Potent Chimeric Antimicrobial Derivatives of the Medicago truncatula NCR247 Symbiotic Peptide

Abstract

In Rhizobium-legume symbiosis, the bacteria are converted into nitrogen-fixing bacteroids. In many legume species, differentiation of the endosymbiotic bacteria is irreversible, culminating in definitive loss of their cell division ability. This terminal differentiation is mediated by plant peptides produced in the symbiotic cells. In Medicago truncatula more than ∼700 nodule-specific cysteine-rich (NCR) peptides are involved in this process. We have shown previously that NCR247 and NCR335 have strong antimicrobial activity on various pathogenic bacteria and identified interaction of NCR247 with many bacterial proteins, including FtsZ and several ribosomal proteins, which prevent bacterial cell division and protein synthesis. In this study we designed and synthetized various derivatives of NCR247, including shorter fragments and various chimeric derivatives. The antimicrobial activity of these peptides was tested on the ESKAPE bacteria; Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli as a member of Enterobacteriaceae and in addition Listeria monocytogenes and Salmonella enterica. The 12 amino acid long C-terminal half of NCR247, NCR247C partially retained the antimicrobial activity and preserved the multitarget interactions with partners of NCR247. Nevertheless NCR247C became ineffective on S. aureus, P. aeruginosa, and L. monocytogenes. The chimeric derivatives obtained by fusion of NCR247C with other peptide fragments and particularly with a truncated mastoparan sequence significantly increased bactericidal activity and altered the antimicrobial spectrum. The minimal bactericidal concentration of the most potent derivatives was 1.6 µM, which is remarkably lower than that of most classical antibiotics. The killing activity of the NCR247-based chimeric peptides was practically instant. Importantly, these peptides had no hemolytic activity or cytotoxicity on human cells. The properties of these NCR derivatives make them promising antimicrobials for clinical use.

Unexplored Arsenals of Legume Peptides With Potential for Their Applications in Medicine and Agriculture

“…While the antimicrobial activity of cationic peptides is generally attenuated by the presence of divalent cations and higher salt concentrations (Hancock and Sahl, 2006), the bactericidal activity of these chimeric peptides was maintained in Mueller Hinton broth. Importantly, these peptides did not have hemolytic activity or cytotoxicity on human cells (Jenei et al, 2020).…”

“…C-or N-terminal fusion of NCR247C with X2 made the chimeric peptides very effective on most strains at 1.6 and 3.1 µM MBCs. The MBCs of these chimeric derivatives were much lower than that of the classical antibiotic carbenicillin, and were comparable or even more effective than levofloxacin, a third generation antibiotic (Jenei et al, 2020). The killing activity of the NCR247-based chimeric peptides occurred within 0.1-5 min.…”

“…Based on the different mode of action and broad spectrum of NCRs it is conceivable that they may also be able to kill these resistant pathogens. The antibacterial activity of chemically synthesized NCR247 and NCR247-derivatives was investigated against ESKAPE strains (Enterococcus faecalis, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa) and E. coli, L. monocytogenes, and S. enterica (Jenei et al, 2020; Table 1). The minimal bactericidal concentration of NCR247 was 3.1 µM against P. aeruginosa and 6.3 µM against S. aureus and E. coli while killing of the other bacteria required higher concentrations.…”

“…Each of these chimeric peptides possessed higher antibacterial efficacy and affected the antimicrobial spectrum. In the case of X1-NCR247C TABLE 1 | The amino acid sequence of NCR247 and its derivatives (A) and the minimal bactericidal concentrations (MBCs) of these peptides and antibiotics (in µM) on different pathogens (B) (from Jenei et al, 2020 the minimal bactericidal concentrations (MBC) varied between 1.6 and 12.5 µM. C-or N-terminal fusion of NCR247C with X2 made the chimeric peptides very effective on most strains at 1.6 and 3.1 µM MBCs.…”

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species

“…NCR247 acts via multiple mechanisms, interacting with the bacterial membranes and by entering the cells with many bacterial proteins and affecting both transcription and translation and inhibiting cell division by interacting with the conserved bacterial FtsZ protein required for septum formation and cell division [ 11 , 12 ]. Shorter chimeric derivatives of NCR247 gained even higher activities and were also able to kill the most resistant and problematic ESKAPE bacteria at low concentrations making them comparable or even superior to third generation antibiotics with the advantage of lack of cytotoxicity on human cells [ 13 ].…”