Therapeutic Potential of Antimicrobial Peptide PN5 against Multidrug-Resistant E. coli and Anti-Inflammatory Activity in a Septic Mouse Model.

Antibiotic-resistant bacteria have become a public health problem. Thus, antimicrobial peptides (AMPs) have been evaluated as substitutes for antibiotics. Herein, we investigated PN5 derived from Pinus densiflora (pine needle). PN5 exhibited antimicrobial activity without causing cytotoxic effects. Based on these results, we examined the mode of action of PN5 against Gram-negative and -positive bacteria. PN5 exhibited membrane permeabilization ability, had antimicrobial stability in the presence of elastase, a proteolytic enzyme, and did not induce resistance in bacteria. Bacterial lipopolysaccharide (LPS) induces an inflammatory response in RAW 264.7 macrophages. PN5 suppressed proinflammatory cytokines mediated by NF-κB and mitogen-activated protein kinase signaling. In C57BL/6J mice treated with LPS and d-galactosamine, PN5 exhibited anti-inflammatory activity in inflamed mouse livers. Our results indicate that PN5 has antimicrobial and anti-inflammatory activities and thus may be useful as an antimicrobial agent to treat septic shock caused by multidrug-resistant (MDR) Escherichia coli without causing further resistance. IMPORTANCE Antibiotic-resistant bacteria are a global health concern. There is no effective treatment for antibiotic-resistant bacteria, and new alternatives are being suggested. The present study found antibacterial and anti-inflammatory activities of PN5 derived from Pinus densiflora (pine needle), and further investigated the therapeutic effect in a mouse septic model. As a mechanism of antibacterial activity, PN5 exhibited the membrane permeabilization ability of the toroidal model, and treated strains did not develop drug resistance during serial passages. PN5 showed immunomodulatory properties of neutralizing LPS in a mouse septic model. These results indicate that PN5 could be a new and promising therapeutic agent for bacterial infectious disease caused by antibiotic-resistant strains.

A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids.

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA. We discovered that the length-variable poly-Arg tail marked distinctly the molecular processes associated with the nanopore-mediated duplexes capture, trapping and unzipping. This enabled the detection of ssDNA targets via the signatures of (poly-Arg)-PNA-cDNA blockade events, rendered most efficient from the ß-barrel entrance of the nanopore, and scaled proportional in efficacy with a larger poly-Arg moiety. We illustrate the approach by sensing synthetic ssDNAs designed to emulate fragments from two regions of SARS-CoV-2 nucleocapsid phosphoprotein N-gene.

Antimicrobial peptide-loaded gold nanoparticle-DNA aptamer conjugates as highly effective antibacterial therapeutics against Vibrio vulnificus.

Vibrio vulnificus causes fatal infections in humans, and antibiotics are commonly used in treatment regimens against V. vulnificus infection. However, the therapeutic effects of antibiotics are limited by multidrug resistance. In this study, we demonstrated that an antimicrobial peptide (AMP), HPA3PHis, loaded onto a gold nanoparticle-DNA aptamer (AuNP-Apt) conjugate (AuNP-Apt-HPA3PHis) is an effective therapeutic tool against V. vulnificus infection in vivo in mice. HPA3PHis induced bacterial cell death through the disruption of membrane integrity of V. vulnificus. The introduction of AuNP-Apt-HPA3PHis into V. vulnificus-infected HeLa cells dramatically reduced intracellular V. vulnificus by 90%, leading to an increase in the viability of the infected cells. Moreover, when V. vulnificus-infected mice were intravenously injected with AuNP-Apt-HPA3PHis, a complete inhibition of V. vulnificus colonization was observed in the mouse organs, leading to a 100% survival rate among the treated mice, whereas all the control mice died within 40 hours of being infected. Therefore, this study demonstrated the potential of an AMP delivered by AuNP-Apt as an effective and rapid treatment option against infection caused by a major pathogen in humans and aquatic animals.