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Few discoveries in modern medicine are as legendary as the tale of in 1928 that an unidentified substance produced by Penicillium notatum mold could inhibit the growth of bacteria. This substance, later named penicillin, was found to have activity against pathogenic bacteria in vivo and became one of the very first antibiotics used to treat patients, including many . In the early years of its use, before efficient production methods had been established, penicillin was such a precious commodity that doctors of patients receiving this therapy so that it could be administered to more patients.

While penicillin is rightly famous for the transformative effect it had on the treatment of bacterial infections (and, consequently, on the field of medicine as a whole), its utility as a tool for understanding bacterial physiology was, perhaps, no less remarkable. It is telling that the enzymes that synthesize the peptidoglycan layer of the bacterial cell wall are named “penicillin-binding proteins (PBPs).” Binding to penicillin is not the primary function of these transpeptidase enzymes; indeed, from the perspective of bacterial cells, such activity is a potentially deadly perversion of their proper role. Yet the name is illustrative of the primacy that penicillin has played in illuminating bacterial structure and function over the course of the 20th century.

Grotesque Swellings: What Penicillin Does to Bacterial Cells

In 1940, described the dramatic of bacterial cells exposed to penicillin at sub-inhibitory concentrations when viewed under a microscope. Even Gram-negative rods such as E. coli, which are relatively impervious to the inhibitory effect of penicillin (except at extremely high concentrations), could be observed to take on “grotesque giant-forms” upon exposure to relatively low concentrations of the drug.

In 1946, the again remarked on the effect of penicillin on the morphology of E. coli, with elegant pen-and-ink drawings that depicted elongation and central swelling of cells exposed to varying concentrations of penicillin. “The morphological changes,” Duguid observed, “…suggest that penicillin in these concentrations interferes specifically with the formation of the outer supporting cell wall, while otherwise allowing growth to proceed until the organism finally bursts its defective envelope and so undergoes lysis.”

Identification of Penicillin-Binding Proteins

At the time, the nature of the bacterial cell wall was still little understood, so investigators like Duguid could not know what molecule penicillin might be interfering with. In 1956, a decade after Duguid’s observations, (based, in part, on data produced from studies using to localize the drug in bacterial cells) that the “penicillin-binding component” of bacteria was part of the cell wall. That same year, the incomparably named R.E. Strange and F.A. Dark reported on “,” which was subsequently identified and named muramic acid.

Also known as N-acetylmuramic acid, muramic acid is 1 of 2 sugars that compose the cross-linked chains of the peptidoglycan layer of the cell wall. These sugar chains are cross-linked by short peptide side chains that end in 2 D-alanine amino acids. In 1965, D.J. Tipper and recognized the structural similarity of penicillin to the D-Ala-D-Ala terminus of these peptides. They proposed that penicillin acts as an analog of this cell wall component and inhibits cross-linking, or transpeptidation, by binding to and blocking the activity of the transpeptidase that normally performs this cross linkage. And what is this enzyme that binds to penicillin (and to bacterial cell wall components that look like penicillin)? Why, a penicillin-binding protein, of course.

Beyond Penicillin: The b-lactam Family

We have since learned that comprise a large family of cytoplasmic membrane proteins that carry out transpeptidation of the peptidoglycan layer, as well as a variety of related tasks essential for cell wall maintenance and cell division. In the nearly 100 years that have passed since the discovery of penicillin, dozens of other compounds in the have been discovered and developed for clinical use. These drugs remain among the safest, most effective, and most widely used antibiotics throughout the world and have been essential in combatting the growing problem of .

Indeed, most new antibiotics that have been introduced over the past decade with broad activity against gram-negative pathogens are b-lactams, often administered in combination with a b-lactamase inhibitor that prevents degradation of the b-lactam by bacterial defense enzymes. These combination drugs derive much of their unique spectrum of activity from the specific PBPs that they target, as well as their ability to resist degradation by b-lactamases.

For gram-positive bacteria, antibiotic development over the past decades has largely centered on non-b-lactam drug classes, most of which are ineffective against gram-negative bacteria because they cannot penetrate the gram-negative outer membrane barrier.

When Alexander Fleming noticed that fateful zone of clearance around the colonies of Penicillium mold on his agar plates, he recognized this as the effect of a medically significant substance. He surely could not have guessed the extent to which that substance would revolutionize our understanding of bacteria as well as our ability, for many decades to come, to prevent human illness and death from these bacteria. Today, b-lactam antibiotics remain among our most effective options to treat highly drug-resistant gram-negative pathogens, but bacteria continue to develop resistance to each new generation of b-lactam and b-lactamase inhibitor. It may take another discovery as fortuitous as Fleming’s to help us to identify a truly novel approach to treating multidrug-resistant infections.