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.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, 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.

Keywords: Helicobacter pylori; RNA degradosome; RNase J; post-transcriptional regulation; subcellular localization; superresolution microscopy.

FIG 1

The two partners of the RNA degradosome of H. pylori, RNase J and RhpA, are associated with the inner membrane in a peripheral manner, independently of ribosomes and RNA, and this association does not depend on RNase Y, flotillin, or RhpA. Cellular compartments of H. pylori B128 strain were separated by fractionation; all samples correspond to the same initial number of bacteria. Experiments were performed in triplicate. TE, total extract fraction; SE, soluble extract fraction; IM, inner membrane fraction; OM, outer membrane fraction. (A) Western blot with antibodies against RNase J and RhpA on the different cellular fractions of wild-type (WT) H. pylori strain B128. (B) Western blot with antibodies against RNase J and RhpA on the different cellular fractions of WT H. pylori upon treatment of the membrane fraction with 6 M urea (T1), 100 mM Na₂CO₃ (T2), or 2 M NaCl (T3); untreated fractions are shown as a control. Lanes 1, 2, and 3 indicate the wash fractions corresponding to treatments T1, T2, and T3, respectively. (C) Western blot with antibodies against RNase J and RhpA and a V5 tag (marking the L9 ribosomal protein) on the different cellular fractions of an L9-V5-expressing H. pylori strain, under untreated conditions and upon treatment with 20 mM EDTA or 1 μg/ml RNase A. (D) Western blot with antibodies against RNase J and RhpA on the different subcellular fractions of WT, Δrny, ΔfloA, and ΔrhpA H. pylori strains.

FIG 2

RNase J and RhpA form foci in H. pylori cells that do not have a polar localization. (A) Representative composite confocal microscopy images of live H. pylori cells expressing RNase J-GFP (green) or RhpA-CFP (cyan). In the RNase J-GFP image, blue indicates DNA (Hoechst 33342); in both images, red indicates membranes (FM4-64). Experiments were performed at least 3 times. (B) Histogram showing the number of foci of RNase J-GFP that are located in each position along the spine of the H. pylori cells.

FIG 3

The number of RNase J-GFP foci per cell is progressively reduced along the H. pylori growth curve. (A) Representative composite confocal microscopy images of the RNase J-GFP-expressing strain at different time points (in hours) along the growth curve. Blue, DNA (labeled with Hoechst 33342); green, RNase J-GFP; red, the membrane (labeled with FM4-64). The experiment was performed in triplicate. (B) Quantification of the amount of foci per cell, normalized by the nucleoid area, along the growth curve. “n” corresponds to the number of cells analyzed for each condition. The median value is represented by a horizontal bar, and the error bars correspond to the interquartile range. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. (C) Western blot (upper panel) and quantification (lower panel) of RNase J-GFP of cells taken at different time points along the growth curve and normalized on total proteins. The experiments were reproduced twice. The differences are not statistically significant (P value of 0.11).

FIG 4

The number of RNase J-GFP foci per cell is affected by antibiotics and in different mutants. “n” corresponds to the number of cells analyzed for each condition. The median value is represented by a horizontal bar, and the error bars correspond to the interquartile range. Experiments were performed in triplicate. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; “ns,” nonsignificant. (A) Quantification of the number of RNase J-GFP foci, normalized by the nucleoid area, in untreated cells and upon treatment with chloramphenicol, rifampicin, or puromycin. (B) Quantification of the number of RNase J-GFP foci, normalized by the nucleoid area, in wild-type cells and in bacteria deleted for the genes encoding RNase Y, flotillin, or RhpA.

FIG 5

Visualization of the foci of RNase J-GFP in exponential and stationary phase by dSTORM superresolution microscopy. Representative single-molecule localization microscopy images show the membrane of H. pylori as visualized after labeling with WGA-AF555 (yellow-red) and the RNase J-GFP foci after labeling with anti-GFP-Cy5 nanobodies (blue-green). The upper panels show the full 3D volume of representative cells, and the lower panels show a 2D longitudinal slice of the bacteria. The X, Y, and Z values in each image indicate the distance between the ticks of the respective axes in the picture. The color gradients red-yellow (for WGA-AF555) and blue-green (for anti-GFP-Cy5 nanobodies) indicate the distance for each dot with respect to the coverslip, as indicated in the corresponding scale bars in each panel. The experiment was performed 3 times.

FIG 6

dSTORM quantification of the RNase J-GFP foci of H. pylori cells in exponential phase (red hatched bars) and stationary phase (blue bars). The experiment was carried out 3 times, and the data correspond to the results presented in Fig. 5. (A) Distribution histogram of the amount of foci per cell in exponential-phase cells (n = 121 cells) and stationary-phase cells (n = 191 cells). Median values determined under both conditions are indicated. (B) Distribution histogram of the number of RNase J-GFP molecules per cell in exponential-phase and stationary-phase cells. Median values determined under both conditions are indicated. (C) Distribution histogram of the number of RNase J-GFP molecules per cluster in exponential-phase and stationary-phase cells (n = 535 clusters in exponential phase and n = 371 clusters in stationary phase). Median values determined under both conditions are indicated. (D) Distribution histogram of the volume of the RNase J-GFP clusters in exponential-phase and stationary-phase cells. Median values determined under both conditions are indicated. (E) Distribution histogram of the volume of H. pylori cells in exponential phase (n = 142 cells) and stationary phase (n = 40 cells). ****, P < 0.0001 (for panels A to E). (F) Bar graph of the proportions of focus-forming and non-focus-forming RNase J-GFP molecules calculated on the basis of mean values in exponential-phase and stationary-phase cells.

FIG 7

Model for the regulation of the RNA degradosome foci in H. pylori. RNase J and RhpA, the two protein components of the RNA degradosome, are associated with the H. pylori inner membrane. Minor proportions of these proteins are associated with translating ribosomes, which could be either cytoplasmic or located at the membrane. RNase J and RhpA assemble into foci at the membrane, probably together. On the basis of our data, we propose a model where foci represent the active form of the RNA degradosome, constituting RNA degradation hubs. Comparatively, the complexes located outside foci would retain little or no activity. In this model, target RNA molecules would be directed to the foci for degradation by an unknown mechanism, providing in addition a spatiotemporal delay that allows for their translation before their degradation. The RNA degradosomes associated with ribosomes could be involved either in rRNA maturation or in coupling between translation and mRNA degradation.