Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Abstract

Commonly used antimicrobials show poor cellular uptake and often have limited access to intracellular targets, resulting in low antimicrobial activity against intracellular pathogens. An efficient delivery system to transport these drugs to the intracellular site of action is needed. Cell-penetrating peptides (CPPs) mediate the internalization of biologically active molecules into the cytoplasm. Here, we characterized two CPPs, ␣1H and ␣2H, derived from the Yersinia enterocolitica YopM effector protein. These CPPs, as well as Tat (trans-activator of transcription) from HIV-1, were used to deliver the antibiotic gentamicin to target intracellular bacteria. The YopM-derived CPPs penetrated different endothelial and epithelial cells to the same extent as Tat. CPPs were covalently conjugated to gentamicin, and CPP-gentamicin conjugates were used to target infected cells to kill multiple intracellular Gram-negative pathogenic bacteria, including Escherichia coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Taken together, CPPs show great potential as delivery vehicles for antimicrobial agents and may contribute to the generation of new therapeutic tools to treat infectious diseases caused by intracellular pathogens.

Drug delivery systems designed to overcome antimicrobial resistance

“…After its endocytic internalization, the conjugate will be released from the endosomes onto the cytosol to exert its ATB action . Over the past years, this approach has been reported as a promising delivery strategy to enhance the permeability and the ATB activity of drugs against intracellular microbes …”

“…In a recent study, Gomarasca et al used two bacterial CPPs derived from the Yersinia enterocolitica (YopM effector proteins), α1H ( 91 ) and α2H ( 92 ), as well as Tat 47‐57 ( trans activator of transcription) ( 93 ) from HIV‐1 to deliver the antibiotic gentamicin ( 94 ) (Figure ), to target intracellular Gram‐negative pathogenic bacteria. In fact, ( 94 ) is a potent aminoglycoside antibiotic against extracellular pathogenic bacteria, but possesses a poor permeability to mammalian cell membranes.…”

“…Gentamicin ( 94 ) was covalently conjugated with the three CPPs through a hetero bifunctional cross‐linker succinimidyl‐4‐( N ‐maleimidomethyl) cyclohexane‐1‐carboxylate (Figure ). For conjugation purposes, the authors added an N ‐terminal cysteine (Cys) residue to the Tat sequence (shorten Tat 47‐57 ), a C ‐terminal Cys to the α1H sequence, and a natural Cys residue was used in the case of α2H …”

“…Gentamicin ( 94 ) was firstly mixed with the cross‐linker SMCC ( 95 ) in PBS, then incubated for 2 hours at room temperature (rt) to form a stable amide bond. After that, the CPPs were added into the mixture and incubated for another 2 hours at rt to form a thioether bond . CPP, cell‐penetrating peptides; PBS, phosphate‐buffered saline; SMCC, succinimidyl‐4‐(N‐maleimidomethyl) cyclohexane‐1‐carboxylate [Color figure can be viewed at wileyonlinelibrary.com]…”

“…The obtained CPP‐gentamicin conjugates ( 96‐98 ) were firstly evaluated against multiple intracellular pathogens including E. coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri (Table ) . The results showed that the CPP‐gentamicin conjugates ( 96‐98 ) were capable of retaining their ATB activity when compared to the parent antibiotic gentamicin.…”