Viral sRNA Unlocks Phage Therapy Potential Against Drug-Resistant Bacteria

As the number of antibiotic-resistant infections continues to rise, scientists are looking to bacteriophages (“phages”), viruses that infect bacteria, as an approach to tackling antibiotic resistance. A new study by researchers at the Hebrew University of Jerusalem has revealed how bacteriophages use a tiny piece of genetic material to hijack bacterial cells and make more copies of themselves.

Focusing on infection of Escherichia coli by phage lambda, a bacteriophage that scientists have been studying for decades, research lead Sahar Melamed, PhD, and colleagues identified a virus-encoded small RNA molecule (sRNA) called phage replication enhancer sRNA (PreS) that acts like a hidden genetic “switch.” The team’s research indicated that this switch rewires bacterial genes to help the virus copy its DNA more efficiently and boost viral replication.

The team said that understanding how phages control bacterial cells is important both for basic science and to help inform future medical applications. By uncovering how phages use tools such as PreS to take control of bacterial cells, the newly reported study provides important basic knowledge that could help scientists design new phage-based therapies targeting drug-resistant bacteria.

Melamed, together with first author Aviezer Silverman and colleagues at the Hebrew University of Jerusalem and at the University of Illinois Urbana-Champaign, reported on their studies in Molecular Cell, in a paper titled “Phage-encoded small RNA hijacks host replication machinery to support the phage lytic cycle.” In their paper, they stated, “These findings uncover an RNA-level regulatory layer in phage-host interactions and demonstrate how a phage-encoded sRNA can hijack host replication machinery to optimize infection.”

Antibiotic resistance is one of the biggest global health threats of our time. It is currently estimated that by 2050, infections caused by antibiotic-resistant bacteria could kill up to 10 million people every year worldwide. As an alternative to relying on antibiotics, one promising strategy being developed is phage therapy, which uses viruses that specifically attack bacteria.

“Phages are the most abundant genetic entities on Earth (≈10³¹), and shape bacterial evolution and ecology, influencing virulence and antibiotic resistance,” the authors wrote. Yet, while phages are major drivers of bacterial population dynamics, “… the significance of post-transcriptional regulation during infection remains largely unexplored.”

Phage lambda is the most extensively characterized temperate phage, the team explained. “Following infection, phages enter either a lytic cycle, producing progeny and lysing the cell, or a lysogenic cycle, integrating into the host genome.” Virulent phages are strictly lytic, the team continued, whereas temperate phages can enter both cycles.

Lambda infects E. coli bacteria, and in the lytic cycle uses the host cell’s resources to replicate its genome via a process called rolling-circle replication, which creates long linear DNA molecules from a circular template. The DNA is then packaged, assembled into virus particles, and released by cell lysis.

For their newly published research, Melamed and colleagues applied a technique known as RNA interaction by ligation and sequencing (RIL-seq) to map the in vivo RNA-RNA interaction network in E. coli during phage lambda infection. “This analysis revealed extensive reprogramming of E. coli–E. coli interactions, phage-specific lambda-lambda interactions, and interkingdom interactions between phage and host RNAs,” they reported.

Among their findings, the investigators discovered that the phage makes a small RNA molecule called PreS, which acts as a molecular switch. Until now, most phage research has focused on viral proteins, but the new discoveries demonstrated that phages also use RNA molecules to quickly reprogram the host cell after the bacterial genes have already been read and bacterial messages (mRNAs) have been made, adding an extra layer of control during infection.

PreS attaches to these important bacterial messages and tweaks them in a way that helps the virus copy its DNA and move more efficiently toward the stage where new viruses are produced and burst out of the cell, killing the bacterium. The researchers found that a key PreS target is a bacterial message that makes DnaN, a protein that plays a central role in copying DNA. By helping the cell make more DnaN, PreS gives the virus a strong head start in the infection process. Interestingly, the results showed that PreS works by changing the shape of the bacterial dnaN message.

Normally, part of this message is tightly folded, which makes it hard for the cell’s protein-making ribosomes to access. PreS binds to this folded region, opens it up, and allows ribosomes to read and translate the message more efficiently. The result is more DnaN protein, faster viral DNA copying, and a stronger infection. PreS regulates essential host genes, including dnaN … This regulation enhances DNA replication and fine-tunes the phage lytic cycle,” the team noted. When the researchers removed PreS or disrupted the spot where it binds, the phage became weaker, multiplied more slowly, and its destructive phase was delayed.

The new discovery is particularly striking because small RNAs have not traditionally been seen as major players in phage biology. Yet PreS is highly conserved in many related viruses, indicating that phages may share a common toolkit of small RNAs that scientists are only beginning to uncover, the authors suggest. “Our findings highlight cross-kingdom sRNA-based regulation, with bacterial sRNAs responding to phage attack, potentially serving as a defense mechanism, and a phage-encoded sRNA directly regulating essential E. coli genes to alter DNA replication and promote infection,” the team concluded.

“This small RNA gives the phage another layer of control,” commented Melamed. “By regulating essential bacterial genes at exactly the right moment, the virus improves its chances of successful replication. What astonished us most is that phage lambda, one of the most intensively studied viruses for more than 75 years, still hides secrets. Discovering an unexpected RNA regulator in such a classic system suggests we have only grasped a single thread of what may be an entirely richer, more intricate tapestry of RNA-mediated control in phages.”

As scientists search for solutions to antibiotic-resistant infections, phages are drawing growing interest as a targeted, flexible therapy. Discoveries such as PreS show that even the smallest viral molecules can have a big impact on whether an infection succeeds. In the long term, this knowledge could help researchers engineer phages that are safer, more predictable, and more powerful in the fight against drug-resistant bacteria.

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