Looking for a needle in the haystack: Expanding the Functional Landscape of Ribosome-Arresting Antimicrobial Peptides

Antimicrobial resistance remains one of the most pressing threats to public health and there is an urgent need to discover new and more effective strategies for treating bacterial infections. In light of this growing crisis, attention has shifted toward alternative classes of antimicrobial agents, including host-derived peptides like the proline-rich antimicrobial peptides (PrAMPs). These small, naturally occurring molecules have shown potential not only because of their broad activity but also due to their unique mechanisms of action. One interesting example is apidaecin which is a peptide produced by honeybees. Unlike most antibiotics, which target the early phases of bacterial translation, apidaecin operates at the very end of the process. It binds inside the ribosome’s exit tunnel and traps release factors (RF1 and RF2) on ribosomes that have just finished synthesizing proteins. This jams the translation machinery and effectively halts the release of subsequently synthesized new proteins. The result is ribosome stalling at stop codons and a ripple effect across the cell, ultimately leading to widespread disruption in protein synthesis. Although apidaecin’s mechanism is unique and holds great promise for drug development, there’s still a lot we don’t know about the extent of its flexibility at the sequence level. Earlier studies have mostly tested the effects of changing single amino acids, but this gives only a partial picture. For real-world applications, especially when designing improved synthetic versions, we need to understand how the peptide behaves when multiple residues are altered at once. Because apidaecin interacts so precisely with the ribosome—a complex and highly ordered structure—even small changes might have unpredictable consequences. This uncertainty has posed major limitations in efforts to optimize the peptide for therapeutic use. Adding to the complexity, bacterial species can differ significantly in how they import peptides, in their internal biochemical environments, and even in the structure of their ribosomes. So, it’s critical to ask: if we start modifying apidaecin’s sequence, will it still work across different bacterial systems? Answering this question has both fundamental and translational significance. On the one hand, it helps us better understand how translation termination operates and how newly made protein leave the ribosome. On the other, it opens the door to customizing apidaecin-like peptides to tackle a wider range of bacterial infections.

To this account, a recent study published in Nucleic Acids Research, mapped the sequence flexibility of apidaecin. The strategy was ambitious: build and screen a large-scale library of apidaecin variants, each containing multiple amino acid substitutions, and evaluate their ability to arrest translation. By combining high-throughput genetic screening with functional biochemical assays, the team identified which regions of the peptide are indispensable for function and which areas can be modified without compromising its activity.

To explore how much structural variation apidaecin can tolerate while still halting bacterial translation, the researchers created a massive library of ~350,000 peptide variants, each containing between two and six amino acid changes. Tightly regulated expression of these peptides in E. coli allowed the team to test their effects directly in living cells. Their first strategy was a depletion-based screen. If a variant can trap release factors on the ribosome, it would stall translation and kill the cell.  They compared the abundance of cells expressing each mutant peptide before and after induction and identified over 15,000 variants that were depleted upon induction which indicates strong antibiotic activity. Afterward, they synthesized a subset and tested with biochemical assays confirming that most apidaecin-like peptides  could indeed stall ribosomes at stop codons, despite the presence of multiple substitutions in the peptide sequence. To find even more of the active peptides, the authors used a complementary approach. They expressed the library of the peptides in an engineered E. coli strain with premature stop codons in essential genes. Upon limited expression of an active peptide, the release factors will be depleted and some ribosomes will be able to bypass premature stop codon restoring the production of the essential protein and allowing cells to form colonies. Peptides from such surviving cells were sequenced, revealing 222 active variants. Many of these showed ribosome-stalling in vitro. Interestingly, the two strategies revealed slightly different sequence preferences.

The C-terminal region remained highly conserved across all active peptides, highlighting its essential role in ribosome arrest. Meanwhile, the N-terminal region was far more flexible, and some wild-type residues were frequently replaced in top-performing variants. Peptides from the readthrough screen retained more proline residues, possibly aiding RF trapping, while depletion-selected peptides tolerated more substitutions, hinting at broader inhibitory effects. It is noteworthy that several of these modified peptides showed improved antimicrobial activity in rich and serum-like media, outperforming wild-type apidaecin.

In conclusion, the new research work offers a compelling look at how antimicrobial peptides like apidaecin interact with the bacterial ribosome and more importantly how much flexibility these peptides have without losing their function. But by testing thousands of variants with multiple substitutions, this work shows that apidaecin’s sequence is more tolerant to substitutions than expected, especially in the N-terminal region which is an important finding. What really makes this study stand out is that some of the engineered peptides didn’t just retain activity—they worked better than the original. In the context of rising antibiotic resistance, that’s a big deal. If we can design peptides that hit a bacterial target in a completely different way than conventional antibiotics, we might be able to stay ahead of evolving resistance. These results suggest we’re not limited to tweaking natural sequences—we can push much further and still get potent antimicrobial effects. There’s also conceptual value here. By exploring how changes to apidaecin affect its ability to stall the ribosome and trap release factors, the authors reveal details about translation termination that aren’t well understood. The fact that some variants promote stop codon readthrough hints at interesting regulatory possibilities, and could even help guide work in synthetic biology or translational control more broadly. Using both negative and positive selection screens added depth to the study. Each approach captured a different slice of the functional landscape, and comparing the two helped highlight which sequence elements are essential and which are more flexible. That kind of information is incredibly useful if you’re trying to design better therapeutic peptides from scratch.

While the Nucleic Acids Research study took a synthetic biology approach—testing how much you can push apidaecin’s sequence and still retain function—this EMBO Reports paper by the same research group approaches the problem from the opposite angle. Instead of engineering new variants, the authors looked to nature, mining insect genomes for apidaecin-like peptides that have evolved naturally. They identified 71 of these peptides, many of which differ quite a bit from the original apidaecin, especially in the N-terminal region. Interestingly, several of them turned out to be significantly more potent than the wild-type peptide—up to 16 times more active in some cases—despite their sequence divergence. What’s especially compelling is that, structurally, these natural variants all seem to rely on the same C-terminal segment to interact with the ribosome. The team confirmed this using high-resolution structural data, which showed that, regardless of upstream variability, the critical C-terminal residues consistently engage the ribosome in a conserved manner. So, while the earlier NAR paper mapped out the boundaries of what can be achieved through deliberate mutation and screening, this study demonstrates how far evolution has already explored that same space—often arriving at highly effective solutions. Together, these two papers are complementary rather than overlapping. One shows what’s possible when we push apidaecin’s structure experimentally; the other shows what’s already been refined in nature. Looking at both, we get a broader and more nuanced view of how these peptides function and evolve, and how they might be harnessed or improved for antimicrobial applications.

The leading authors in these two papers are Drs. Weiping Huang and Chetana Baliga and the work was done at the University of Illinois at Chicago in the lab co-run by Nora Vázquez-Laslop and Alexander Mankin. Both of the lead authors are now faculty members on their own and continue working on antibacterial peptides with the goal of finding new peptide antibiotics that could target a wide range of the human- animal- and plant pathogens.