Structural biology reveals secrets of DEV phage infection

Viruses that infect bacteria are the most common biological entities on the planet. For example, a recent simple study of 92 shower heads and 36 toothbrushes from American bathrooms identified more than 600 types of bacterial viruses, commonly called bacteriophages or phages. A teaspoon of coastal seawater contains about 50 million phages.

Despite the fact that phages are practically undetected, they do not cause harm to humans. Instead, these viruses are becoming increasingly popular as biologics to kill pathogenic bacteria, especially those associated with antibiotic-resistant infections.

In a study published in the journal Natural communications, Gino Cingolani, PhD, of the University of Alabama at Birmingham, and Federica Briani, PhD, of the University of Milan, Milan, Italy, described the complete molecular structure of phage DEV. DEV infects and lyses Pseudomonas aeruginosa bacteria, opportunistic pathogens of cystic fibrosis and other diseases. DEV is part of an experimental phage cocktail developed to eradicate P. aeruginosa infection in preclinical studies.

A feature of DEV is the presence of a 3,398 amino acid virion-associated RNA polymerase within the capsid, which is released into the bacterium upon infection. Surprisingly, Cingolani and Briani’s study showed that virion-associated RNA polymerase is part of the genome ejection engine that pulls phage DNA out of its head after the phage has attached to and penetrated the surface of the Pseudomonas bacterium using its tail fibers. outer and inner cell membranes using a tail tube.

“We argue that the design principles of the DEV ejection device are conserved across phages of the Schitoviridae,” Cingolani said. “As of October 2024, more than 220 shield virus genomes have been sequenced and are available in a publicly accessible database. Because these genomes are largely unannotated and many open reading frames have unknown functions, our work paves the way for easy identification of structural components when a new Schitoviridae phage is discovered.”

The Schitoviridae family of phages “represent some of the most biologically understudied bacterial viruses that are increasingly being used in phage therapy,” Cingolani said. “We use structural biology to decipher the building blocks and map gene products. This is vital when the amino acid sequence is evolving too quickly for conventional phylogenetic analysis.”

The researchers used localized cryoelectron microscopy reconstruction, biochemical methods, and genetic knockouts to describe the complete molecular architecture of DEV, whose DNA genome has 91 open reading frames including a giant virion-associated RNA polymerase. “This vRNAP is part of a three-gene operon that is conserved across all Schitoviridae genomes that we analyzed,” Cingolani said. “We propose that these three proteins are introduced into the host to form a genome ejection engine that spans the cell envelope.”

The structure of DEV and many other phages resembles a miniature version of Neil Armstrong’s 1969 moonship, with a large head, or capsid, that contains the genome and fiber-like legs that support the phage as it lands on the surface of the bacteria, preparing to be killed. infect a living bacterial cell.

The researchers determined the structures of all DEV protein capsid factors and tail components involved in host attachment. Using genetic experiments, they showed that the long tail fibers of DEV are required for infection of P. aeruginosa, but are not required for infection of P. aeruginosa mutants that lack the O-antigen on the lipopolysaccharide surface. Typically, viruses attach to various cell surface molecules during the first stage of infection.

Although this study provides several still images of phage structure, researchers do not fully understand DEV infection. They envision three stages to this infection process.

In the first stage, as one DEV phage drifts in isolation, its flexible, long-tailed fibers oscillate, increasing the likelihood of contact with a surface Pseudomonas lipopolysaccharide molecule. After the first touch, all five fibers attach, tethering the phage perpendicular to the outer surface of the bacterium.

In the second stage, a short tail fiber, which also acts as a tail plug, touches the secondary Pseudomonas receptor and a mechanical signal releases the tail plug.

Until this point, three proteins called gp73, gp72 and gp71 were stored inside the phage head near its tail, their shape changing dramatically as they left the phage head. In the third stage, when the plug is removed, three proteins are released from the head into the bacterial cell wall. The lead protein gp73 changes its shape to form an outer membrane pore with a hollow center. Below this, gp72 folds back into a hollow tube enclosing the Pseudomonas periplasm—the space between the outer membrane of the bacterium and its inner membrane. Finally, gp71 crosses the inner membrane and becomes a large RNA polymerase motor in the bacterial cytoplasm, which pulls phage DNA through the gp73 and gp72 hollow channels into the Pseudomonas cell.

Cingolani, a professor in the Department of Biochemistry and Molecular Genetics, recently came to UAB to lead the new Center for Integrative Structural Biology, approved this summer by the University of Alabama Board of Regents. The center will help UAB researchers study the three-dimensional structures of biological macromolecules such as proteins and nucleic acids to decipher their functions and mechanisms of action.

Integrative structural biology seeks to visualize a complete movie of how macromolecules function, using multiple techniques to view molecular structures and how they interact with each other. The main activity of JSC Center for Integrative Structural Biology will be the study of biological problems associated with infections, inflammation, immunity, cancer and neurodegeneration.

Co-authors of the DEV study, with Cingolani and Briani, “Integrative structural analysis of Pseudomonas phage DEV reveals genome ejection engine,” are Ravi K. Locareddy, UAB Department of Biochemistry and Molecular Genetics; Chun-Feng David Howe, Rutgers, The State University of New Jersey, Piscataway, NJ; Francesca Forti and David S. Horner, Università degli Studi di Milano; Stefano M. Iglesias and Fenglin Li, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; and Mikhail Pavlenok and Michael Niederweis, Department of Microbiology, JSC.

Support came from National Institutes of Health grants GM100888, GM140733, and OD030457; and Fondazione per la richerca sulla Fibrosi cistica-Associazione Trentina Fibrosi Cistica ODV In ricordo di Pio Nicolini, FFC grant no. 15/2021.