Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

Molecular Insights into Conformational Heterogeneity and Enhanced Structural Integrity of Helicobacter pylori DNA Binding Protein Hup at Low pH.

The summarized amalgam of internal relaxation modulations and external forces like pH, temperature, and solvent conditions determine the protein structure, stability, and function. In a free-energy landscape, although conformers are arranged in vertical hierarchy, there exist several adjacent parallel sets with conformers occupying equivalent energy cleft. Such conformational states are pre-requisites for the functioning of proteins that have oscillating environmental conditions. As these conformational changes have utterly small re-arrangements, nuclear magnetic resonance (NMR) spectroscopy is unique in elucidating the structure-dynamics-stability-function relationships for such conformations. Helicobacter pylori survives and causes gastric cancer at extremely low pH also. However, least is known as to how the genome of the pathogen is protected from reactive oxygen species (ROS) scavenging in the gut at low pH under acidic stress. In the current study, biophysical characteristics of H. pylori DNA binding protein (Hup) have been elucidated at pH 2 using a combination of circular dichroism, fluorescence, NMR spectroscopy, and molecular dynamics simulations. Interestingly, the protein was found to have conserved structural features, differential backbone dynamics, enhanced stability, and DNA binding ability at low pH as well. In summary, the study suggests the partaking of Hup protein even at low pH in DNA protection for maintaining the genome integrity.

An inter-switch between hydrophobic and charged amino acids generated druggable small molecule binding pocket in chemokine paralog CXCL3.

Multigene families such as chemokines arose as a result of gene duplication events, followed by mutations and selection. GRO chemokines are three duplicated CXCL genes, comprising of CXCL1, CXCL2 and CXCL3 proteins. Comparative structural analysis of the two closely related paralog chemokines CXCL2 and CXCL3 in the current study indicated a variable electrostatic surface between them, and a specific hydrophobic pocket on the surface of CXCL3 that can bind naphthalene derivatives. Combined fluorescence and NMR analyses revealed that CXCL3 monomer can specifically bind to ANS (8-Anilinonaphthalene-1-sulfonic acid) with a stoichiometry of 1:1 by involving the residues belonging to the structural elements 310 helix and the α-helix. A close observation of the surfaces of these paralogs suggested that such a hydrophobic pocket is a resultant of inter-switch between a charged and a hydrophobic residue on the primary sequence of the two paralog proteins. Interestingly, the hydrophobic pocket is in the vicinity of GAG binding region of CXCL3, a molecular determinant in leukocyte trafficking. Such unique pockets/patches on specific chemokine surfaces can be exploited to design the naphthalene/small molecule based inhibitors against GAG binding to regulate their molecular interactions during the onset and progression of various types of cancers and inflammatory diseases.

Molecular cloning and biophysical characterization of CXCL3 chemokine.

CXCL3 is a neutrophil activating chemokine that belongs to GRO subfamily of CXC chemokines. GRO chemokine family comprises of three chemokines GRO α (CXCL1), GROß (CXCL2), and GRO γ (CXCL3), which arose as a result of gene duplication events during the course of chemokine evolution. Although primary sequences of GRO chemokines are highly similar, they performs several protein specific functions in addition to their common property of neutrophil trafficking. However, the molecular basis for their differential functions has not well understood. Although structural details are available for CXCL1 and CXCL2, no such information regarding CXCL3 is available till date. In the present study, we have successfully cloned, expressed, and purified the recombinant CXCL3. Around 15mg/L of pure recombinant CXCL3 protein was obtained. Further, we investigated its functional divergence and biophysical characteristics such as oligomerization, thermal stability and heparin binding etc., and compared all these features with its closest paralog CXCL2. Our studies revealed that, although overall structural and oligomerization features of CXCL3 and CXCL2 are similar, prominent differences were observed in their surface characteristics, thus implicating for a functional divergence.