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The Role of Bacteriophages in Fighting Antibiotic Resistance

Bacteriophages, the viruses that exclusively infect bacteria, are the most numerous biological entities on Earth. A recent investigation involving 92 showerheads and 36 toothbrushes from a variety of American households revealed the presence of over 600 distinct types of these bacterial viruses. To illustrate their abundance, just one teaspoon of coastal seawater can contain approximately 50 million phages.

Although often overlooked, bacteriophages play a crucial role in biotechnology and medicine. They do not pose health risks to humans and are increasingly being recognized for their potential to combat pathogenic bacteria, particularly those resistant to antibiotics.

A significant advancement in this field was reported in a study published in Nature Communications, where researchers Gino Cingolani, Ph.D., from the University of Alabama at Birmingham, and Federica Briani, Ph.D., from the Università degli Studi di Milano, China, analyzed the complete molecular structure of phage DEV. This specific bacteriophage targets and lyses Pseudomonas aeruginosa, a common pathogen associated with cystic fibrosis and several other conditions. DEV is a key element in an experimental cocktail designed to treat preclinical P. aeruginosa infections.

One intriguing characteristic of DEV is its 3,398-amino acid virion-associated RNA polymerase, which is released into the bacterial host upon infection. Cingolani and Briani’s findings suggest that this polymerase is integral to a motor-like mechanism that facilitates the ejection of the phage’s DNA once it attaches to a bacterial cell. The process begins when the phage’s tail fibers adhere to the surface of Pseudomonas, followed by penetration of the bacterial membranes via the tail structure.

“We propose that the design principles of the DEV ejection apparatus are likely conserved among all Schitoviridae phages,” stated Cingolani. “As of October 2024, more than 220 Schitoviridae genomes are sequenced and cataloged in public databases. Our research lays the groundwork for recognizing structural elements when new phages from this family are identified.”

The Schitoviridae family is one of the least explored groups of bacteriophages, yet it is gaining traction in therapeutic applications. “Through structural biology, we aim to unravel the fundamental building blocks and gene products involved in these viruses,” explained Cingolani. This research is particularly important as the rapid evolution of amino acid sequences may complicate traditional phylogenetic methodologies.

Employing cryo-electron microscopy and various biochemical approaches, the research team achieved a comprehensive depiction of DEV’s structural components, which includes 91 open-reading frames, one of which encodes the gigantic RNA polymerase. “This virion-associated RNA polymerase forms part of a conserved three-gene operon across various Schitoviridae genomes we examined,” Cingolani noted. “We believe these proteins work together to create a genome ejection motor that bridges the bacterial cell envelope.”

Structurally, DEV and similar phages evoke the image of Neil Armstrong’s lunar lander, possessing a considerable head, or capsid, that safeguards the genomic material, coupled with leg-like appendages that assist in the phage’s adhesion to bacterial surfaces prior to invasion.

Moreover, the researchers identified all protein components in DEV essential for host attachment, revealing that its long tail fibers are critical for Pseudomonas infections. However, these fibers were rendered unnecessary against bacterial mutants lacking specific surface structures. Viral infection generally initiates upon binding to various cell surface receptors.

While the study captures still images of DEV’s structure, the researchers acknowledge that the dynamic process of phage infection remains partly unexplored. They conceptualize the infection in three distinct phases.

In the initial phase, a solitary DEV phage navigates through its environment, with its flexible long tail fibers adapting to enhance contact with Pseudomonas surface molecules. Upon establishing contact, the five tail fibers anchor the phage closely against the bacterial outer surface.

The second phase involves a short tail fiber, which acts as a tail plug, interacting with a secondary receptor on the Pseudomonas, triggering the release of this plug through a mechanical signal.

During the third phase, three proteins—gp73, gp72, and gp71—are expelled from the phage head into the bacterial envelope. The lead protein, gp73, restructures to form a pore in the outer membrane, while gp72 takes shape as a tube extending through the bacterial periplasm. Finally, gp71 infiltrates the inner membrane, transforming into an RNA polymerase motor that facilitates the transfer of the phage DNA into the bacterial cytoplasm through the recently formed channels.

Cingolani, who recently joined UAB as head of the new Center for Integrative Structural Biology, aims to advance research into the three-dimensional configurations of biological macromolecules, including proteins and nucleic acids. The center, recently approved by the University of Alabama System Board of Trustees, will focus on visualizing molecular functions amidst various biological challenges such as infections, inflammation, cancer, and neurodegeneration.

This integrative structural biology approach aspires to synthesize a holistic portrayal of macromolecular interaction dynamics via a combination of methodologies to fully understand their functional mechanisms.

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