RESUMO
INTRODUCTION: Antimicrobial peptides (AMPs) are valuable alternatives to traditional antibiotics, possess a variety of potent biological activities and exhibit immunomodulatory effects that alleviate difficult-to-treat infections. Clarifying the structure-activity relationships of AMPs can direct the synthesis of desirable peptide therapeutics. OBJECTIVES: In this study, the lipopolysaccharide-binding domain (LBD) was identified through machine learning-guided directed evolution, which acts as a functional domain of the anti-lipopolysaccharide factor family of AMPs identified from Marsupenaeus japonicus. METHODS: LBDA-D was identified as an output of this algorithm, in which the original LBDMj sequence was the input, and the three-dimensional solution structure of LBDB was determined using nuclear magnetic resonance. Furthermore, our study involved a comprehensive series of experiments, including morphological studies and in vitro and in vivo antibacterial tests. RESULTS: The NMR solution structure showed that LBDB possesses a circular extended structure with a disulfide crosslink at the terminus and two 310-helices and exhibits a broad antimicrobial spectrum. In addition, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed that LBDB induced the formation of a cluster of bacteria wrapped in a flexible coating that ruptured and consequently killed the bacteria. Finally, coinjection of LBDB, Vibrio alginolyticus and Staphylococcus aureus in vivo improved the survival of M. japonicus, demonstrating the promising therapeutic role of LBDB for treating infectious disease. CONCLUSIONS: The findings of this study pave the way for the rational drug design of activity-enhanced peptide antibiotics.
RESUMO
Medium-chain-length (mcl)-polyhydroxyalkanoates (PHAs), elastomeric polyesters synthesized by Genus Pseudomonas bacteria, generally have many different monomer components. In this study, PHAs biosynthesized by four type strains of Pseudomonas (P. putida, P. citronellolis, P. oleovorans, and P. pseudoalcaligenes) and a typical PHA producer (P. putida KT2440) were characterized in terms of the monomer structure and composition by gas chromatography-mass spectrometry (GC-MS) analysis. With a thiomethyl pretreatment of PHA methanolysis derivatives, two unsaturated monomers, 3-hydroxy-5-dodecenoate (3H5DD) and 3-hydroxy-5-tetradecenoate (3H5TD), were identified in mcl-PHAs produced by P. putida and P. citronellolis. The quantitative analysis of PHA monomers was performed by employing GC-MS with methanolysis derivatives, and the results coincided with those obtained by performing nuclear magnetic resonance spectroscopy. Only poly(3-hydroxybutyrate) was detected from the P. oleovorans and P. pseudoalcaligenes type strains. These analytical results would be useful as a reference standard for phenotyping of new PHA-producing bacteria.
