“Metaphilic” Cell-Penetrating Polypeptide-Vancomycin Conjugate Efficiently Eradicates Intracellular Bacteria via a Dual Mechanism

[Image: see text] Infections by intracellular pathogens are difficult to treat because of the poor accessibility of antibiotics to the pathogens encased by host cell membranes. As such, a strategy that can improve the membrane permeability of antibiotics would significantly increase their efficiency against the intracellular pathogens. Here, we report the design of an adaptive, metaphilic cell-penetrating polypeptide (CPP)–antibiotic conjugate (VPP-G) that can effectively eradicate the intracellular bacteria both in vitro and in vivo. VPP-G was synthesized by attaching vancomycin to a highly membrane-penetrative guanidinium-functionalized metaphilic CPP. VPP-G effectively kills not only extracellular but also far more challenging intracellular pathogens, such as S. aureus, methicillin-resistant S. aureus, and vancomycin-resistant Enterococci. VPP-G enters the host cell via a unique metaphilic membrane penetration mechanism and kills intracellular bacteria through disruption of both cell wall biosynthesis and membrane integrity. This dual antimicrobial mechanism of VPP-G prevents bacteria from developing drug resistance and could also potentially kill dormant intracellular bacteria. VPP-G effectively eradicates MRSA in vivo, significantly outperforming vancomycin, which represents one of the most effective intracellular antibacterial agents reported so far. This strategy can be easily adapted to develop other conjugates against different intracellular pathogens by attaching different antibiotics to these highly membrane-penetrative metaphilic CPPs.