how it is transported through the tail tube.
"These are new insights," Hatfull said. "There are a lot of questions remaining." But Hatfull, Freeman, and researchers across the world can now start thinking seriously about beginning to engineer phages to recognize different bacteria.
"Before, we wouldn't have stood a chance. And now, doing this is going to become completely routine."
Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award R35GM131729 and by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award K99Al173544. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
