Abstract
Tachyplesin is a type of cationic β-hairpin antimicrobial peptide discovered in horseshoe crab approximately 30 years ago that is well known for both its potential antimicrobial activities against multidrug-resistant bacteria and its cytotoxicity to mammalian cells. Though its physical interactions with artificial membranes have been well studied, details of its physiological mechanism of action the physiological consequences of its action remain limited. By using the DNA-binding fluorescent dye propidium iodide to monitor membrane integrity, confocal microscopy to assess the intracellular location of FITC-tagged tachyplesin, and RNA sequencing of the differentially expressed genes in four Gram-negative bacteria (Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa) treated with lethal or sublethal concentrations of tachyplesin, we found that compared with levofloxacin-treated bacteria, tachyplesin-treated bacteria showed significant effects on the pathways underlying unsaturated fatty acid biosynthesis. Notably, RNA levels of the conserved and essential 3-ketoacyl carrier protein reductase in this pathway (gene FabG) were elevated in all of the four bacteria after tachyplesin treatment. In vitro tests including surface plasmon resonance and enzyme activity assays showed that tachyplesin could bind and inhibit 3-ketoacyl carrier protein reductase, which was consistent with molecular docking prediction results. As unsaturated fatty acids are important for membrane fluidity, our results provided one possible mechanism to explain how tachyplesin kills bacteria and causes cytotoxicity by targeting membranes, which may be helpful for designing more specific and safer antibiotics based on the function of tachyplesin.
Highlights
Tachyplesin is a type of cationic β-hairpin antimicrobial peptide (AMP) discovered from horseshoe crab hemocytes approximately 30 years ago (Nakamura et al, 1988; Miyata et al, 1989; Kawano et al, 1990; Muta et al, 1990)
The first is that compared with membrane rupture activities, the membrane translocation activity of tachyplesin is more relevant to its antimicrobial potential, which is demonstrated by the fact that linear analogs of tachyplesin without disulfide formation could cause more serious membrane disruptions than a cyclic peptide, which formed pores during the translocation process but showed weaker membrane translocation ability and weaker antimicrobial activity (Matsuzaki et al, 1993, 1997; Doherty et al, 2006, 2008)
1% and 2% hemolysis was observed when tachyplesin III was applied at concentrations of 50 and 100 mg/L, respectively, and hemolysis accelerated as the concentration of
Summary
Tachyplesin is a type of cationic β-hairpin antimicrobial peptide (AMP) discovered from horseshoe crab hemocytes approximately 30 years ago (Nakamura et al, 1988; Miyata et al, 1989; Kawano et al, 1990; Muta et al, 1990). The other proposed that the antiparallel beta-sheet structure formed by disulfides helped with tachyplesin binding to DNA and might play roles in bacteria killing (Yonezawa et al, 1992) Though the former model seems to be more prevalent, reports concerning this issue are mainly depictions of the physical interactions between this peptide and artificial lipid bilayers, and more biological details are still needed (Doherty et al, 2006, 2008). The other conclusion is that membrane translocation activity is necessary but not sufficient for tachyplesin to kill bacteria, which is based on the reports that compared to tachyplesin alone, poly(ethylene glycol) (PEG)grafted (PEGylated) tachyplesin showed similar membrane translocation activity but significantly weakened antimicrobial activity (Imura et al, 2007; Han and Lee, 2013) These phenomena imply that tachyplesin may play an intracellular role
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