It’s a dog-eat-dog world out there, even for those that live on a microscopic scale. Bacteria, battling to survive against invaders, have devised various defense mechanisms over billions of years. In turn, phages — viruses that attack bacteria — have craftily come up with a few evasive maneuvers of their own.
“It’s an arms race,” says biophysicist Elizabeth Villa, a Howard Hughes Medical Institute (HHMI) Investigator at the University of California, San Diego (UC San Diego). “There’s very complex biology in the fight between bacteria and phages.”
In 2017, Villa and her collaborator Joe Pogliano discovered one of the more curious counter-defense strategiesexternal link, opens in a new tab, employed by a group of viruses called jumbo phages. When the phages enter bacterial cells, they assemble a special ‘nucleus-like’ shell around their viral DNA, thus preserving their ability to replicate and eventually take over the host bacterium.
“We saw a closed compartment made from a single layer of protein,” says Villa. However, the images obtained at that time were too fuzzy to determine the protein’s exact identity and overall shape.
But now, new research from Villa’s team, published August 3, 2022, in Nature, fills in those missing gaps. The nuclear shell, they discovered, consists primarily of a previously undescribed protein called chimallin, which forms a quadrangular mesh around the phage DNA.
Unique and useful
Jumbo phages occur in diverse environments, ranging from seawater to cheese. In the world of phages, they stand apart from all others — firstly for their size (with more 200 kilobase pairs, they’re at least three times bigger than the average phage), and secondly for their unique counter-defense systems.
“Most phages have specific proteins that inhibit a specific nuclease from the bacterial host,” says Villa’s postdoctoral fellow, Thomas Laughlin, who is co-first author on the new paper. “But the jumbo phage appears to make a giant barrier between any nuclease and its DNA.”
“Their basic cell biology is insanely weird,” adds Villa, who has been studying the phages for the past eight years.
Additionally, because “human cells and bacteria use many similar mechanisms to control viral infection,” understanding phage biology may one day lead to new treatments for drug-resistant bacterial infections and other useful clinical applications, says Philip Kranzuschexternal link, opens in a new tab, a microbiologist at Harvard Medical School who was not involved in the new study.
A funny fishnet
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 cryo-electron tomography (cryo-ET), a visualization technique that Laughlin describes as akin to a CT scan.
The benefit of cryo-ET is that it allows for the chimallin protein to be observed in its native state, says Laughlin, but the drawback is its limited resolution. To refine their view down to the atomic scale, they collaborated with Amar Deep and Kevin Corbettexternal link, opens in a new tab to purify a single phage nuclear shell and observe it in vitro using a related but different technique: cryo-electron microscopy (cryo-EM). They were then able to take the information gleaned and create a high-resolution structural model of chimallin.
The dual visualization approach was well-suited to the task at hand, says structural biologist and cryo-EM expert Eva Nogales, an HHMI Investigator at the University of California, Berkeley who was not involved with the new research. “It’s a truly beautiful example of the synergy of both methodologies.”
From their experiments, Villa’s team discovered that chimallin — a previously undescribed protein — comprises most of the nuclear shell. “We named it after the chimalli, a shield carried by ancient Aztec warriors,” explains Villa. “Because of its role in protecting the phage genome against host defenses.”
The nuclear shell forms rapidly once the jumbo phage invades a host bacterium, adopting a structure resembling a fishnet with a square mesh. “It’s unusual because normally in biology, at all scales, you see hexagons as it’s the closest stacking you can get,” says Villa, who points to honeycombs as the quintessential example. “It’s really hard to form a closed surface with squares.”
Laughlin, who was the first in her lab to observe chimallin’s unusual structure, “initially thought it was wrong.” But he verified his findings by doing a deep dive into the literature and doubling back to the original data. “It was surprising but, in hindsight, made a lot of sense,” he says.
As the phage replicates, its genome grows up to 100 times in volume. The square mesh offers the nuclear shell sufficient pliability to accommodate for this expansion. “It’s pretty cool,” says Villa. “Biology always finds a way.”
Her team is now studying what triggers the shell to form. They’re also working to identify which proteins, other than chimallin, allow for the selective transport of materials to and from the shell. One day, they hope to be able 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 Shellexternal link, opens in a new tab.” Nature. Published online August 3, 2022. doi: 10.1038/s41586-022-05013-4