Identification of HPr kinase/phosphorylase inhibitors: novel antimicrobials against resistant Enterococcus faecalis.

Enterococcus faecalis, a gram-positive bacterium, is among the most common nosocomial pathogens due to its limited susceptibility to antibiotics and its reservoir of the genes coding for virulence factors. Bacterial enzymes such as kinases and phosphorylases play important roles in diverse functions of a bacterial cell and, thus, are potential antibacterial drug targets. In Gram-positive bacteria, HPr Kinase/Phosphorylase (HPrK/P), a bifunctional enzyme is involved in the regulation of carbon catabolite repression by phosphorylating/dephosphorylating the histidine-containing phosphocarrier protein (HPr) at Ser46 residue. Deficiencies in HPrK/P function leads to severe defects in bacterial growth. This study aimed at identifying novel inhibitors of E. faecalis HPrK/P from a commercial compound library using structure-based virtual screening. The hit molecules were purchased and their effect on enzyme activity and growth of resistant E. faecalis was evaluated in vitro. Furthermore, docking and molecular dynamics simulations were performed to study the interactions of the hit compounds with HPrK/P. Among the identified hit molecules, two compounds inhibited the phosphorylation of HPr as well as significantly reduced the growth of resistant E. faecalis in vitro. These identified potential HPrK/P inhibitors open new research avenues towards the development of novel antimicrobials against resistant Gram-positive bacteria.

Interplay of gut microbiota and oxidative stress: Perspective on neurodegeneration and neuroprotection.

Background: Recent research on the implications of gut microbiota on brain functions has helped to gather important information on the relationship between them. Pathogenesis of neurological disorders is found to be associated with dysregulation of gut-brain axis. Some gut bacteria metabolites are found to be directly associated with the increase in reactive oxygen species levels, one of the most important risk factors of neurodegeneration. Besides their morbid association, gut bacteria metabolites are also found to play a significant role in reducing the onset of these life-threatening brain disorders. Aim of Review: Studies done in the recent past raises two most important link between gut microbiota and the brain: "gut microbiota-oxidative stress-neurodegeneration" and gut microbiota-antioxidant-neuroprotection. This review aims to gives a deep insight to our readers, of the collective studies done, focusing on the gut microbiota mediated oxidative stress involved in neurodegeneration along with a focus on those studies showing the involvement of gut microbiota and their metabolites in neuroprotection. Key Scientific Concepts of Review: This review is focused on three main key concepts. Firstly, the mounting evidences from clinical and preclinical arenas shows the influence of gut microbiota mediated oxidative stress resulting in dysfunctional neurological processes. Therefore, we describe the potential role of gut microbiota influencing the vulnerability of brain to oxidative stress, and a budding causative in Alzheimer's and Parkinson's disease. Secondly, contributing roles of gut microbiota has been observed in attenuating oxidative stress and inflammation via its own metabolites or by producing secondary metabolites and, also modulation in gut microbiota population with antioxidative and anti-inflammatory probiotics have shown promising neuro resilience. Thirdly, high throughput in silico tools and databases also gives a correlation of gut microbiome, their metabolites and brain health, thus providing fascinating perspective and promising new avenues for therapeutic options.

Vitamin K2 Modulates Organelle Damage and Tauopathy Induced by Streptozotocin and Menadione in SH-SY5Y Cells.

Vitamin K2, known for its antioxidative and anti-inflammatory properties, can act as a potent neuroprotective molecule. Despite its action against mitochondrial dysfunction, the mechanism underlying the links between the protective effects of vitamin K2 and endoplasmic reticulum (ER) stress along with basal levels of total tau protein and amyloid-beta 42 (Aß42) has not been elucidated yet. To understand the neuroprotective effect of vitamin K2 during metabolic complications, SH-SY5Y cells were treated with streptozotocin for 24 h and menadione for 2 h in a dose-dependent manner, followed by post-treatment of vitamin K2 for 5 h. The modulating effects of vitamin K2 on cell viability, lactate dehydrogenase release, reactive oxygen species (ROS), mitochondrial membrane potential, ER stress marker (CHOP), an indicator of unfolded protein response (UPR), inositol requiring enzyme 1 (p-IRE1α), glycogen synthase kinase 3 (GSK3α/ß), total tau and Aß42 were studied. Results showed that vitamin K2 significantly reduces neuronal cell death by inhibiting cytotoxicity and ROS levels and helps in the retainment of mitochondrial membrane potential. Moreover, vitamin K2 significantly decreased the expression of CHOP protein along with the levels and the nuclear localization of p-IRE1α, thus showing its significant role in inhibiting chronic ER stress-mediated UPR and eventually cell death. In addition, vitamin K2 significantly down-regulated the expression of GSK3α/ß together with the levels of total tau protein, with a petite effect on secreted Aß42 levels. These results suggested that vitamin K2 alleviated mitochondrial damage, ER stress and tauopathy-mediated neuronal cell death, which highlights its role as new antioxidative therapeutics targeting related cellular processes.

