Investigations on the membrane interaction of C-terminally amidated esculentin-2 HYba1 and 2 peptides against bacteria.

The membrane interaction and damage caused by C-terminally amidated esculentin-2 peptides identified from the frog skin is illustrated in the present study using Staphylococcus aureus and Vibrio cholerae. Double staining with fluorescent probes SYTOX and DAPI proved the concentration-dependent bacterial membrane damage induced by the peptides. It was found that the sub-MIC of both peptides induced transient pores on the bacterial membrane. These peptides also caused depolarisation on the bacterial membrane during their interaction. The physical changes on bacterial cells like blebbing, elongation, fusion, and so forth upon peptide treatment were visualized through SEM images. The antimicrobial activity of the peptides against S. aureus and V. cholerae was not altered at physiological concentrations of divalent and monovalent cations, which is advantageous in a therapeutic context. The increase of MIC against V. cholerae at higher concentrations of Mg2+ and Ca2+ (>5 µM) is due to the concentration-dependent antagonism exhibited by these ions for the cation binding sites on the bacterial membrane, which facilitates the process of 'self-promoted uptake.' The study emphasizes to utilize the ability of these peptides to produce transient pores at sub-MICs in combinatorial therapy.

Computational analysis of sodium-dependent phosphate transporter SLC20A1/PiT1 gene identifies missense variations C573F, and T58A as high-risk deleterious SNPs.

SLC20A1/PiT1 is a sodium-dependent inorganic phosphate transporter, initially recognized as the retroviral receptor for Gibbon Ape Leukemia Virus in humans. SNPs in SLC20A1 is associated with Combined Pituitary Hormone Deficiency and Sodium Lithium Counter transport. Using in silico techniques, we have screened the nsSNPs for their deleterious effect on the structure and function of SLC20A1. Screening with sequence and structure-based tools on 430 nsSNPs, filtered 17 nsSNPs which are deleterious. To evaluate the role of these SNPs, protein modeling and MD simulations were performed. A comparative analysis of model generated with SWISS-MODEL and AlphaFold shows that many residues are in the disallowed region of Ramachandran plot. Since SWISS-MODEL structure has a 25-residue deletion, the AlphaFold structure was used to perform MD simulation for equilibration and structure refinement. Further, to understand perturbation of energetics, we performed in silico mutagenesis and ΔΔG calculation using FoldX on MD refined structures, which yielded SNPs that are neutral (3), destabilizing (12) and stabilizing (2) on protein structure. Furthermore, to elucidate the impact of SNPs on structure, we performed MD simulations to discern the changes in RMSD, Rg, RMSF and LigPlot of interacting residues. RMSF profiles of representative SNPs revealed that A114V (neutral) and T58A (positive) were more flexible & C573F (negative) was more rigid compared to wild type, which is also reflected in the changes in number of local interacting residues in LigPlot and ΔΔG. Taken together, our results show that SNPs can lead to structural perturbations and impact the function of SLC20A1 with potential implications for disease.Communicated by Ramaswamy H. Sarma.

Identification and functional characterisation of Esculentin-2 HYba peptides and their C-terminally amidated analogs from the skin secretion of an endemic frog.

Here, we report the identification, functional characterisation, and the effect of C-terminal amidation on the activity profile of two novel Esculentin-2 peptides (Esculentin-2 HYba1 and Esculentin-2 HYba2). The parent peptides and their analogs exhibited potent activity against the tested Gram-positive and Gram-negative bacteria. The effect of amidation was evident in the activity profile of fish pathogens and killing kinetics. The analogs showed a 10-fold decrease in MIC, and the killing time was reduced to 10-15 minutes. The hemolytic potential was unaltered upon amidation. The selectivity index revealed that these peptides are more selective to bacteria than mammalian cells. Cytotoxicity against Hep3B cells reveals their potential to destroy cancer cells; they showed potential inhibition compared to anticancer drug silymarin. The study also highlights the need for further truncations and modifications of esculentin peptides for developing them as lead drug molecules.

A Review of the Mechanism of Action of Amphibian Antimicrobial Peptides Focusing on Peptide-Membrane Interaction and Membrane Curvature.

Research interests on amphibian antimicrobial peptides (AMPs) are currently increasing because of their capability to combat microorganisms from both terrestrial and aquatic environments, which are the warehouses of human pathogens. The most remarkable feature of AMPs are their mechanism of action, primarily targeted to anionic membranes. Researchers have postulated many models to describe peptide- membrane interaction, which leads to membrane permeation/intracellular targeting. Despite these models information regarding the relationship between membrane curvature and peptidemembrane interaction is scarce. This relationship could be clearly depicted using the two-state model and interfacial activity model. In the review, we discuss in detail the two state and interfacial activity models and explain the influence of membrane curvature on peptide binding and the membrane interaction of curvature-sensitive peptides. In addition, the models proposed to explain the mechanism of action of membrane lytic and non-lytic AMPs are also reviewed.