A Caltech-led team of biochemists has homed in on an underexplored small transporter called MurJ that is a vital part of the pathway bacteria use to build their chain-mail-like cell wall. An essential component of the cell wall, called peptidoglycan, provides the strength that allows bacteria to resist pressure. Using advanced tools, the scientists have determined the common mechanism used by three different bacteria-killing viruses to block MurJ from doing its job. The findings reveal a novel target for designing new antibiotics.
The scientists report their findings in the February 26 issue of the journal Nature. The lead author of the paper is Yancheng Evelyn Li, a graduate student in the lab of Bil Clemons , the Arthur and Marian Hanisch Memorial Professor of Biochemistry at Caltech, who is the corresponding author.
"Evolution is powerful, and in bacteria, resistance to antibiotics develops quickly. This means that we now deal with bacteria that are resistant to all the medicines that we have," Clemons says. "In the USA alone, tens of thousands of people die every year from antibiotic-resistant bacterial infections, and that number is rising rapidly. We need new antibiotics to combat this."
Scientists have long been interested in the cellular pathway that builds peptidoglycan, aptly known as the peptidoglycan biosynthesis pathway, as an antimicrobial target. "Peptidoglycan is a unique feature of bacteria, and that makes it an attractive antibiotic target," Clemons says.
Many details of the peptidoglycan biosynthesis pathway are known and have been leveraged as targets for antibiotics. The first pharmaceutical, discovered by Alexander Fleming in the middle part of the last century, was the antibiotic penicillin. It and its derivatives, such as amoxicillin, target a late step in this pathway to kill bacteria.
In bacteria, three key proteins-MraY, MurG, and MurJ-facilitate the transfer and transport of peptidoglycan's building blocks from within the cell across the inner membrane barrier. If any of the three proteins fail, peptidoglycan cannot be made, and bacteria die, making them exciting targets for antibiotic discovery. Scientists know a lot about these proteins, but, as noted by Clemons, many basic mechanistic questions remain unanswered.
While the benefits of inhibiting these proteins are clear, there are currently no medicines that target them. However, Clemons says, "We do know that we can find small molecules, either derived from nature or synthesized in chemical libraries, that will inhibit these proteins. Excitingly, recent discoveries have shown that bacteriophages have figured out how to target this pathway."
The survival of viruses that target bacteria, called bacteriophages, or phages, depends on their ability to enter the bacterial cell, make copies of themselves, and then leave to spread as widely as possible. "Getting back out means that they have to get past the peptidoglycan layer. Because it acts like chainmail, the phages get stuck if they can't break through it," Clemons explains.
The Clemons lab has turned some of its focus to single-stranded DNA and RNA phages, tiny phages with small genomes that require simple methods for killing bacteria. In 2023, the lab published a paper in Science about one such phage, φX174 , that has a long history at Caltech.
The weapons these small phages use to kill bacteria are protein antibiotics called single-gene lysis proteins, or Sgls (pronounced like "sigils"). Most recently, Li and Clemons have focused on Sgls that target MurJ for antibiotic discovery. MurJ is a flippase, a protein that "flips" peptidoglycan building blocks across the cellular membrane so they can be used to build the peptidoglycan chain. Collaborators had already shown that two Sgls, SglM and SglPP7-which are unrelated and produced by two different phages-both cause bacterial death by inhibiting MurJ.
In the current work, Li used Caltech's Beckman Institute Biological and Cryogenic Transmission Electron Microscopy (Cryo-EM) Resource Center to reveal how these two Sgls inhibit MurJ's flipping activity. Flippases, like MurJ, work by alternating the access of the molecules they transport between the two sides of the membrane without ever making an opening in the membrane. For MurJ, binding of the peptidoglycan precursor within the cell triggers a structural change that effectively moves the molecule outside the cell. Li found that both Sgls bind to a groove in the flippase that prevents the protein from making these structural changes.
"It is clear that both of these Sgls bind to MurJ in an outward-facing conformation, locking it into this position," Li says. That is exciting to researchers because the outward-facing conformation of MurJ is accessible to the surrounding environment. In theory, that makes it easier to target with antibiotics than an internal-facing conformation.
Clemons says the discovery is shocking for another reason. "These peptides, which have no evolutionary links to each other, have both figured out how to target MurJ in a very similar way. These are two examples of convergent evolution, in which different evolutionary paths arrive at the same solution. We were surprised!"
The researchers add that because viruses evolve rapidly, there is likely an endless supply of phages that will all have Sgls. Because phages are easy to find, mining these viral genomes can lead to new biological discoveries and new antibiotic targets. In the Nature paper, the scientists did just that with a new phage. Working with a collaborator, they identified a new Sgl, called SglCJ3 (from a genome sequence that is predicted to be a phage and is called Changjiang3), for cryo-EM analysis. Li resolved the structure of SglCJ3 bound to MurJ and found that it also binds in the same outward-facing conformation of MurJ.
"This is a third genome that evolved a distinct peptide to inhibit the same target in a similar way," Clemons says. "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. This demonstrates the power of basic biology to help us solve problems in medicine. Our path is set on leveraging Sgl discovery, and we hope to continue to be supported to turn these concepts into realities."
The paper is titled "Convergent MurJ flippase inhibition by phage lysis proteins." Along with Clemons and Li, additional authors are Caltech graduate student Grace F. Baron; and Francesca S. Antillon, Karthik Chamakura, and Ry Young of Texas A&M University. 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, jointly sponsored by Texas A&M AgriLife.
