Bacteriophage-Encoded Small RNAs: Emerging Tools for Phage Therapy and Antibacterial Intervention

Phage therapy: Revisiting an old solution

Phage therapy, the use of phages as antibacterial agents, has reemerged as a promising strategy to combat the escalating threat of multidrug-resistant bacterial infections, driven by the high specificity and self-amplifying nature of these viruses. Current clinical applications focus primarily on lytic phages that target drug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (Yao et al., 2025) (Fig. 1A). These efforts aim to demonstrate that combining phage cocktails with antimicrobial drugs can improve the elimination of severe infections. But in hindsight, this task may be more challenging than thought, as bacteria can become resistant to phages due to strong evolutionary pressure. The need to think outside the box is essential, as continued conceptual and experimental innovation will be key to identifying and overcoming evolutionary and mechanistic barriers, ultimately enabling the robust and durable implementation of phage therapy.

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FIG. 1.

Strategies for phage- and sRNA-based antibacterial interventions. (A) Conventional phage therapy relies on lytic phages to eliminate bacterial pathogens, but rapid emergence of phage resistance can limit clinical durability. (B) Phages engineered to deliver regulatory sRNAs, such as PreS, can reprogram bacterial networks to promote phage propagation and improve therapeutic performance. (C) Synthetic sRNAs delivered by plasmids or alternative non-lytic, non-fully functional phage-based systems can modulate bacterial physiology. For example, they can reduce virulence or increase susceptibility to conventional antibiotic treatments. Figure was generated with assistance from NotebookLM (Google), based on the authors’ data.

Leveraging phage-encoded sRNAs for therapeutic design

When designing phage therapy interventions, the starting point is typically a clinically relevant bacterial pathogen that is resistant to conventional antibiotics and persists despite aggressive medical treatment. Thus, therapeutic phages are selected to be strictly lytic, highly efficient, and narrowly specific to the target bacterium (Sawa et al., 2024; Strathdee et al., 2023). In this context, phage-encoded sRNAs represent an intrinsic regulatory layer that can reshape infection outcomes at the post-transcriptional level. Once the molecular targets and mechanisms of these sRNAs are defined, they offer opportunities to enhance phage performance, either by engineering their expression directly within the therapeutic phage genome or by co-delivering them via auxiliary genetic elements such as plasmids or synthetic RNA constructs. In principle, sRNAs that promote lytic development, enhance phage DNA replication, such as PreS (Fig. 1B) (Silverman et al., 2025), or suppress bacterial anti-phage defenses could increase therapeutic efficacy. This effect may be especially important under clinical conditions in which bacterial growth rates are low (Gonzalez and Aranda, 2023) and high phage dosing is essential to achieve enough phage load at the site of infection (Fig. 1C) (Abedon, 2023). Together, these considerations support the view of phage-encoded sRNAs as modular regulatory components that can be leveraged to improve phage efficacy in therapeutic settings.

RNA-based regulation offers several inherent advantages over protein-based control mechanisms. Since sRNAs act through base-pairing interactions at the post-transcriptional level, they can rapidly modulate existing gene expression programs without requiring translation or the synthesis of new regulatory proteins. A single sRNA can simultaneously influence multiple target transcripts, enabling coordinated fine-tuning of host physiology rather than the establishment of entirely new regulatory pathways (Papenfort and Melamed, 2023; Silverman and Melamed, 2025). In the context of phage infection, such multitarget regulation allows one sRNA to enhance phage fitness through multiple mechanisms by shifting host gene expression toward a more favorable intracellular state. This mode of regulation provides a fast, efficient, and versatile strategy for optimizing phage infection dynamics.

