Three Unrelated Viruses All Evolved the Same Way to Kill Bacteria - and That Points to a Drug Target

Antibiotic-resistant bacteria kill tens of thousands of Americans each year, and that number is climbing. The pharmaceutical toolkit is not keeping pace. One reason is that identifying genuinely new targets - proteins essential to bacteria but absent from human cells - is slow, expensive work. A study published in Nature from Caltech suggests a shortcut may exist: let viruses do the target validation first.

The work, led by Bil Clemons, the Arthur and Marian Hanisch Memorial Professor of Biochemistry at Caltech, centers on a protein called MurJ. It is a flippase - a membrane protein that transports the building blocks of bacterial cell walls across the inner membrane. Without MurJ, bacteria cannot construct their cell walls and die. No approved antibiotic currently targets it.

What Phages Already Figured Out

Bacteriophages, viruses that infect bacteria, have a problem: after they replicate inside a bacterial cell, they need to escape. The bacterial cell wall - a layer of peptidoglycan that acts, in Clemons's description, like chainmail - stands in the way. Some phages solve this by evolving small proteins that punch holes in the wall from inside. These proteins, called single-gene lysis proteins or Sgls, kill bacteria by disrupting the same peptidoglycan biosynthesis pathway that antibiotics have long targeted.

Clemons's lab has focused specifically on Sgls from small single-stranded DNA and RNA phages, organisms with minimal genomes that need simple, efficient mechanisms for bacterial killing. Graduate student Yancheng Evelyn Li used cryo-electron microscopy at Caltech's Beckman Institute Biological and Cryogenic Transmission Electron Microscopy Resource Center to determine precisely how two known Sgls - called SglM and SglPP7, produced by two entirely different phages with no evolutionary relationship to each other - manage to kill bacteria by targeting MurJ.

Locked in the Same Position

MurJ works through alternating access: it binds a peptidoglycan precursor on the inside face of the membrane, changes shape to expose that precursor on the outside, releases it, and resets. Li's cryo-EM structures revealed that both SglM and SglPP7 bind to a groove on MurJ's outward-facing conformation, physically preventing the protein from completing the shape change required to reset. The flippase gets stuck. Building blocks cannot cross the membrane. Cell wall synthesis stops.

"It is clear that both of these Sgls bind to MurJ in an outward-facing conformation, locking it into this position," Li said. The outward-facing surface of MurJ is accessible from outside the cell, which matters for drug design - compounds that cannot penetrate bacterial membranes might still reach and block this site.

The structural similarity between two proteins with no shared evolutionary history was striking enough on its own. Clemons characterized it as convergent evolution - two different phages arriving at the same molecular solution through independent paths. But the finding became more significant when the team identified a third example.

A Third Independent Solution

Working with collaborators, the team identified a new Sgl from a phage genome sequence called Changjiang3. The protein, designated SglCJ3, had no sequence relationship to either SglM or SglPP7. Li resolved the cryo-EM structure of SglCJ3 bound to MurJ and found the same result: the protein bound the outward-facing conformation of the flippase and locked it in place.

Three unrelated proteins, from three separate evolutionary lineages, converging on the same site and the same mechanism for killing bacteria. "It is the first strong evidence that evolution identifies MurJ as a great target for killing bacteria, which means we should follow evolution's lead and develop therapeutics that target MurJ," Clemons said.

The argument from convergent evolution is more than rhetorical. When multiple independent evolutionary experiments arrive at the same answer, it suggests the target has properties that make it genuinely vulnerable - in this case, that the outward-facing conformation is accessible, that locking it is lethal, and that the binding groove can accommodate diverse protein structures. Those same properties should, in principle, accommodate small-molecule drugs.

What Comes Next

The study is structural and mechanistic rather than directly therapeutic - it identifies a target and characterizes how it can be blocked, but does not produce a drug candidate. Moving from these structures to an actual antibiotic would require screening for or designing small molecules that bind the same groove, then testing whether they kill bacteria in culture, then assessing selectivity and toxicity, then animal studies. The Clemons lab describes its current path as "leveraging Sgl discovery" - mining diverse phage genomes for additional proteins that target the same or related sites, which can inform what a small-molecule inhibitor might need to look like.

Because phage genomes are easy to obtain and phages evolve rapidly, this approach could continue generating new biological probes of MurJ and related proteins for some time.

Additional authors on the Nature paper include Caltech graduate student Grace F. Baron and Texas A&M researchers Francesca S. Antillon, Karthik Chamakura, and Ry Young. The work was supported by the Chan Zuckerberg Initiative, the National Institutes of Health, the G. Harold and Leila Y. Mathers Foundation, and the Center for Phage Technology at Texas A&M.