Expression of the VapD protein by Helicobacter pylori during intracellular infection

TL;DR

This study shows that the VapD protein is produced by Helicobacter pylori when it infects human cells, supporting its role in persistent infection.

Contribution

First direct evidence of VapD protein expression during H. pylori intracellular infection.

Findings

VapD expression was detected exclusively in AGS cells infected with H. pylori strain 26695.

Confocal microscopy confirmed intracellular localization of VapD coinciding with H. pylori within AGS cells.

A high proportion of infected AGS cells showed VapD signal, confirming protein-level production.

Abstract

Helicobacter pylori can persist intracellularly, offering protection against the immune system and antimicrobial treatments, which promote chronic infection. Previous studies revealed that the virulence-associated protein D (vapD) gene is transcriptionally induced during H. pylori intracellular infection and was associated with bacterial persistence, as deletion of vapD impairs bacterial intracellular survival. However, whether VapD protein is expressed and localized during intracellular infection had not been demonstrated. The aim of this study was to detect the VapD protein when H. pylori is inside eukaryotic cells. Polyclonal antibodies against VapD and immunofluorescence microscopy were used to detect the VapD expression in co-cultures of H. pylori strain 26695 and AGS cells. The H. pylori strain Tx30a, which lacks the vapD gene, was used as a negative control. VapD expression was detected exclusively in AGS cells infected with strain 26695. Confocal microscopy confirmed the intracellular localization of the VapD signal, which coincided with the localization of H. pylori within AGS cells. In addition, a high proportion of H. pylori-infected AGS cells were positive for VapD signal. Together, these results provide first direct evidence of VapD expression during H. pylori intracellular infection and extend previous transcriptional and genetic studies by confirming VapD production at the protein level, supporting its association with the intracellular state of H. pylori.

Figures

PCR amplification of the vapD gene in H. pylori strains 26695 and Tx30a. The PCR product of the vapD gene is 282 bp in size. The vacA gene S region, used as a loading control, yields a product of 259 bp in H. pylori 26695 strain (S1 genotype) and 286 bp in H. pylori Tx30a strain (S2 genotype).

Detection of VapD and H. pylori via an immunofluorescence assay. (a, g) Phase-contrast images of AGS cells infected with H. pylori strains 26695 (VapD-positive) or Tx30a (VapD-negative), respectively. (b, h) AGS cell nuclei stained with DAPI. (c, i) VapD labelling. (d, j) H. pylori labelling. (e, k) Merge of AGS cells and H. pylori. (f, l) Merge of VapD and H. pylori. Images were acquired at 100× magnification.

Confocal images of H. pylori strain 26695-AGS cells co-culture. (a) Phase-contrast image of AGS cells infected with H. pylori 26695. (b) Confocal single z-plane image showing VapD detection. (c) Confocal single z-plane image showing H. pylori detection. (d) Orthogonal projection of the confocal z-stack, including transversal (xz) and sagittal (yz) projections. (e) 3D cross-sectional reconstruction from the z-stack images. The z-stack corresponds to a thickness of 3.99 µm, and images were acquired at 63× magnification. The AGS cell nucleus is stained with DAPI (blue).

Proportion of AGS cells infected with H. pylori strain 26695 and expressing VapD. The detection was performed by counting the number of cells positive for VapD and/or H. pylori. Results are presented as the mean percentage of VapD- and/or H. pylori-positive cells±sd across 14 optical fields from 2 independent experiments. Statistical significance was assessed using the Kruskal–Wallis test followed by the Dunnett’s post hoc test.

Equations

Full text

Data Summary

All the data generated or analysed during this study are included in this published article and its supplementary information files.

Introduction

Helicobacter pylori is a bacterium capable of persisting within cells for extended periods. This intracellular phenotype offers the bacterium advantage by protecting it from the immune response and antimicrobial treatment, thereby contributing to chronic infection and the persistence of the bacteria within gastric epithelial cells [12].

