Engraulisin: A novel marine derived cell penetrating peptide with activity against drug resistant bacteria.

Cell penetrating peptides (CPP) with their intrinsic ability to penetrate plasma membranes facilitate intracellular uptake of various macromolecules. Although a substantial number of CPPs have been reported over the last three decades, the number is still inadequate when compared to the theoretically feasible peptides with similar physicochemical composition. Marine organisms, due to their hostile environment, are an immense source of several high-valued therapeutically relevant peptides. Various marine derived antibacterial, antimycotic and anticancer peptides have demonstrated improved activity in comparison to peptides of terrestrial origin. While a significant number of marine bioactive peptides exist, cell penetrating peptides from marine organisms remain unravelled. In this study, we report Engraulisin from Engraulis japonicus, a computationally derived novel cell penetrating peptide of marine origin. Engraulisin manifest successful uptake in mammalian cells at 5 µM concentration with negligible cytotoxicity observed through MTT assay. Analysis of its cellular uptake mechanism revealed significant inhibition at 4 °C suggesting endocytosis as the major route of cellular entry. Interestingly, the novel peptide also demonstrated selective antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Additionally, molecular dynamics simulation with POPC and POPG bilayer system unveiled significance of positively charged residues in forming a stable membrane interaction. Engraulisin represents a novel marine-derived cell penetrating peptide which can be explored for cellular delivery of pharmaceutically relevant molecules.

An efficient computational protocol for template-based design of peptides that inhibit interactions involving SARS-CoV-2 proteins.

The RNA-dependent RNA polymerase (RdRp) complex of SARS-CoV-2 lies at the core of its replication and transcription processes. The interfaces between holo-RdRp subunits are highly conserved, facilitating the design of inhibitors with high affinity for the interaction interface hotspots. We, therefore, take this as a model protein complex for the application of a structural bioinformatics protocol to design peptides that inhibit RdRp complexation by preferential binding at the interface of its core subunit nonstructural protein, nsp12, with accessory factor nsp7. Here, the interaction hotspots of the nsp7-nsp12 subunit of RdRp, determined from a long molecular dynamics trajectory, are used as a template. A large library of peptide sequences constructed from multiple hotspot motifs of nsp12 is screened in-silico to determine sequences with high geometric complementarity and interaction specificity for the binding interface of nsp7 (target) in the complex. Two lead designed peptides are extensively characterized using orthogonal bioanalytical methods to determine their suitability for inhibition of RdRp complexation. Binding affinity of these peptides to accessory factor nsp7, determined using a surface plasmon resonance (SPR) assay, is slightly better than that of nsp12: dissociation constant of 133nM and 167nM, respectively, compared to 473nM for nsp12. A competitive ELISA is used to quantify inhibition of nsp7-nsp12 complexation, with one of the lead peptides giving an IC50 of 25µM . Cell penetrability and cytotoxicity are characterized using a cargo delivery assay and MTT cytotoxicity assay, respectively. Overall, this work presents a proof-of-concept of an approach for rational discovery of peptide inhibitors of SARS-CoV-2 protein-protein interactions.

The Slowing Rate of CpG Depletion in SARS-CoV-2 Genomes Is Consistent with Adaptations to the Human Host.

