By Phoebe Ingraham Renda
Photography by Rayni Shiring, University of Pittsburgh
James Conway, professor and chair, Department of Structural Biology, University of Pittsburgh School of Medicine, presents a three-millimeter grid similar to those used to hold vitrified phage capsid samples for analysis in a cryogenic electron microscope.
To use science to our benefit, we need to know the basics. For James Conway, professor of structural biology and chair of the Department of Structural Biology at the University of Pittsburgh School of Medicine, and his research team, answering a basic question like “how?” requires a deep dive—down to the atomic scale.
Holding a 3D-printed model of a bacteriophage (a virus that specifically infects bacteria), Conway prefaced the explanation of his team’s recent research discovery with a simple statement: “This is a basic science study.”
That statement captures a profound truth—his team’s findings lay the base for the next frontier in reliably using bacteriophages as biomedical tools, which have shown inconsistent success.
“It's easy to lose sight of the value of basic research because the payoff is often not immediate,” says Conway, as he underscored that many medical advances, such as antibiotics and genetic engineering, would not have been developed if their basic biological origin had not been discovered long before. “If people had not been studying bacteriophages in the ’50s and ’60s, we wouldn't have eventually discovered the CRISPR system.”
The CRISPR system, one of several antibacteriophage immune systems that bacteria have developed, was discovered in 1987. But it wasn’t until 26 years later, in 2013, that it was first used for genome editing. Its developers received a Nobel Prize in 2020. Now known as CRISPR/Cas9 technology, it is readily used to perform gene editing to treat gene-related illnesses, such as sickle cell disease.
In a similar manner, Conway says that understanding what regulates the size of bacteriophage capsids—the protein shell that holds the virus’s DNA—from a structural biology standpoint holds valuable down-the-road significance for biomedicine. It could be key to improving their therapeutic utility.
In a study published in Nature Communications on Nov. 23, Anna Belford (first author and molecular biophysics and structural biology PhD candidate), Conway (corresponding author) and their lab colleagues detailed, for the first time, how Pseudomonas aeruginosa phage D3—a virus that infects only P. aeruginosa bacteria—controls the size of its capsid.
Conway lab team members standing next to the cryogenic electron microscope inside Pitt's Biomedical Science Tower 3, featuring (left to right) Alexis Huet, staff researcher; James Conway, professor and chair; and Robert Duda, staff researcher, all in the Department of Structural Biology, School of Medicine, University of Pittsburgh. Lab members not pictured include Anna Belford, graduate student and lead author, and Josh Maurer, research technician.
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Bacteriophages, or simply “phages,” have two general anatomies, tailed and nontailed. Tail or not, both types of phages have an icosahedral capsid—akin to a soccer ball’s stitching pattern—that houses the virus’s DNA. These capsids, Conway explains, are all built from similar building blocks, like uniform Lego pieces, but capsid sizes somehow range from small (60 pieces) to massive (3,120 pieces) depending on the phage type.
To investigate the mechanisms governing capsid size, Conway’s team characterized P. aeruginosa phage D3 capsid assembly by structurally analyzing its procapsid (pre-DNA packaging) and mature capsid (post-DNA packaging) protein structures using cryogenic electron microscopy.
“Turns out that there’s a part that we’ve not seen in detail before that gets cleaved off after procapsid assembly,” says Conway. That part is called the assembly domain.
Their research shows, in exquisite detail, how assembly domain proteins form a scaffold that governs what capsid size is built around it. Different versions of those assembly domains appear to govern the size-specific conformations. These proteins restrict the ways capsid building blocks—called capsomers—can connect, forcing them into specific orientations. After the capsid has been built, a more extensive network of protein interactions solidifies the structure so that the assembly scaffold can be removed, creating the hollow capsid that’s ready for DNA packaging.
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Phages are already being used to treat some antibiotic-resistant bacterial infections, in what is called phage therapy. However, phage therapy is often used as a last resort and still involves trial and error because a lot of basic questions about how these phages function remain, says Conway. While the direct biomedical connection is not blatantly clear now, this aspect of capsid size regulation may have valuable implications for biomedicine, including improved phage design and application of bacteriophages as alternatives to antibiotics, as P. aeruginosa—the bacteria this phage infects—is a “high-priority pathogen” according to the World Health Organization. And the phage's structure and assembly pathway are both similar to that of the herpes virus, which affects humans.
“Understanding how these things work may help us engineer things like vaccines or other potential antibiotics and understand why current biomedical application attempts have failed,” says Conway. “Without structural information, you're shooting in the dark.”