Several facultative intracellular bacterial species, including nontypeable Haemophilus influenzae (NTHi) [3], Rhodococcus equi [45], Dichelobacter nodosus [6] and Neisseria gonorrhoeae [7], possess the virulence-associated protein D (vapD) gene, which has been proposed to contribute to bacterial protection against stress or to intracellular persistence. H. pylori also harbours the vapD gene, which was first described in 2012 as encoding a small 11.2 kDa protein with endoribonuclease activity related to Cas2 proteins [8]; however, its biological role was not defined at the time.

Previous studies conducted by our group focused on determining whether the vapD gene is associated with the survival and persistence of H. pylori within gastric epithelial cells. Initial findings showed that H. pylori strain 26695 can persist intracellularly for up to 108 h, acquiring a coccoid morphology after 36 h while remaining metabolically active. During this period, vapD transcription was detected throughout the course of infection [2]. In the same study, vapD transcription was also detected in gastric biopsies from patients with different gastric pathologies. More recently, we demonstrated that deletion of vapD results in a progressive decrease in bacterial survival within AGS, whereas no growth differences were observed between wt and mutant H. pylori strains when cultured in synthetic media, indicating vapD is not required for extracellular bacterial growth under these conditions [9]. Together, these findings support an association between vapD expression and the intracellular state of H. pylori. However, whether VapD is produced and localized at the protein level during intracellular infection has not been experimentally demonstrated. Addressing this gap is essential to validate VapD as a molecular component associated with the intracellular lifestyle of H. pylori.

The aim of this study was, therefore, to detect the VapD protein expression and determine its localization when H. pylori is inside eukaryotic cells, using polyclonal antibodies against VapD. By providing direct evidence of VapD expressions specifically in intracellular bacteria, this work extends previous transcriptional and genetic findings and lays the groundwork for future investigations to better understand the role of VapD during intracellular infection.

Methods

Anti-VapD antibodies and Western blot

Polyclonal antibodies were produced by ProteoGenix SAS (Schiltigheim, France) using the previously generated recombinant VapD protein [10] as the antigen. Antibody specificity was validated by Western blot against the recombinant VapD protein (Fig. S1, available in the online Supplementary Material).

Western blotting was performed as follows: recombinant VapD protein at concentrations of 0.25, 0.5, 1.0 and 2.0 µg was separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 3% (w/v) skimmed milk in 1× PBS and incubated for 1 h at room temperature with gentle shaking. The membrane was then incubated with anti-VapD polyclonal antibodies, followed by a horseradish peroxidase-conjugated anti-rabbit IgG (H+L) secondary antibody (Santa Cruz Biotechnology Inc., USA), both at a dilution of 1:2500 for 1 h each with gentle shaking at 4 °C. Between incubations, the membrane was washed three times with 1× PBS containing 0.1% (v/v) Tween-20. Immunoreactive bands were visualized using SuperSignal™ West Pico PLUS chemiluminescent substrate (Thermo Fisher Scientific, MA, USA) and detected with a C-DiGit® 131 blot scanner (LI-COR, NE, USA).

H. pylori and AGS cell co-culture

Co-cultures of human gastric adenocarcinoma cell lines [AGS (ATCC CRL-1739)] and H. pylori strains 26695 (ATCC 700392) and Tx30a (ATCC 51932) were grown in Dulbecco’s modified Eagle’s medium (Gibco/BRL, USA) supplemented with 5% FBS (Biowest, México), 100 IU ml^−1^ ampicillin, 100 IU ml^−1^ streptomycin, 0.2% NaHCO_3_, 10 mM HEPES and 2 mM glutamine on Petri dishes with coverslips in a 5% CO_2_ atmosphere for 48 h. H. pylori strains were added at an m.o.i. of 100 bacteria per cell.

