Conjugation of Cell-Penetrating Peptides to Antimicrobial Peptides Enhances Antibacterial Activity

Abstract

Antimicrobial peptides (AMPs), essential elements in host innate immune defenses against numerous pathogens, have received considerable attention as potential alternatives to conventional antibiotics. Most AMPs exert broad-spectrum antimicrobial activity through depolarization and permeabilization of the bacterial cytoplasmic membrane. Here, we introduce a new approach for enhancing the antibiotic activity of AMPs by conjugation of a cationic cell-penetrating peptide (CPP). Interestingly, CPP-conjugated AMPs elicited only a 2- to 4-fold increase in antimicrobial activity against Gram-positive bacteria, but showed a 4- to 16-fold increase in antimicrobial activity against Gram-negative bacteria. Although CPP–AMP conjugates did not significantly increase membrane permeability, they efficiently translocated across a lipid bilayer. Indeed, confocal microscopy showed that, while AMPs were localized mainly in the membrane of Escherichia coli, the conjugates readily penetrated bacterial cells. In addition, the conjugates exhibited a higher affinity for DNA than unconjugated AMPs. Collectively, we demonstrate that CPP–AMP conjugates possess multiple functional properties, including membrane permeabilization, membrane translocation, and DNA binding, which are involved in their enhanced antibacterial activity against Gram-negative bacteria. We propose that conjugation of CPPs to AMPs may present an effective approach for the development of novel antimicrobials against Gram-negative bacteria.

Molecular Basis of the Anticancer and Antibacterial Properties of CecropinXJ Peptide: An In Silico Study

“…As opposed to common AMPs, many CPPs are able to enter the cell without damaging the membrane [ 7 , 8 , 32 ] and exert their killing action inducing apoptosis or targeting intracellular macromolecules such as DNA, RNA, ribosomes or organelles such as mitochondria [ 11 , 31 , 109 , 125 , 126 , 127 , 128 ], as it is the case for MPPs [ 11 ]. This mechanism is used by CPPs such as coprisin, some magainins or cecropins and postulated for CXJ [ 4 , 5 ].…”

“…MPPs, a special class of CPPs able to target mitochondria, have displayed remarkable properties for medical applications [ 31 ]. They have been shown to cause apoptosis in cancer cells [ 6 ] and to enter the cell without necessarily damaging the plasmatic membrane [ 7 , 8 , 32 ]. Once inside, they can target the mitochondrion acting as membrane-disrupting agents or inducing apoptosis with different mechanisms [ 31 ].…”

Bistable Bacterial Growth Dynamics in the Presence of Antimicrobial Agents

“…It was recently established that when cell-penetrating peptides are employed in conjunction with AMPs, the MIC is reduced [ 43 , 44 , 45 ]. We do expect, as we established with all other non-linear antimicrobial mechanisms of action, that IE will be identified also when such combinations are employed.…”

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance

“…Cell penetrating peptides (CPPs) themselves have been harnessed to improve the antimicrobial activity of AMPs. Interestingly, these CPPs often negligibly affect the cytotoxicity of the AMPs while improving their antimicrobial activity and specificity, particularly when targeting Gram-negative bacteria [140,141]. Hydrophilic polypeptides (e.g., XTEN and PAS) have also gained much interest in recent years as alternatives to polymers for protein conjugation [26], though no examples of AMP conjugation have yet been published.…”