It’s In Our Bones

Art Courtesy of Gia Cabral.

Bumps, whiteheads, rashes, scars: acne vulgaris is the most prevalent skin disease in the world, affecting the physical and mental health of 9.4 percent of the global population. This figure includes a whopping eighty-five percent of people between twelve and twenty-four years old. Cutibacterium acnes is the major cause of acne. It is a Gram-positive bacterium, meaning it has a thicker cell wall and causes different infections than Gram-negative bacteria. C. acnes coexists with healthy skin follicles and pores, but certain strains lead to acne and other complications, such as eye inflammation after tissue implantation. To treat these infections, dermatologists often prescribe a class of antibiotics called “tetracycline,” which include doxycycline, minocycline, and sarecycline. 

Tetracycline-class antibiotics are a group of medications used in the treatment of bacterial infectious diseases. They target the ribosome, an organelle that translates genetic information from RNA sequences into amino acid chains, which then fold into proteins. Tetracycline is a naturally-occurring antibiotic isolated from actinomycetes, a soil bacteria, and was approved by the FDA for medical use in 1954. Doxycycline and minocycline, termed second-generation tetracyclines, were approved in 1967 and 1971, respectively, thanks to their improved stability and pharmacological efficacy. Almost half a century later, in 2018, sarecycline was introduced as a third-generation tetracycline-class antibiotic. While doxycycline and minocycline kill all types of bacteria—meaning they are broad-spectrum antibiotics—sarecycline has narrow-spectrum activity against only certain Gram-positive bacteria, including C. acnes

Christopher Bunick, associate professor of dermatology at the Yale School of Medicine, noticed the importance of this novel antibiotic and decided to look into its molecular mechanism. “Because of our expertise in protein synthesis, ribosome structure, and dermatology, we couldn’t resist taking on this project,” said Ivan Lomakin, an associate research scientist in the Bunick lab. Lomakin, Bunick, and their collaborators analyzed how sarecycline interacts with the C. acnes ribosome using cryogenic electron microscopy (cryo-EM), and published the results in Nucleic Acids Research. They discovered that sarecycline binds to the C. acnes ribosome at two different sites, which had not been observed in structures with other tetracyclines or model bacteria. They also discovered two novel ribosomal proteins and demonstrated their antimicrobial properties independent of the ribosomal complex. This study ultimately confirmed that sarecycline may be a more effective treatment option for C. acnes infections. 

Why bind to ribosomes? 

Since protein synthesis is an indispensable part of life, many antibiotics inhibit the activity of bacterial ribosomes, and sarecycline is no exception. As a complex—an assembly of multiple subunits that carry out a function together—the ribosome is made of ribosomal RNA (rRNA) associated with certain ribosomal proteins. Translation, the process it catalyzes, involves messenger RNAs (mRNAs) and transfer RNAs (tRNAs). The mRNA contains information ‘copied’ from the DNA that the ribosome must ‘read and translate.’ The tRNA contains nucleotides that recognize specific, three-base long mRNA sequences and the amino acid that corresponds to this sequence. The ribosome consists of large and small subunits, which are responsible for different steps of the process. The small subunit binds to the mRNA strand and identifies the correct tRNA molecule for every three bases within the decoding center. The large subunit then catalyzes the addition of the new amino acid to the growing polypeptide (protein) chain, which exits the ribosome through a structure called the nascent peptide exit tunnel (NPET). This process is how cells make proteins. 

Structural biologists have characterized the binding mechanisms of many antibiotics with ribosomes of model bacteria like E. coli and Thermus thermophilus. For first- and second-generation tetracyclines, existent structures all suggest that they bind to the decoding center of the small ribosomal subunit, inhibiting mRNA-tRNA interactions and slowing protein synthesis. The interaction patterns that Lomakin and colleagues characterized with cryo-EM agree with models of other tetracyclines. They show that the stacking of three hydrophobic layers stabilizes the binding of sarecycline in the decoding center of the C. acnes ribosome and inhibits tRNA arrival. 

