Clovibactin - a game-changing novel antibiotic with no resistance

By Neha MathurReviewed by Lily Ramsey, LLMSep 6 2023

In a recent article published in the Cell Journal, researchers reported the discovery of a new antibiotic, clovibactin.

Clovibactin was isolated from an uncultured Gram-negative β-proteobacterium (E. terrae ssp. carolina) found in sandy soil from North Carolina. Additionally, they reported the unusual structure and mode of action of clovibactin.

Study: An antibiotic from an uncultured bacterium binds to an immutable target. Image Credit: Jaromond/Shutterstock.com

Background

The 1940s to 60s were considered the golden age of antibiotic discovery. The discovery of streptomycin, vancomycin, or tetracycline enabled the practice of modern medicine.

They were discovered through screening of natural product scaffolds, e.g., Actinomycetes bacteria; however, conventional antibiotic screening methods have become redundant and are unlikely to facilitate the discovery of new antibiotics. 

Furthermore, the development and spread of antimicrobial resistance (AMR) has decreased the effectiveness and life of antibiotics, which, so far, have helped combat infectious diseases and enable complex surgical endeavors, such as organ transplantation.

It is, thus, vital to pursue the untapped potential of antibiotic-producing bacteria using novel approaches.

The recently emerged iChip technology has made new natural product scaffolds accessible, such as uncultured bacteria representing ∼99% of all microbial species. This led to the discovery of new antibiotics, lassomycin and teixobactin. Indeed, uncultured bacteria are a rich source for the sustained discovery of next-generation antibiotics.

About the study

In the present study, researchers sub-cultured colonies detected after 12 weeks of incubation on agar plates overlaid with Staphylococcus aureus.

Bioassay-guided fractionation of the extract from these microbial colonies yielded Kalimantan, a previously known antibiotic originating from Pseudomonas and Alcaligenes.

Initially, kalimantacin was more abundant in the extract; however, when the researchers disrupted the first gene in the kalimantacin/batumin operon, viz. bat1, it reduced kalimantacin production below detectable levels.

Further fermentation yielded a novel depsi-peptide compound like teixobactin with a unique mass of 903.5291 [M+H]+, which the researchers named clovibactin. 

They used a combination of mass spectrometry (MS), solid-state nuclear magnetic resonance (NMR), and atomic force microscopy to resolve the structure of clovibactin. In addition, they confirmed its stereochemistry by Marfey’s analysis.

Study findings 

Structurally, clovibactin featured two D-amino acids, d-alanine, and d-glutamic acid, in its four amino acid long linear N terminus and D-3-hydroxy asparagine, a unique amino acid residue.

Sequencing of the E. terrae ssp. carolina genome revealed 19 predicted biosynthetic gene clusters (BGCs) in clovibactin, and BLASTN alignment revealed 72% identity between the clovibactin and teixobactin BGCs.

Clovibactin was active against Bacillus subtilis, unlike kalimantacin. To exert its antibiotic effects, it blocked cell wall synthesis by binding the pyrophosphate (PPi) moiety of multiple cell wall lipid precursors, including undecaprenyl phosphate (C₅₅PP), lipid II, and lipid III_WTA (wall teichoic acid). 

Clovibactin molecules antiparallelly arranged themselves to selectively bind to the PPi moiety of lipid precursors, resulting in a supramolecular complex that subsequently oligomerized into a stable higher-order fibrillar assembly using its short N terminus acts as oligomerization domain. These supra-structures appear to be an essential part of the killing mechanism of clovibactin.

However, a detailed structural analysis of clovibactin could only uncover how it manages to bind PPi of lipid II tightly and selectively.

Another striking feature of clovibactin was its exceptional ability to cause cell lysis in a mechanistically distinct manner from teixobactin.

Conclusion

The discovery of clovibactin is an encouraging development for several reasons.

First, clovibactin was isolated from a previously uncultivated soil bacterium when the drug discovery pipeline had substantially thinned.

More importantly, it avoided AMR, a leading cause of mortality worldwide, by targeting the PPi moiety of essential lipid precursors of microbial cell walls using an unusual hydrophobic interface. 

The study finding enhances the understanding of antibiotics evolved to avoid AMR; more importantly, this data could inform the design of other drug compounds with a clinically long lifespan.

In future studies, advanced ssNMR methods enabling selective isotope labeling of clovibactin could help establish the precise supramolecular arrangement of clovibactin-lipid II observed in this study.