Exploring the Therapeutic Potential of Scorpion-Derived Css54 Peptide Against Candida albicans.

Candida albicans (C. albicans) is one of the most common opportunistic fungi worldwide, which is associated with a high mortality rate. Despite treatment, C. albicans remains the leading cause of life-threatening invasive infections. Consequently, antimicrobial peptides (AMPs) are potential alternatives as antifungal agents with excellent antifungal activity. We previously reported that Css54, found in the venom of Centrurodies suffusus suffusus (C. s. suffusus) showed antibacterial activity against zoonotic bacteria. However, the antifungal activity of Css54 has not yet been elucidated. The objective of this study was to identify the antifungal activity of Css54 against C. albicans and analyze its mechanism. Css54 showed high antifungal activity against C. albicans. Css54 also inhibited biofilm formation in fluconazole-resistant fungi. The antifungal mechanism of action of Css54 was investigated using membrane-related assays, including the membrane depolarization assay and analysis of the membrane integrity of C. albicans after treatment with Css54. Css54 induced reactive oxygen species (ROS) production in C. albicans, which affected its antifungal activity. Our results indicate that Css54 causes membrane damage in C. albicans, highlighting its value as a potential therapeutic agent against C. albicans infection.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

Cathelicidin-related antimicrobial peptide mediates skeletal muscle degeneration caused by injury and Duchenne muscular dystrophy in mice.

BACKGROUND: Cathelicidin, an antimicrobial peptide, plays a key role in regulating bacterial killing and innate immunity; however, its role in skeletal muscle function is unknown. We investigated the potential role of cathelicidin in skeletal muscle pathology resulting from acute injury and Duchenne muscular dystrophy (DMD) in mice. METHODS: Expression changes and muscular localization of mouse cathelicidin-related antimicrobial peptide (Cramp) were examined in the skeletal muscle of normal mice treated with chemicals (cardiotoxin and BaCl2 ) or in dystrophic muscle of DMD mouse models (mdx, mdx/Utrn+/- and mdx/Utrn-/- ). Cramp penetration into myofibres and effects on muscle damage were studied by treating synthetic peptides to mouse skeletal muscles or C2C12 myotubes. Cramp knockout (KO) mice and mdx/Utrn/Cramp KO lines were used to determine whether Cramp mediates muscle degeneration. Muscle pathophysiology was assessed by histological methods, serum analysis, grip strength and lifespan. Molecular factors targeted by Cramp were identified by the pull-down assay and proteomic analysis. RESULTS: In response to acute muscle injury, Cramp was activated in muscle-infiltrating neutrophils and internalized into myofibres. Cramp treatments of mouse skeletal muscles or C2C12 myotubes resulted in muscle degeneration and myotube damage, respectively. Genetic ablation of Cramp reduced neutrophil infiltration and ameliorated muscle pathology, such as fibre size (P < 0.001; n = 6) and fibrofatty infiltration (P < 0.05). Genetic reduction of Cramp in mdx/Utrn+/- mice not only attenuated muscle damage (35%, P < 0.05; n = 9-10), myonecrosis (53%, P < 0.05), inflammation (37-65%, P < 0.01) and fibrosis (14%, P < 0.05) but also restored muscle fibre size (14%, P < 0.05) and muscle force (18%, P < 0.05). Reducing Cramp levels led to a 63% (male, P < 0.05; n = 10-14) and a 124% (female, P < 0.001; n = 20) increase in the lifespan of mdx/Utrn-/- mice. Proteomic and mechanistic studies revealed that Cramp cross-talks with Ca2+ signalling in skeletal muscle through sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase1 (SERCA1). Cramp binds and inactivates SERCA1, leading to the activation of Ca2+ -dependent calpain proteases that exacerbate DMD progression. CONCLUSIONS: These findings identify Cramp as an immune cell-derived regulator of skeletal muscle degeneration and provide a potential therapeutic target for DMD.

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.

PEP27-2, a Potent Antimicrobial Cell-Penetrating Peptide, Reduces Skin Abscess Formation during Staphylococcus aureus Infections in Mouse When Used in Combination with Antibiotics.

PEP27, a 27-amino acid (aa) peptide secreted by Streptococcus pneumoniae, is an autolytic peptide that functions as a major virulence factor. To develop a clinically applicable antimicrobial peptide (AMP), we designed PEP27 analogs with Trp substitutions to enhance its antimicrobial activity compared to that of PEP27. Particularly, PEP27-2 showed strong antimicrobial activity against a wide variety of bacteria, including multidrug-resistant (MDR) bacteria. It was found that the antimicrobial activity of PEP27-2 was increased by substituting Trp for the aa at the middle position of PEP27. We found that PEP27-2 acts as an effective cell-penetrating peptide in bacterial and mammalian cells. Here, we proved that subcutaneous infection with MDR Staphylococcus aureus induced skin lesions such as skeletal muscle damage, deep inflammation, and necrosis of the overlaying dermis in mice. Combination treatment with antibiotics revealed synergistic effects, remarkably reducing abscess size and improving the bacteria removal rate from the infection site. Moreover, PEP27-2-antibiotic combination treatment reduced inflammation, lowering levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, inducible NO synthase (iNOS), and cyclooxygenase (COX-2) in skin abscess tissue. The results suggest that the PEP27-2 peptide is a promising therapeutic option for combating MDR bacterial strains by enhancing antibiotic penetration and protecting against MDR bacteria.

A phage protein-derived antipathogenic peptide that targets type IV pilus assembly.

Phage-inspired antibacterial discovery is a new approach that recruits phages in search for antibacterials with new molecular targets, in that phages are the biological entities well adapted to hijack host bacterial physiology in favor of their own thrive. We previously observed that phage-mediated twitching motility inhibition was effective to control the acute infections caused by Pseudomonas aeruginosa and that the motility inhibition was attributed to the delocalization of PilB, the type IV pilus (TFP) assembly ATPase by binding of the 136-amino acid (aa) phage protein, Tip. Here, we created a series of truncated and point-mutant Tip proteins to identify the critical residues in the Tip bioactivity: N-terminal 80-aa residues were dispensable for the Tip activity; we identified that Asp82, Leu84, and Arg85 are crucial in the Tip function. Furthermore, a synthetic 15-aa peptide (P1) that corresponds to Leu73 to Ala87 is shown to suffice for PilB delocalization, twitching inhibition, and virulence attenuation upon exogenous administration. The transgenic flies expressing the 15-aa peptide were resistant to P. aeruginosa infections as well. Taken together, this proof-of-concept study reveals a new antipathogenic peptide hit targeting bacterial motility and provides an insight into antibacterial discovery targeting TFP assembly.