It’s a dog-and-dog world out there, even for those who live on a microscopic scale. Bacteria, fighting for survival against invaders, have devised various defense mechanisms over billions of years. In turn, phages — viruses that attack bacteria — have surreptitiously devised a few evasive maneuvers of their own.
“It’s an arms race,” said biophysicist Elizabeth Villa, a researcher at the Howard Hughes Medical Institute (HHMI) at the University of California, San Diego (UC San Diego). “There is a very complex biology in the battle between bacteria and phages.”
In 2017, Villa and her collaborator Joe Pogliano discovered one of the more curious counter-defense strategies, used by a group of viruses called jumbo phages. When the phages invade bacterial cells, they assemble a special ‘core-like’ shell around their viral DNA, preserving their ability to replicate and eventually take over the host bacteria.
“We saw a closed compartment made of a single layer of protein,” Villa says. However, the images obtained at the time were too vague to determine the exact identity and overall shape of the protein.
But now, new research from Villa’s team, published August 3, 2022, in Nature, fills those missing gaps. The nuclear shell, they found, consists mainly of a previously undescribed protein called chimallin, which forms a quadrangular mesh around the phage DNA.
Unique and convenient
Jumbo phages are found in a variety of environments, from seawater to cheese. In the world of phages, they stand out from all others – firstly, because of their size (at more than 200 kilobase pairs, they are at least three times larger than the average phage), and secondly, because of their unique counter-defense systems.
“Most phages have specific proteins that inhibit a specific nuclease of the bacterial host,” said Villa’s postdoctoral fellow, Thomas Laughlin, who is co-first author of the new paper. “But the jumbo phage seems to form a giant barrier between a nuclease and its DNA.”
“Their basal cell biology is insanely weird,” adds Villa, who has been studying the phages for the past eight years.
In addition, because “human cells and bacteria use many similar mechanisms to control viral infections,” understanding phage biology may one day lead to new treatments for drug-resistant bacterial infections and other beneficial clinical applications, said Philip Kranzusch, a microbiologist at Harvard University. Medical School that was not involved in the new study.
A funny fishing net
To characterize the jumbo phage’s nuclear shell, Villa and her collaborators at UC San Diego infected the bacteria Pseudomonas chlororaphis with the phage 201phi2-1. They then observed the infected cells using cryoelectron tomography (cryo-ET), a visualization technique Laughlin describes as akin to a CT scan.
The advantage of cryo-ET is that the chimallin protein can be observed in its native state, Laughlin says, but the disadvantage is the limited resolution. To refine their vision down to the atomic scale, they teamed up with Amar Deep and Kevin Corbett to purify a single nuclear shell from a phage and observe it in vitro using a related but different technique: cryo-electron microscopy (cryo -EM). They were then able to use the collected information and create a high-resolution structural model of chimallin.
“We named it after the chimalli, a shield worn by ancient Aztec warriors because of its role in protecting the phage genome from the host’s defenses.”
Elizabeth Villa, HHMI researcher at the University of California, San Diego
The dual visualization approach was well suited to the task ahead, says structural biologist and cryo-EM expert Eva Nogales, an HHMI researcher at the University of California, Berkeley who was not involved in the new research. “It is a truly wonderful example of the synergy of both methods.”
From their experiments, Villa’s team found that chimallin — a previously undescribed protein — comprises most of the nuclear shell. “We named it after the chimallic, a shield carried by ancient Aztec warriors,” explains Villa. “Due to its role in protecting the phage genome from host defenses.”
The nuclear shell forms quickly once the jumbo phage invades a host bacteria and takes on a structure that resembles a square-mesh fishing net. “It’s unusual because normally in biology, at all scales, you see hexagons because it’s the densest stack you can get,” says Villa, citing honeycombs as the ultimate example. “It’s very difficult to form a closed surface with squares.”
Laughlin, who was the first in her lab to observe chimallin’s unusual structure, “at first thought it was wrong.” But he verified his findings by taking a deep dive into the literature and returning to the original data. “It was surprising, but in hindsight very logical,” he says.
As the phage replicates, its genome grows up to 100 times in volume. The square mesh size provides the nuclear shell with sufficient flexibility to accommodate this expansion. “It’s pretty cool,” Villa says. “Biology always finds a way.”
Her team is now studying what creates the shell. They are also trying to identify which proteins, besides chimallin, enable the selective transport of materials to and from the shell. One day they hope to create synthetic phages for use in antibacterial drug therapy.
As Villa says, “What we found is just the beginning of the story, rather than the end.”
Thomas G. Laughlin et al. “Architecture and Self-Assembly of the Jumbo Bacteriophage Nuclear Shell.” Nature. Published online Aug 3, 2022. doi: 10.1038/s41586-022-05013-4