Besides the canonical binding site, researchers were surprised to discover that sarecycline has a second binding site (SBS) on the C. acnes large ribosomal subunit, in the NPET. Here, sarecycline blocks the NPET and is thought to suppress the growth of the peptide chain. This binding site is specific to the C. acnes ribosome, as X-ray crystallography of sarecycline in complex with the Thermus thermophilus ribosome—also performed by the Bunick Lab—showed only the canonical binding site. Based on indirect experiments, researchers speculated that sarecycline could probably bind to both sites with similar affinities, and the sarecycline molecule in the SBS may assist with function in the canonical binding site. 

The paper compared this binding interaction with an antibiotic from another structural family, tetracenomycin X, which was recently shown to bind to the E. coli ribosome at a similar site. The team postulated that certain uridine bases were important in this interaction, and mutations from uridine to another base, cytosine, would confer resistance. However, Lomakin is not too worried about bacteria gaining sarecycline resistance, compared to other tetracyclines. “For sarecycline, we have an additional site on the large ribosomal subunit, encoded by a different gene than the small ribosomal subunit. The probability of simultaneously getting mutations on both of them is the multiplication of getting mutations on each, so it is very low,” Lomakin said. 

Potential Antimicrobial Properties

Using cryo-EM, the researchers managed to capture the first high-resolution structure of the C. acnes ribosome. They also examined the ribosomal proteins attached to the ribosomal RNA and explored how the C. acnes ribosome’s catalytic mechanisms could differ from other known bacterial models. This knowledge would help inform the future design of antibiotics against C. acnes

But of particular interest to the team were the bacterial small ribosomal subunit protein 22 (bS22) and the bacterial large ribosomal subunit protein 37 (bL37). These proteins are normally part of the ribosome and help synthesize proteins, but independent of the complex, they have displayed antimicrobial properties. C. acnes exists in normal skin, so researchers wonder if it helps defend the skin against other pathogenic bacteria. “From a dermatology perspective, we know C. acnes lives in our follicles and pilosebaceous units as a commensal organism—only certain strains of it are pathogenic for acne. We are trying to probe whether or not C. acnes has a natural defense mechanism against Staphylococcus aureus, which is a major pathogenic skin organism,” Bunick said. They discovered that the bS22 could inhibit the growth of E. coli and S. aureus while the bL37 only affected S. aureus but not E. coli

The next step down the antimicrobial properties path was to understand whether these proteins were present independent of the ribosome. Usually, ribosomal proteins form a complex with ribosomal RNAs to catalyze protein synthesis, but additional functions outside of ribosomes are possible. Bunick proposes that C. acnes may either secrete these peptides directly or release them when they die and lose their membranes. 

Sarecycline: The Better Acne-biotic?

Sarecycline was introduced in 2018 as a drug with higher specificity and fewer side effects than minocycline and doxycycline. However, primarily due to pricing issues, it only has about six percent of the prescription market for oral tetracycline in dermatology. To Bunick, sarecycline deserves more recognition. “At least to my knowledge, it is the only FDA-approved drug that targets two active centers of the ribosomes currently,” he said. 

The human gastrointestinal tract contains many beneficial Gram-negative bacteria. Since sarecycline is more specific to certain Gram-positive bacteria and spares Gram-negative bacteria, it causes fewer side effects in the gastrointestinal tract while effectively targeting Gram-positive C. acnes. Doxycycline is photosensitive, so certain users may experience rashes, itching, or severe sunburn, while sarecycline is not. Sarecycline is also less hydrophobic, meaning a decreased possibility of diffusing through the blood-brain barrier and causing dizziness, vertigo, or tubular disturbance. The Bunick lab partners with Almirall, the pharmaceutical company that licenses and sells sarecycline in the United States. “Our laboratory [work] has been predominantly keratins, intermediate filaments, and the skin barrier for over a decade. But [learning about sarecycline] presented a unique opportunity as an entryway into a new area of research understanding the molecular mechanisms of dermatology drugs. Sometimes the science leads you to where you need to be,” Bunick said. He does not rule out the possibility of developing a fourth-generation tetracycline. This paper was the first to publish a C. acnes ribosomal structure. Given the new information about multiple active sites, it is possible to further optimize the structure of the antibiotic to achieve more precise treatment of the infection behind your bumps and whiteheads.