The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human alpha-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

What We Already Know about This Topic What This Article Tells Us That Is New Background Pyroptosis, a type of proinflammatory programmed cell death, drives cytokine storm. Caspase-11–dependent macrophage pyroptosis contributes to mortality during sepsis. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling can amplify interleukin-1β secretion in endotoxin-induced inflammation. Here, we hypothesized that S1PR2 signaling increases caspase-11–dependent macrophage pyroptosis and worsens Gram-negative sepsis outcome. Methods A Gram-negative sepsis model was induced through intraperitoneal injection of Escherichia coli. Primary peritoneal macrophages isolated from wild-type, S1pr2-deficient (S1pr2-/-), or nucleotide-binding oligomerization domain-like receptor protein-3–deficient mice were treated with E. coli. Caspase-11 activation, macrophage pyroptosis, and Ras homolog gene family, member A-guanosine triphosphate levels were assessed in those cells. Additionally, monocyte caspase-4 (an analog of caspase-11) expression and its correlation with S1PR2 expression were determined in patients with Gram-negative sepsis (n = 11). Results Genetic deficiency of S1PR2 significantly improved survival rate (2/10 [20%] in wild-type vs. 7/10 [70%] in S1pr2-/-, P = 0.004) and decreased peritoneal macrophage pyroptosis (pyroptosis rate: 35 ± 3% in wild-type vs. 10 ± 3% in S1pr2-/-, P < 0.001). Decreased caspase-11 activation in S1PR2 deficiency cells contributed to the reduced macrophage pyroptosis. In addition, RhoA inhibitor abrogated the amplified caspase-11 activation in wild-type or S1PR2-overexpressing cells. In patients with Gram-negative sepsis, caspase-4 increased significantly in monocytes compared to nonseptic controls and was positively correlated with S1PR2 (r = 0.636, P = 0.035). Conclusions S1PR2 deficiency decreased macrophage pyroptosis and improved survival in E. coli sepsis. These beneficial effects were attributed to the decreased caspase-11 activation of S1PR2-deficient macrophages. S1PR2 and caspase-11 may be promising new targets for treatment of sepsis.

Gram-negative sepsis has become one of major increasing medical burdens globally, which is subjected to growing antibiotic resistance problem and the relatively delayed development of new antibiotics. LL-37, the only type of Cathelicidin identified in humans, has diverse biological activities including direct bactericidal action, regulation of inflammation and LPS-neutralization. The KR-12 peptide is the smallest portion of LL-37 with antibacterial action, which has been shown that could be modified into more effective antimicrobials. Here, we synthesize two myristoylated derivatives of KR-12, Myr-KR-12N and Myr-KR-12C, which can spontaneously form nanoparticles when mixed with deionized water. We show that myristoylated KR-12 derivatives possess a broad-spectrum and more powerful bactericidal activity through interrupting the membranes of bacteria. Myr-KR-12N rescues mice from lethal sepsis induced by E. coli, even more potent rescuing activity than meropenem. We also demonstrate that myristoylated KR-12 nanobiotic can significantly bind with LPS and inhibit the inflammation in vitro and Myr-KR-12N rescue mice from LPS-induced sepsis in vivo, even more potent rescuing activity than polymyxin B. Toxic experiments indicate that neither Myr-KR-12N nor Myr-KR-12C nanobiotics exhibits meaningful hemolytic activity, liver and kidney injury. We thus developed a novel nanobiotic with dual bactericidal and LPS-neutralization properties, which may provide good insights for clinical translation of antimicrobial peptides and the creation of new antibiotics.

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