ABSTRACT
Antimicrobial peptides are prominent components of the plant immune system acting against a wide variety of pathogens. Legume plants from the inverted repeat lacking clade (IRLC) have evolved a unique gene family encoding nodule-specific cysteine-rich NCR peptides acting in the symbiotic cells of root nodules, where they convert their bacterial endosymbionts into non-cultivable, polyploid nitrogen-fixing cells. NCRs are usually 30-50 amino acids long peptides having a characteristic pattern of 4 or 6 cysteines and highly divergent amino acid composition. While the function of NCRs is largely unknown, antimicrobial activity has been demonstrated for a few cationic Medicago truncatula NCR peptides against bacterial and fungal pathogens. The advantages of these plant peptides are their broad antimicrobial spectrum, fast killing modes of actions, multiple bacterial targets, and low propensity to develop resistance to them and no or low cytotoxicity to human cells. In the IRLC legumes, the number of NCR genes varies from a few to several hundred and it is possible that altogether hundreds of thousands of different NCR peptides exist. Due to the need for new antimicrobial agents, we investigated the antimicrobial potential of 104 synthetic NCR peptides from M. truncatula, M. sativa, Pisum sativum, Galega orientalis and Cicer arietinum against eight human pathogens, including ESKAPE bacteria. 50 NCRs showed antimicrobial activity with differences in the antimicrobial spectrum and effectivity. The most active peptides eliminated bacteria at concentrations from 0.8 to 3.1 µM. High isoelectric point and positive net charge were important but not the only determinants of their antimicrobial activity. Testing the activity of shorter peptide derivatives against Acinetobacter baumannii and Candida albicans led to identification of regions responsible for the antimicrobial activity and provided insight into their potential modes of action. This work provides highly potent lead molecules without hemolytic activity on human blood cells for novel antimicrobial drugs to fight against pathogens.
ABSTRACT
Streptomyces sp. RAB12 having potential to produce novel actinomycin group compounds was isolated from soil samples collected from CSIR-Indian Institute of Chemical Technology, Hyderabad, India, garden premises using International Streptomycetes Project (ISP) protocols. The 16S rRNA sequence of the strain RAB12 exhibited identity with Streptomyces sp. 13647M and the sequence was deposited in NCBI under the accession number KY 203650 while the strain RAB12 was deposited in The Microbial Type Culture Collection and Gene Bank (MTCC) with accession number MTCC 12747. Cell-free extract of this novel strain revealed two bioactive principles viz., RSP 01 and RSP 02. HR-MS analysis indicated a molecular mass of 1269.61 and 1270.63 m/z g/mol for RSP 01 and RSP 02, respectively. Proton 1H, 13C NMR, 2D NMR and mass spectroscopy analysis revealed a similar fingerprint to that actinomycin D except for a peak at δH3.59 J (1H NMR) and δ 208.88 (13C NMR) for RSP 01 compound suggesting the presence of keto carbonyl at 5-oxo position on the proline moiety which is absent in actinomycin D. Purified RSP 02 depicted a similarity with RSP 01 except a peak in the 1H proton NMR at δH 3.81 J. HR-ESI mass spectra confirmed the molecular formulae for RSP 01 and RSP 02 as C62H84N12O17 and C62H86N12O17, respectively. Antimicrobial activity profile revealed higher antimicrobial activity against bacterial strains (Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis) and Candida albicans compared to standard actinomycin D. MIC and MBC for RSP 01 were observed to be 0.0039 and 0.0078 (µg/ml) against C. albicans, while for actinomycin D, it was found to be 0.031 and 0.62 (µg/ml), respectively indicating a tenfold higher potency. Thus, these RSP 01 and RSP 02 compounds from Streptomyces sp. RAB12 may be promising candidates for industrial and clinical applications.