Immunofluorescence assay

The co-cultures were washed three times with 1× PBS and fixed with 2% paraformaldehyde in 1× PBS (pH 7.2) for 20 min at room temperature. The samples were washed with 1× PBS and permeabilized with 0.5% Triton X-100 for 5 min at 4 °C. The cells were subsequently incubated with anti-VapD at a dilution of 1:100 in 1× PBS and anti-H. pylori at a dilution of 1:200 (Biocare, USA) overnight at 4 °C. The cells were washed with 1× PBS and incubated with anti-mouse Alexa Fluor 594 (Life Technologies, USA) and anti-rabbit Alexa Fluor 488 (Life Technologies) antibodies, both of which were developed in goats and diluted 1:100 in 1× PBS, for 2 h at room temperature. Finally, the cells were washed with 1× PBS, incubated with DAPI for 1 min, washed and mounted with mounting medium for fluorescence (Vectashield Antifade Mounting Medium, Vector Laboratories). The samples were observed under a Nikon Eclipse E600 epifluorescence microscope (Nikon, USA) or Zeiss LSM 800 Confocal Laser Scanning Microscope (Zeiss, Germany).

Percentage of H. pylori-infected cells expressing VapD

To determine the percentage of infected cells, the total number of cells (infected and uninfected) was counted across 14 optical fields from 2 independent experiments. Within the same fields, cells infected with H. pylori (red label) and those expressing VapD (green label) were also counted. The proportion of H. pylori-infected cells expressing VapD was calculated as follows:

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Cell counts were performed using a Nikon Eclipse E600 epifluorescence microscope (Nikon) at a 40× objective.

The results of the comparison among cells infected with H. pylori expressing VapD, the cells infected with H. pylori only and uninfected cells were reported as mean±sd. Data were evaluated for normality using the Shapiro–Wilk test. Because the data did not meet normality assumptions, the nonparametric Kruskal–Wallis test was performed, and Dunnett’s test was performed as a post hoc test; P<0.05 was considered statistically significant. Statistical analysis and graphs were conducted using RStudio v3.2.2.

Results

To assess the expression and localization of the VapD protein in H. pylori during intracellular infection, we performed an immunofluorescence assay on co-cultures of *H. pylori-AGS cells at 48 h post-inoculation. As a negative control, we used the * H. pylori strain Tx30a, which lacks the vapD gene (Fig. 1).

PCR amplification of the vapD gene in H. pylori strains 26695 and Tx30a. The PCR product of the vapD gene is 282 bp in size. The vacA gene S region, used as a loading control, yields a product of 259 bp in H. pylori 26695 strain (S1 genotype) and 286 bp in H. pylori Tx30a strain (S2 genotype).

The epifluorescence assay in co-cultures of AGS cells with strains 26695 (Fig. 2a–f) or Tx30a (Fig. 2g–l) showed that both strains localized within the cytoplasm of AGS cells (Fig. 2d, e, j, k). However, VapD expression was detected only in cells infected with strain 26695 (Fig. 2c, f) and not in those infected with Tx30a (Fig. 2i, l). Immunofluorescence assays using secondary antibody only were also performed in cells infected with strain 26695 (Fig. S2), confirming the specificity of the polyclonal antibodies against VapD.

Detection of VapD and H. pylori via an immunofluorescence assay. (a, g) Phase-contrast images of AGS cells infected with H. pylori strains 26695 (VapD-positive) or Tx30a (VapD-negative), respectively. (b, h) AGS cell nuclei stained with DAPI. (c, i) VapD labelling. (d, j) H. pylori labelling. (e, k) Merge of AGS cells and H. pylori. (f, l) Merge of VapD and H. pylori. Images were acquired at 100× magnification.

Confocal microscopy was performed to further confirm the co-localization of H. pylori strain 26695 and VapD within AGS cell (Fig. 3a–e). Orthogonal projection (Fig. 3d) and 3D z-stack reconstruction (Fig. 3e) verified that the VapD protein co-localizes with the bacteria inside AGS cells.

