Cloacaenodin, an Antimicrobial Lasso Peptide with Activity against Enterobacter


Enterobacter species commonly reside commensally in the human and animal GI tract and in the environment on decaying matter, in soil, and in sewage, certain species like E. cloacae have been the causative agents of many nosocomial outbreaks. With the emergence of Enterobacter strains displaying resistance to many last-resort antibiotics like carbapenems, these concerning pathogens call for a renewed interest in development of new treatments. The Enterobacter cloacae complex consisting of closely related species that are commonly isolated as clinical specimens, most notably E. cloacae and E. hormaechei. Concerningly, many of these pathogens have natural resistance to β-lactam antibiotics and possess various carbapenemase genes. Colistin resistance has also been increasingly found in Enterobacter infections. With the rise of multidrug resistance in Enterobacter isolates, new compounds are sorely needed for future treatments.

A rapidly growing list of lasso peptides exhibit antimicrobial activity by several different mechanisms. These peptides are named after their unusual threaded shape, resembling a slipknot, and are biosynthesized from a ribosomal precursor peptide via the action of two enzymes, a protease and lasso cyclase. The compact, threaded structure of lasso peptides shields portions of the amide backbone, often rendering the peptides protease resistant. With advances in genomic sequencing, we can now find and predict new lasso peptides and other ribosomally synthesized and post-translationally modified peptides (RiPPs) that may not be detected from cultivation of the native species in the lab, enabling discovery of potential new drug compounds.

Many lasso peptides display focused spectra of activity, presenting a route to target specific pathogens without disturbing commensal bacteria. From past investigations with lasso peptides such as ubonodin, citrocin, klebsidin, microcin J25 , and capistruin,  it was noticed that these compounds often target strains that are phylogenetically similar to the producer, potentially serving as a mechanism for competition in microbial communities. We thus hypothesized that lasso peptide biosynthetic gene clusters (BGCs) found in pathogen-related species would be more likely to have antimicrobial activity against clinically relevant pathogens (a guilt-by-association approach), presenting an effective way to screen and prioritize genome mining hits.

In a discovery with implications for the drug-resistance crisis, Princeton Engineering researchers have isolated a compound that kills bacteria that can cause incurable infections. The compound, called cloacaenodin, is a short, slip-knotted chain of amino acids known as a lasso peptide, encoded by gut-dwelling bacteria as a defense mechanism. Peptides do all kinds of things in the body and have been used in a wide range of medical treatments. This peptide works by attacking rival bacteria, and it’s a very potent killer, according to A. James Link, professor of chemical and biological engineering.  When released, the peptide hooks into a target cell’s RNA-producing enzymes and shuts down basic cell functions. It targets an especially fearsome group of pathogens belonging to the genus Enterobacter, has identified as a primary driver in an accelerating global crisis: bacterial infections that increasingly do not respond to conventional antibiotics.

The research team has discovered several peptides in this same class—structured with a ring knotted to a tail that threads back down through the ring, like a lasso in a rodeo trick that show promising antibacterial properties. According to the authors, cloacaenodin is unique because it can kill clinically relevant drug-resistant strains, making it a promising subject for antibiotic development. The finding also suggests peptide-mining and synthetic biology techniques could reveal more antimicrobial compounds with strong drug-development potential, an essential step in quelling the growing superbug crisis.

While cloacaenodin shows strong antibacterial properties, it’s only the first of many steps to a new treatment. Determining a compound’s safety is difficult and expensive, and moving from initial testing through the regulatory process takes a minimum of 10 years. The researchers said that, historically, some peptides have proven toxic to the kidneys, curbing their use in drugs. But peptides with bacterial-selective activity that don’t harm animal cells will likely lack this toxicity. But this new compound shows promising antibacterial properties and the researchers have only just begun to consider what comes next. They plan to start by testing it in animal infection models to confirm that it can clear the infection and that it is safe for animal cells. More broadly, however, this compound’s discovery suggests that Link and his team have developed a peptide-mining toolkit that will turn up many other interesting compounds in the future, and there is no telling where that will lead.

Cloacaenodin, an Antimicrobial Lasso Peptide with Activity against Enterobacter - Medicine Innovates

About the author

A. James Link
Professor of Chemical and Biological Engineering
Princeton University

the Link lab has been interested in applying the tools of protein engineering and bioconjugate chemistry to engineer peptides and proteins with conformational constraints. Conformational constraints within a polypeptide can lead to improvements in properties such as protease stability, thermostability, and binding affinity. With the development of bioorthogonal chemistry in the early 2000’s, the toolbox for conformational constraints expanded. We have used a combination of protein engineering and azide-alkyne click chemistry to carry out “protein stapling” on small proteins (Abdeljabbar et al. Chem. Comm. 2014). We have also used olefin metathesis chemistry to constrain short engineered peptides that function to turn on apoptosis in cells (Link and Zhang, US patent 9,464,125). We have also looked to nature for inspiration in strategies for conformationally constraining peptides. This has led to our program on lasso peptides, an ever expanding class of natural products defined by their slipknot-like topology. Our group started out working on the antimicrobial lasso peptide microcin J25 as an interesting substrate for engineering by directed evolution (Pan et al. JACS 2011) and with unnatural amino acids (Piscotta et al. Chem. Comm 2015). We became interested in the variety of different lasso peptides present in nature and developed the first algorithm for large-scale genome mining of lasso peptides (Maksimov et al. PNAS 2012). We continue to look to lasso peptides as a source of new antibiotics, interesting new enzymology, and even as building blocks for molecular machines.


Drew V. Carson, Monica Patiño, Hader E. Elashal, Alexis Jaramillo Cartagena, Yi Zhang, Megan E. Whitley, Larry So, Angelo K. Kayser-Browne, Ashlee M. Earl, Roby P. Bhattacharyya, and A. James Link, Cloacaenodin, an Antimicrobial Lasso Peptide with Activity against Enterobacter, ACS Infectious Diseases (2022). DOI: 10.1021/acsinfecdis.2c00446

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