The recently characterized sRNA PreS (Silverman et al., 2025) exemplifies this potential by enhancing phage replication through post-transcriptional activation of the bacterial dnaN. As dnaN sequences are highly conserved among related bacterial hosts (Silverman et al., 2025), PreS-mediated regulation is likely to be effective across multiple phage-host systems, increasing the likelihood that this sRNA can be broadly exploited to boost phage DNA replication. Importantly, PreS is likely only one example within a much larger, yet unexplored, repertoire of phage-encoded sRNAs. The identification and incorporation of PreS-like sRNAs into therapeutic phage platforms may therefore represent a targeted approach to increase phage productivity, accelerate bacterial clearance, and improve treatment outcomes in infections where standard phage therapy alone is insufficient.

sRNAs as precision anti-virulence tools

Phage-encoded sRNAs operate according to principles similar to those of bacterial regulatory sRNAs that shape pathogenicity and host adaptation, exploiting existing post-transcriptional regulatory networks to rapidly and coordinately remodel gene expression with minimal metabolic cost. Thus, insights gained from decades of research on bacterial sRNAs in pathogenic contexts provide a valuable framework for understanding how phage-derived sRNAs can be repurposed or engineered to modulate infection outcomes. This parallel further supports the idea that phages may co-opt or refine regulatory strategies already embedded within bacterial physiology, rather than relying solely on de novo protein-based control mechanisms. sRNAs are already well established as central regulators in pathogenic bacteria across diverse taxa, including S. aureus, Salmonella enterica, Listeria monocytogenes, and Vibrio species, where they modulate virulence, stress adaptation, and host interaction (Adams and Storz, 2020; Felden and Augagneur, 2021). The base-pairing nature of sRNA-mRNA interactions means they can simultaneously coordinate multiple virulence pathways with minimal metabolic cost, allowing pathogens to swiftly adapt during infection. This regulatory role positions sRNAs as attractive targets and tools for therapeutic intervention: disrupting virulence-associated sRNAs or mimicking their regulatory effects could slow down infections. This approach aims to disarm pathogens rather than kill them directly, potentially reducing the selective pressure for resistance.

More specifically, in S. aureus, sRNAs such as RNAIII, SprD, and RsaA regulate quorum sensing, toxin production, and immune evasion, enabling the transition from colonization to invasive disease (Novick et al., 1993; Pichon and Felden, 2005; Romilly et al., 2014). Similarly, S. enterica employs sRNAs, including IsrM and PinT, to fine-tune pathogenicity island expression and coordinate invasion with intracellular survival (Romilly et al., 2014; Westermann et al., 2016). In L. monocytogenes, multiple sRNAs such as Rli27, Rli31, and Rli50 are required for stress resistance, intracellular persistence, and full virulence in animal models (Mraheil et al., 2011; Quereda et al., 2014). Population-level behaviors relevant to infection are also governed by sRNAs, exemplified by the Qrr family in V. cholerae, which links quorum sensing to virulence gene expression and biofilm formation (Feng et al., 2015). Collectively, these examples highlight sRNAs as rapid, energy-efficient regulators that shape infection dynamics in diverse pathogens, underscoring their potential as targets or tools for anti-virulence and RNA-based therapeutic strategies.

Future directions and challenges

Growing evidence of antibiotic-resistant bacteria urges the scientific community to search for alternative strategies that modulate bacterial fitness and pathogenicity without relying exclusively on bactericidal approaches. As sRNAs increasingly emerge as central regulators of cellular decision-making, they represent a promising yet largely untapped class of molecular tools for biomedical and biotechnological applications. While PreS was originally discovered in phage lambda, it is likely only one example within a much broader and still largely unexplored repertoire of phage-encoded sRNAs. Systematic discovery efforts combining comparative genomics, RNA-RNA interactome mapping, and dual RNA-seq approaches during infection will be essential to uncover this hidden regulatory layer. Beyond discovery, key challenges lie in functional characterization and therapeutic integration. Determining the host range, target specificity, and evolutionary robustness of phage-encoded sRNAs will be critical for assessing their translational potential. Moreover, successful implementation in phage therapy will require careful consideration of expression timing, dosage control, and dependence on host RNA chaperones such as Hfq or alternative RNA-binding proteins. Addressing these challenges will not only refine our understanding of phage-host regulatory networks but may also enable the rational design of next-generation phage therapeutics that leverage RNA-based regulation to enhance efficacy, robustness, and clinical reliability.