Targeted Modification of a Novel Amphibian Antimicrobial Peptide from Phyllomedusa tarsius to Enhance Its Activity against MRSA and Microbial Biofilm

Abstract

Antimicrobial peptides (AMPs) in the skin secretions of amphibians are fundamental components of a unique defense system that has evolved to protect these hosts from microbial invasion. Medusins constitute a recently-discovered AMP family from phyllomedusine leaf frog skin and exhibit highly-conserved structural characteristics. Here, we report a novel medusin, medusin-PT, from the skin secretion of the Tarsier Leaf Frog, Phyllomedusa tarsius. The mature peptide was initially identified from its cloned biosynthetic precursor-encoding cDNA as obtained by the rapid amplification of cDNA ends (RACE) method. Reverse-phase HPLC and tandem mass spectrometry confirmed both the presence of medusin-PT in the skin secretion and its primary structure. In a range of bioassays, medusin-PT exhibited antimicrobial activity against only the Gram-positive bacterium Staphylococcus aureus at 64 μg/ml. However, after directed changes to enhance the cationicity and amphipathicity of the peptide structure, three analog showed more potent antimicrobial activity against several additional bacteria including the antibiotic-resistant bacterium MRSA. In addition, these analog exhibited activity against microbial biofilm (minimum biofilm inhibitory and eradication concentrations of 32 μg/ml and over 64 μg/ml, respectively). These data provide evidence that medusins might be promising candidates as novel antibiotic leads and that the targeted modification of a natural AMP can both improve its efficacy so as to provide new insights into antibiotic design and development.

Identification and target-modifications of temporin-PE: A novel antimicrobial peptide in the defensive skin secretions of the edible frog, Pelophylax kl. esculentus

“…Then the fraction containing the same molecular weight of the putative mature peptide was analyzed by MS/MS fragmentation sequencing. The experimental procedures for RP-HPLC, MALDI-TOF and LC-MS/MS have been described previously [18].…”

“…Meanwhile, another analog was designed by coupling a TAT (-GRKKRRQRRR-) at the C-terminus of temporin-PE, namely temporin-PEb, in order to enhance the cell penetrating activity as well as investigate the potential intracellular targets of temporin-PE [20]. All peptides were chemically-synthesized by solid phase Fmoc chemistry [18].…”

“…The helical wheels and the physiochemical parameters of peptides were predicted using the HeliQuest tool [25,26]. The secondary structures of all peptides were examined by circular dichroism (CD) and analyzed by the K2D3 web server as described before [18].…”

“…The minimal inhibitory concentrations (MICs) of peptides were evaluated by the standard microdilution assay described previously [18]. Six different microorganism strains, The HC 50 here is the concentration of a peptide inducing 50% erythrocyte hemolysis.…”

Modification Targeting the “Rana Box” Motif of a Novel Nigrocin Peptide From Hylarana latouchii Enhances and Broadens Its Potency Against Multiple Bacteria

“…Minimum biofilm eradication concentration (MBEC) assay was carried out with a modified microtiter plate method as described previously (Gao et al, 2017). …”