Plant-Derived Natural Biomolecule Picein Attenuates Menadione Induced Oxidative Stress on Neuroblastoma Cell Mitochondria.

Several bioactive compounds are in use for the treatment of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Historically, willow (salix sp.) bark has been an important source of salisylic acid and other natural compounds with anti-inflammatory, antipyretic and analgesic properties. Among these, picein isolated from hot water extract of willow bark, has been found to act as a natural secondary metabolite antioxidant. The aim of this study was to investigate the unrevealed pharmacological action of picein. In silico studies were utilized to direct the investigation towards the neuroprotection abilities of picein. Our in vitro studies demonstrate the neuroprotective properties of picein by blocking the oxidative stress effects, induced by free radical generator 2-methyl-1,4-naphthoquinone (menadione, MQ), in neuroblastoma SH-SY5Y cells. Several oxidative stress-related parameters were evaluated to measure the protection for mitochondrial integrity, such as mitochondrial superoxide production, mitochondrial activity (MTT), reactive oxygen species (ROS) and live-cell imaging. A significant increase in the ROS level and mitochondrial superoxide production were measured after MQ treatment, however, a subsequent treatment with picein was able to mitigate this effect by decreasing their levels. Additionally, the mitochondrial activity was significantly decreased by MQ exposure, but a follow-up treatment with picein recovered the normal metabolic activity. In conclusion, the presented results demonstrate that picein can significantly reduce the level of MQ-induced oxidative stress on mitochondria, and thereby plays a role as a potent neuroprotectant.

Natural ligand-receptor mediated loading of siRNA in milk derived exosomes.

siRNA based therapeutics have become the next frontier in molecular medicine. Though exosomes emerge as a promising drug delivery vehicle for siRNAs, significant hurdle remains in finding safe and effective loading methods. Traditional methods of loading exogenous siRNAs in exosomes are marked by certain limitations like siRNA aggregation, toxicity to the cells and their high experimental cost. As an electroporation and lipofection free approach, we show that the molecular conjugate of bovine lactoferrin with polyl-l-ysine electrostatically interacts with negatively charged siRNA, wherein lactoferrin as a ligand is captured by the GAPDH present in exosomes, loading siRNA in an effortless manner. This method exhibited transfection efficiency, colocalization percentage and colocalization threshold similar to electroporation. Furthermore, efficient uptake of exosomes loaded with siRNA via conjugate in recipient cells was observed. Our current study univocally establishes chemical free and non-mechanical method for the encapsulation and intercellular delivery of siRNA for wider therapeutic applications.

Role of non-PTS dependent glucose permease (GlcU) in maintaining the fitness cost during acquisition of nisin resistance by Enterococcus faecalis.

Nisin is used for food preservation due to its antibacterial activity. However, some bacteria survive under the prevailing conditions owing to the acquisition of resistance. This study aimed to characterize nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and investigate their fitness cost. FE-SEM, biofilm and cytochrome c assay were used for characterization. Growth kinetics, HPLC, qPCR and western blotting were performed to confer their fitness cost. Results revealed that nisin-resistant E. faecalis were morphologically different from sensitive strain and internalize more glucose. However, no significant difference was observed in the growth pattern of the resistant strain compared to that of the sensitive strain. A non-phosphotransferase glucose permease (GlcU) was found to be associated with enhanced glucose uptake. Conversely, Mpt, a major phosphotransferase system responsible for glucose uptake, did not play any role, as confirmed by gene expression studies and western blot analysis of HPr protein. The phosphorylation of His-15 residue of HPr phosphoprotein was reduced, while that of the Ser-46 residue increased with progression in nisin resistance, indicating that it may be involved in the regulation of pathogenicity. In conclusion, resistance imposes a significant fitness cost and GlcU plays a key role in maintaining the fitness cost in nisin-resistant variants.

Small Interfering RNA in Milk Exosomes Is Resistant to Digestion and Crosses the Intestinal Barrier In Vitro.

Milk is not only a composite of nutrients but emerged as a source of exosomes acting as a promising drug delivery vehicle for small interfering RNA (siRNA). siRNA is known for its immense therapeutic potential but has various physiological limitations, including stable delivery. To investigate the suitability of siRNA for physiological stability and oral delivery, we encapsulated scrambled Alexa Fluor (AF)-488 siRNA in milk whey exosomes using lipofection and evaluated stability against the digestive processes along with its uptake and transepithelial transport by intestinal epithelial cells. Milk exosomal siRNA were found resistant to different digestive juices, including saliva, gastric, bile, and pancreatic juices, in vitro and were internalized by Caco-2 cells. The stable delivery of exosomal AF-488 siRNA along with its transepithelial transport was confirmed by fluorescence microscopy and fluorescence intensity measurements. In summary, the encapsulation of siRNA in milk exosomes resists harsh digestive processes, improving intestinal permeability and payload protection.