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High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide.

ABSTRACT: The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.

SUBMITTER: Cho HS

PROVIDER: S-EPMC8073543 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cathelicidins, a family of host defence peptides in vertebrates, play an important role in the innate immune response, exhibiting antimicrobial activity against many bacteria, as well as viruses and fungi. This work describes the design and synthesis of shortened analogues of porcine cathelicidin PMAP-36, which contain structural changes to improve the pharmacokinetic properties. In particular, 20-mers based on PMAP-36 (residues 12-31) and 13-mers (residues 12-24) with modification of amino acid residues at critical positions and introduction of lipid moieties of different lengths were studied to identify the physical parameters, including hydrophobicity, charge, and helical structure, required to optimise their antibacterial activity. Extensive conformational analysis, performed by CD and NMR, revealed that the substitution of Pro25-Pro26 with Ala25-Lys26 increased the α-helix content of the 20-mer peptides, resulting in broad-spectrum antibacterial activity against Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus epidermidis strains. Interestingly, shortening to just 13 residues resulted in only a slight decrease in antibacterial activity. Furthermore, two sequences, a 13-mer and a 20-mer, did not show cytotoxicity against HaCat cells up to 64 µM, indicating that both derivatives are not only effective but also selective antimicrobial peptides. In the short peptide, the introduction of the helicogenic α-aminoisobutyric acid forced the helix toward a prevailing 310 structure, allowing the antimicrobial activity to be maintained. Preliminary tests of resistance to Ser protease chymotrypsin indicated that this modification resulted in a peptide with an increased in vivo lifespan. Thus, some of the PMAP-36 derivatives studied in this work show a good balance between chain length, antibacterial activity, and selectivity, so they represent a good starting point for the development of even more effective and proteolysis-resistant active peptides.

Dataset Information

High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide.

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