A novel lasso peptide platform for targeted, protease-resistant antimicrobial therapy against multidrug-resistant Enterobacter

Background

The field of antimicrobial therapy is facing a critical challenge due to the rapid emergence and spread of multidrug-resistant (MDR) bacterial pathogens, particularly within healthcare settings. Among these, members of the Enterobacter cloacae complex (ECC) have become increasingly problematic, as they are responsible for a significant proportion of hospital-acquired infections and are frequently associated with resistance to last-line antibiotics such as carbapenems. The growing prevalence of carbapenem-resistant Enterobacter strains has rendered many conventional antibiotics ineffective, leading to higher morbidity, mortality, and healthcare costs. This urgent clinical need has driven the search for novel antimicrobial agents with unique mechanisms of action and improved stability profiles, especially those capable of selectively targeting problematic pathogens while minimizing disruption to the beneficial microbiota.

Current approaches to combating MDR Enterobacter infections rely heavily on broad-spectrum antibiotics, which not only contribute to the development of resistance but also disrupt the natural microbial balance in patients. Many existing antimicrobial peptides, while promising due to their potent activity, suffer from rapid degradation by proteases in the body, limiting their therapeutic potential and shelf-life. Additionally, most available antibiotics lack specificity, leading to off-target effects and collateral damage to non-pathogenic bacteria. The limited stability and selectivity of current treatments underscore the need for new antimicrobial solutions that can withstand proteolytic degradation, maintain efficacy against resistant strains, and offer a narrow spectrum of activity to reduce unintended consequences.

Technology

Cloacaenodin is a newly discovered lasso peptide produced by species within the Enterobacter cloacae complex, featuring a distinctive slipknot-like structure. This peptide consists of a 9-membered macrolactam ring formed between its first and ninth amino acids, through which the peptide tail is threaded and locked in place by bulky aromatic residues, resulting in a highly stable, protease-resistant conformation. The biosynthetic gene cluster responsible for cloacaenodin production was identified through genome mining and is encoded by a rare, plasmid-borne operon. Cloacaenodin is efficiently produced in E. coli using recombinant DNA technology, and its unique topology is confirmed by NMR spectroscopy. The peptide exhibits potent, narrow-spectrum antimicrobial activity against clinically significant Enterobacter strains, including carbapenem-resistant isolates, with minimum inhibitory concentrations in the low micromolar range. Its antimicrobial action is linked to inhibition of bacterial RNA polymerase, and only the threaded, lassoed form retains biological activity.

What sets cloacaenodin apart is its combination of structural novelty, target specificity, and exceptional stability. Unlike typical linear or even other lasso peptides, cloacaenodin’s 9-membered ring and C-terminal serine are critical for maintaining its threaded conformation, which confers remarkable resistance to proteolytic degradation and ensures prolonged activity in biological environments. Its selectivity for Enterobacter species, sparing beneficial bacteria, addresses a major challenge in antimicrobial therapy by reducing collateral damage to the microbiome. Furthermore, cloacaenodin remains stable under physiological conditions and can be stored indefinitely in lyophilized form, overcoming common limitations of peptide-based therapeutics. The comprehensive structure-activity studies and robust production platform position cloacaenodin as a differentiated candidate for development against multidrug-resistant hospital pathogens, highlighting the power of genome mining in uncovering next-generation antibiotics.

Benefits

• Potent and selective antimicrobial activity against multidrug-resistant Enterobacter strains, including carbapenem-resistant isolates
• High resistance to proteolytic degradation, enhancing in vivo stability and shelf-life
• Unique lasso peptide structure ensures stability and activity under physiological conditions
• Narrow-spectrum targeting preserves beneficial microbiota by sparing non-target bacteria
• Heterologous production in E. coli enables scalable recombinant manufacturing
• Potential for standalone therapy or combination with existing antibiotics to combat resistant infections
• Stable in lyophilized form and under cold storage, facilitating pharmaceutical formulation and storage

Applications

• Treatment of drug-resistant Enterobacter infections
• Hospital-acquired infection therapeutics
• Targeted microbiome-sparing antibiotics
• Combination therapy with existing antibiotics

Additional info

Cloacaenodin is a novel lasso peptide from Enterobacter cloacae, characterized by a unique threaded slipknot-like structure with a 9-membered macrolactam ring. Produced recombinantly, it exhibits exceptional proteolytic resistance. This peptide selectively targets and inhibits bacterial RNA polymerase, demonstrating potent antimicrobial efficacy against carbapenem-resistant Enterobacter strains, offering a solution for multidrug-resistant infections.