Depletion of CpG dinucleotides in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes has been linked to virus evolution, host-switching, virus replication, and innate immune responses. Temporal variations, if any, in the rate of CpG depletion during virus evolution in the host remain poorly understood. Here, we analyzed the CpG content of over 1.4 million full-length SARS-CoV-2 genomes representing over 170 million documented infections during the first 17 months of the pandemic. Our findings suggest that the extent of CpG depletion in SARS-CoV-2 genomes is modest. Interestingly, the rate of CpG depletion is highest during early evolution in humans and it gradually tapers off, almost reaching an equilibrium; this is consistent with adaptations to the human host. Furthermore, within the coding regions, CpG depletion occurs predominantly at codon positions 2-3 and 3-1. Loss of ZAP (Zinc-finger antiviral protein)-binding motifs in SARS-CoV-2 genomes is primarily driven by the loss of the terminal CpG within the motifs. Nonetheless, majority of the CpG depletion in SARS-CoV-2 genomes occurs outside ZAP-binding motifs. SARS-CoV-2 genomes selectively lose CpGs-motifs from a U-rich context; this may help avoid immune recognition by TLR7. SARS-CoV-2 alpha-, beta-, and delta-variants of concern have reduced CpG content compared to sequences from the beginning of the pandemic. In sum, we provide evidence that the rate of CpG depletion in virus genomes is not uniform and it greatly varies over time and during adaptations to the host. This work highlights how temporal variations in selection pressures during virus adaption may impact the rate and the extent of CpG depletion in virus genomes.

Inhibitory Mechanism on Combination of Phytic Acid with Methanolic Seed Extract of Syzygium cumini and Sodium Chloride over Bacillus subtilis.

The antibiotic resistance in bacteria responsible for causing community and health care-associated infection displayed a major threat to global health. Use of broad-spectrum antibiotics for the treatment of various ailments poses serious side effects. In the present research, we investigated the combined role of 2% phytic acid with 2% methanolic seed extract of Syzygium cumini and 0.5% sodium chloride for inhibition of Bacillus subtilis and Pseudomonas aeruginosa and found it to be efficient over B. subtilis. The zone of inhibition by present mixture was found to be 2.9 ± 0.0004 and 1.9 ± 0.0006 cm against Bacillus subtilis and P. aeruginosa in comparison to individual component. Mixture was found more potent against B. subtilis and selected for further study. The underlying mechanism involved in inhibitory action of this mixture was determined by Scanning electron microscope, DNA fragmentation and propidium iodide staining. Scanning electron microscopy revealed that inhibition of B. subtilis by this mixture is mainly due to the disruption of bacterial cell membrane, leakage of internal cellular content which ultimately leads to the death of bacterial cells. DNA fragmentation showed apoptotic hallmark through degradation caused by mixture against B. subtilis at various time intervals. Likewise, PI staining also revealed the disruption of bacterial membrane by the mixture as the PI gives fluorescence after binding with DNA. The present study concludes that inhibitory potential of this mixture is mainly due to disruption of bacterial cell membrane, degradation of DNA and creation of pores in the membrane. The mixture could be used for inhibition of food pathogen B. subtilis.

Antimicrobial action of methanolic seed extracts of Syzygium cumini Linn. on Bacillus subtilis.

Phytochemicals of Syzygium cumini are used for the treatment of various diseases as a traditional medicine but the mechanism behind their action is not well reported. Antimicrobial activity of methanolic seed extract of S. cumini was done by agar well diffusion assay on Bacillus subtilis and its zone of inhibition was found to be 20.06 mm in comparison to control having no zone of inhibition. MIC of S. cumini was found to be 0.3 mg/ml. Genomic DNA degradation of B. subtilis reveals apoptosis and FE-scanning electron microscope indicates cell wall cracking on several intervals of time. Results of propidium iodide staining showed few bacterial cells were stained in control; however population of stained cells increased after exposing them for varying period of time. Flow cytometric kinetic data analysis on the membrane permeabilization in bacterial cell showed the significant contribution of antimicrobial potential of the seed extract on antimicrobial-induced permeabilization. In silico analysis revealed two components of S. cumini methanolic extract to be active against four enzymes (PDB ID-1W5D, 4OX3, 3MFD and 5E2F) which are crucial for plasma membrane synthesis in B. subtilis. Moreover lupeol showed highest binding energy for macromolecule 1W5D and 4OX3 forming one hydrogen bond each whereas stigmasterol showed the highest binding energy for macromolecule 3MFD and 5E2F forming four hydrogen bonds and alkyl bonds respectively. It demonstrates that methanolic seed extracts of S. cumini could be used for inhibition of food born infection caused by B. subtilis and also an alternative of prevalent antibiotics.