Bactericidal effect of Neem (Azadirachta indica) leaf extract on Helicobacter pylori

Aim: The aim of the present study was to investigate the antibacterial effect of ethanolic extract of neem (Azadirachta indica) leaf against Gram-negative, gastric pathogen, Helicobacter pylori (H. pylori). Methodology: Extracts of neem leaf were prepared in different solventslike hexane, dichloromethane, chloroform, ethyl acetate, acetone and ethanol. Antibacterial activity was estimated in terms of zone of inhibition by performing Agar cup diffusion assay. Depending on the diameter of zone of inhibition, ethyl acetate, acetone and ethanol extract of neem leaves were selected for Thin Layer Chromatography. The presence of photochemicals were detected using iodine fumigation. Elution Assay was done to detect the bioactive components of the ethanol extract. Results: Out of sixsolvents used, ethanol extract of neem leavesshowed the maximum zone of inhibition against H. pylori. TLC separation of ethyl acetate, acetone and ethanol extract of plant products showed dark brown bands of phytochemicals on silica-gel G 60 plates. The contact bioautography assay showed a zone of 15 mm. Elution assay and agar cup bioassay was performed against H. pylori and the loading spot showed a zone of 11 mm. Interpretation: The findings of the present study revealed the anti-bacterial potency of ethanolic extracts of neem (Azadirachta indica) leaf against Gram-negative gastric pathogen H. pylori. The ethanolic extract of neem leaf can be used as an effective natural remedy in combating H. pylori infection.

The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

ABSTRACT Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity. IMPORTANCE Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori. Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria.

The RNase J-based RNA degradosome is compartmentalized in the gastric pathogen Helicobacter pylori

Post-transcriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation and some were reported to be compartmentalized inside these organelle-less cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD-box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and super-resolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes, by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into non-polar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote foci formation in H. pylori. Finally, RNase J-GFP molecules and foci in cells were quantified by 3D-single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as was previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity.ImportanceHelicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, that causes up to 800.000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is post-transcriptional regulation that, in H. pylori, largely relies on a multi-protein molecular machine, an RNA-degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated to the membrane of H. pylori. Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in the organelle-less bacteria.