Conjugation of a Cell-Penetrating Peptide to the Lasso Peptides Citrocin and Microcin J25 Enhances Antimicrobial Activity

A subset of lasso peptides function as antibiotics against Gram-negative bacteria by inhibition of the bacterial RNA polymerase (RNAP), including microcin J25 (MccJ25) and citrocin. These peptides interact with specific transporters in the inner and outer membranes of bacteria to gain access to the cytoplasm. The reliance on transporters limits the breadth of spectrum of these lasso peptide antibiotics and opens up paths to drug resistance. Here, we detail a strategy to overcome the transport limitations of lasso peptides: covalent conjugation of the lasso peptide to a cell penetrating peptide (CPP). Citrocin-CPP and MccJ25-CPP conjugates were synthesized by attaching the CPP to the loop of the lasso peptide and tested for antimicrobial activity against the ESKAPE pathogens. Citrocin-CPP and MccJ25-CPP have antimicrobial activity against all four Gram-negative ESKAPE pathogens, a broader spectrum of activity than the parent lasso peptides. Citrocin-CPP and MccJ25-CPP also exhibit more potent activity than the parent lasso peptides, with minimum inhibitory concentrations in the high nanomolar to single micromolar range. The uptake of citrocin-CPP and MccJ25-CPP was also studied in Escherichia coli revealing that the conjugates are internalized via both transporter-dependent and transporter-independent pathways.