Researchers have detailed the complete molecular architecture of the phage DEV, which specifically targets and destroys Pseudomonas aeruginosa bacteria. This bacterium is known to cause opportunistic infections in cystic fibrosis and various other illnesses. DEV is a key component of a pioneering phage mixture designed to eliminate P. aeruginosa infections in early-stage research. Viruses that attack bacteria, known as phages, represent the most prevalent biological entities on Earth and are being increasingly utilized as treatments to combat antibiotic-resistant bacterial infections.
Bacterial viruses, or phages, are the most prevalent form of life on Earth. A recent straightforward study that looked at 92 showerheads and 36 toothbrushes from bathrooms in the U.S. identified over 600 different types of bacteriophages. In fact, a single teaspoon of coastal seawater contains around 50 million phages.
Although phages often go unnoticed, they are harmless to humans. Instead, these viruses are becoming more recognized as potential treatments to eliminate harmful bacteria, especially those that are resistant to standard antibiotics.
In a publication found in the journal Nature Communications, Gino Cingolani, Ph.D., from the University of Alabama at Birmingham, alongside Federica Briani, Ph.D., from the Università degli Studi di Milano in Italy, presented the complete molecular structure of phage DEV. DEV not only infects but also destroys Pseudomonas aeruginosa, which is a significant opportunistic pathogen in cystic fibrosis and numerous other conditions. This phage is a part of an experimental cocktail that aims to address P. aeruginosa infections in preliminary studies.
A unique characteristic of DEV is the presence of a 3,398-amino acid virion-associated RNA polymerase, which is released into the bacterium during the infection process. Cingolani and Briani’s findings unexpectedly indicated that this RNA polymerase is a component of a mechanism that ejects the phage’s DNA from its head once it attaches to a Pseudomonas bacterium and penetrates its outer and inner membranes using its tail structure.
“We believe that the design principles of the DEV DNA ejection system are likely shared among all Schitoviridae phages,” noted Cingolani. “By October 2024, more than 220 Schitoviridae genomes have been sequenced and can be accessed in public databases. Many of these genomes are not fully annotated, and several of their open-reading frames lack defined functions. Our research sets the stage for easier identification of structural elements when discovering new Schitoviridae phages.”
The Schitoviridae family of phages represents some of the least studied bacterial viruses and are increasingly being used in phage therapy. “We are employing structural biology to unveil the basic building blocks and outline gene products,” explained Cingolani. “This understanding is critical when dealing with rapidly evolving amino acid sequences that are challenging for conventional phylogenetic analysis.”
The researchers employed cryo-electron microscopy, biochemical techniques, and genetic modifications to detail the entire molecular structure of DEV, which has a DNA genome composed of 91 open-reading frames, including the large virion-associated RNA polymerase. “This virion-associated RNA polymerase is part of a three-gene operon present across all Schitoviridae genomes we’ve examined,” Cingolani added. “We suggest that these three proteins are released into the host to create a mechanism that ejects the genome through the bacterial cell envelope.”
The structure of phages like DEV resembles a tiny version of Neil Armstrong’s lunar lander from 1969, featuring a large head or capsid that encases the genome and long, leg-like fibers that anchor the phage onto the surface of bacteria before it infects the living bacterial cell.
The researchers also mapped out the structures of all proteins in the capsid and tail of DEV involved in attaching to the host. Their genetic experiments established that the long tail fibers of DEV are crucial for infecting P. aeruginosa, but are unnecessary for infecting P. aeruginosa mutants that lack the O-antigen in their surface lipopolysaccharide. Generally, viruses latch onto various cell surface molecules as the crucial first step of infection.
Despite providing several snapshots of the phage’s structure, the researchers do not have a comprehensive understanding of the DEV infection process. They have conceptualized three phases of this infection procedure.
In the first phase, as a solitary DEV phage moves independently, its flexible long tail fibers adjust to enhance the likelihood of contacting a lipopolysaccharide on Pseudomonas. After making initial contact, all five fibers secure themselves to hold the phage closely against the surface of the bacterium.
The second phase involves a short tail fiber functioning as a tail plug that contacts a secondary receptor on the Pseudomonas, triggering a mechanical signal that releases this tail plug.
Up to this moment, three proteins—gp73, gp72, and gp71—have been stored inside the phage head near its tail, but their shapes will change drastically upon exiting the phage. In the third phase, once the plug is out, these three proteins are ejected into the bacterial cell envelope. The leading protein, gp73, reshapes itself to create a pore in the outer membrane. Beneath this, gp72 transforms into a hollow structure that stretches across the Pseudomonas’s periplasm—the area between the outer and inner membranes of the bacteria. Lastly, gp71 crosses the inner membrane and alters into an extensive RNA polymerase motor within the bacterial cytoplasm to pull the phage DNA through the hollow channels formed by gp73 and gp72 and into the Pseudomonas cell.
Cingolani, a professor in the Department of Biochemistry and Molecular Genetics, has recently taken the reins of the new Center for Integrative Structural Biology at UAB, which was approved by the University of Alabama System Board of Trustees earlier this summer. This center will facilitate UAB researchers in investigating three-dimensional structures of biological macromolecules, like proteins and nucleic acids, to understand their functions and mechanisms.
Integrative structural biology aims to visualize a comprehensive view of how macromolecules operate, exploiting different approaches to examine molecular structures and their interactions. The UAB Center for Integrative Structural Biology will primarily focus on biological issues related to infection, inflammation, immunity, cancer, and neurodegeneration.
