Bacteria stay safe within their walls only it these walls are properly built and maintained. And so bacteria are vulnerable to antibiotics that can interfere with the bacterial cell wall’s brick and mortar—large sugar molecules and clusters of amino acids. Bacterial masonry, Harvard scientists report, has an overlooked vulnerability. Specifically, a recently discovered class of wall-building proteins displays a cavity, one that may be targeted by new antibiotics.

These proteins—which are called the shape, elongation, division, and sporulation (SEDS) proteins—have been spared any attacks because little is known about their molecular structures. But the protection the SEDS have enjoyed by having stayed in the shadows may soon be lost.

A bacterial wall–building protein has been shown to have an overlooked vulnerability, a cavity that can be perturbed and potentially targeted by newly developed broad-spectrum antibiotics. The blue balls are wall-making proteins,  yellow represents a newly synthesized bacterial cell wall, and green represents “scaffolding” proteins. [Janet Iwasa, for Harvard Medical School]

The Harvard scientists indicated that they have extending earlier work that let them determine that the prototypical SEDS protein, RodA, is a peptidoglycan polymerase. In new work, the scientists have pinpointed a weakness in RodA’s makeup. This new work has been detailed in the journal Nature, in an article entitled “Structure of the Peptidoglycan Polymerase RodA Resolved by Evolutionary Coupling Analysis.”

“…we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement,” wrote the article’s authors. “The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered.”

The protein, the authors continued, contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. This cavity caught the scientists’ attention because it faces the outer surface of the protein. The size and shape of the cavity, along with the fact that it is accessible from the outside, make it a particularly appealing drug target.

“What makes us excited is that this protein has a fairly discrete pocket that looks like it could be easily and effectively targeted with a drug that binds to it and interferes with the protein's ability to do its job,” said study co-senior author David Rudner, Ph.D., professor of microbiology and immunobiology at Harvard Medical School.

In a set of experiments, researchers altered the structure of RodA in two bacterial species—the textbook representatives of the two broad classes that make up most of disease-causing bacteria. One of them was Escherichia coli, which belongs to a class of organisms with a double-cell membrane known as Gram-negative bacteria, so named due to a reaction to staining test used in microbiology. The other bacterium was Bacillus subtilis, a single-membrane organism that belongs to so-called Gram-positive bacteria.

Mutagenesis experiments in B. subtilis and E. coli, the scientists indicated, show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. When the researchers induced even mild alterations to the structure of RodA's cavity, the protein lost its ability to perform its work. E. coli and B. subtilis cells with disrupted RodA structure rapidly enlarged and became misshapen, eventually bursting and leaking their contents.

“Our latest findings reveal the molecular structure of RodA and identify targetable spots where new antibacterial drugs could bind and subvert its work,” noted study senior investigator Andrew Kruse, Ph.D., associate professor of biological chemistry and molecular pharmacology at Harvard Medical School. “Taken together,” the authors of the Nature article elaborated, “the high degree of sequence conservation, intolerance to mutation and catalytic essentiality of residues surrounding the central cavity confirm that this portion of the protein has a critical role in peptidoglycan polymerization, which makes it a prime target for the development of antibiotics.”

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