Inactivation of Frog Virus 3 and Channel Catfish Virus by Esculentin-2P and Ranatuerin-2P, Two Antimicrobial Peptides Isolated from Frog Skin

Abstract

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.

Folded Structure and Insertion Depth of the Frog-Skin Antimicrobial Peptide Esculentin-1b(1–18) in the Presence of Differently Charged Membrane-Mimicking Micelles

“…In both cases, the software predictions for the almost central residue G8 had a low consensus (and were not included in Figure 1), suggesting a flexible kink that divides the peptide sequence into two helical fragments. However, in the case of pure DPC micelles, the residue S4, which is exactly in the middle of the N-terminal fragment, was predicted to have / values not compatible with an -helix (Figure 1c), thus suggesting a regular secondary structure only for the C-terminal fragment. Figure 2 summarizes the sequential NOEs unambiguously identified for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) in the presence of either DPC or DPC/SDS micelles. Figure S1 show the H-HN region of the NOESY spectra.…”

“…This particular DPC/SDS molar ratio was chosen to be close to that between zwitterionic and negatively charged lipids typical for the inner membrane of Gram-negative bacteria. 25 Table S1 reports the 1 H and 13 C resonance assignments for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) in the presence of either DPC or DPC/SDS micelles, obtained through a set of two-dimensional homo-and heteronuclear experiments, namely, DQF-COSY, TOCSY, NOESY, and HSQC. Figures 1a and 1b show the deviations of the observed chemical shifts from the reference "random coil" values for 1 H and 13 C, respectively.…”

“…1,2 Clinical impact of antibiotic resistance is immense, with increasing length of hospital stay, costs and mortality. 3,4 Antimicrobial peptides (AMPs) have attracted the interest of the scientific community as promising candidates for the development of a new class of antimicrobial drugs.…”

“…23 A globally amphipathic structure was found, with a helical conformation for the N-terminal portion (encompassing the first 12 residues) and an unfolded C-terminal fragment. 23 However, as the H 2 O/TFE solvent is an isotropic environment, such a structural model for Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) should be taken with caution. Despite promoting peptide folding by favoring intramolecular H-bonds, indeed, H 2 O/TFE mixtures inherently lack the intrinsic anisotropic character of a lipid bilayer.…”

“…In particular, both zwitterionic and negatively charged micelles have been employed to model eukaryotic and prokaryotic plasma membrane, respectively, as bacterial membranes are usually characterized by a higher content of negatively charged lipids than the former. 6,12,13 The insertion depth of Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) into the micelles was estimated by using Mn 2+ ions as a paramagnetic probe. The results clearly confirm the helical folding of Esc (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and show how binding and folding are prominent only in the presence of a negatively charged membrane.…”