Confocal images of H. pylori strain 26695-AGS cells co-culture. (a) Phase-contrast image of AGS cells infected with H. pylori 26695. (b) Confocal single z-plane image showing VapD detection. (c) Confocal single z-plane image showing H. pylori detection. (d) Orthogonal projection of the confocal z-stack, including transversal (xz) and sagittal (yz) projections. (e) 3D cross-sectional reconstruction from the z-stack images. The z-stack corresponds to a thickness of 3.99 µm, and images were acquired at 63× magnification. The AGS cell nucleus is stained with DAPI (blue).

The proportion of AGS cells infected with H. pylori and expressing VapD was analysed. Out of 447 AGS cells counted, a high percentage were infected with H. pylori (82.33%). Among all AGS cells examined, 65.1±14.73% were positive for both H. pylori and VapD, whereas 17.23±5.77% were positive for H. pylori only. The remaining 17.67±2.62% of AGS cells were uninfected (Fig. 4).

Proportion of AGS cells infected with H. pylori strain 26695 and expressing VapD. The detection was performed by counting the number of cells positive for VapD and/or H. pylori. Results are presented as the mean percentage of VapD- and/or H. pylori-positive cells±sd across 14 optical fields from 2 independent experiments. Statistical significance was assessed using the Kruskal–Wallis test followed by the Dunnett’s post hoc test.

Discussion

Some H. pylori strains exhibit an intracellular phenotype, allowing them to remain inside cells for extended periods, which contributes to their resistance to antibiotic treatment and fosters chronic infection [1211]. Like other intracellular micro-organisms, H. pylori must develop survival strategies to cope with the stress exerted by the intracellular environment. Recent findings have shown that the vapD gene is transcriptionally induced during intracellular infection and the deletion of vapD impairs H. pylori persistence within gastric cells [9]. In that study, a vapD deletion mutant of H. pylori strain 26695 (HpΔvapD) retained its ability to invade AGS cells but exhibited a markedly reduced intracellular bacterial load compared with the wt strain, suggesting an association between vapD and intracellular survival and persistence. Notably, no growth differences were observed between wt and mutant strains under synthetic culture conditions, suggesting that vapD is not required for bacterial survival outside the intracellular niche.

In the present study, we extend these previous transcriptional and genetic observations by providing direct evidence of VapD protein expression. VapD was specifically detected in AGS cells infected with H. pylori strain 26695, and a high proportion of cells infected with the bacterium exhibited VapD signal. These findings demonstrate that VapD production is tightly associated with the intracellular state of H. pylori infection.

Kwon et al. reported that VapD from H. pylori is a protein with purine-specific endoribonuclease activity related to the Cas2 family [8], although its biological function remains unknown. In NTHi, the Cas2-like VapD protein is part of a toxin–antitoxin (TA) system and exhibits endoribonuclease activity capable of cleaving mRNA to downregulate bacterial metabolism [1213]. This activity promotes a dormant state under stress conditions, enhancing the survival and virulence of NTHi during infection. Consistent with this, deletion of the vapD gene significantly decreased the survival of NTHi co-cultured with respiratory epithelial cells [3].

Studies in Legionella pneumophila and Mycobacterium tuberculosis have shown that Cas proteins have functions beyond phage/plasmid immunity. For example, in L. pneumophila, Cas2 proteins regulate the expression of another Legionella genes, including the small heat shock protein C2, which is required for thermal tolerance and optimal intracellular infection in Acanthamoeba castellanii [1415], whereas in M. tuberculosis, Cas2 proteins may regulate the transcription of stress-response genes and contribute to long-term bacterial persistence within the host [16].

VapD is considered an evolutionary intermediate between Cas2 proteins and toxins from the TA systems. It retains RNase activity that likely cleaves ribosome-associated mRNA and may be involved in the induction of bacterial dormancy [17]. In H. pylori, VapD does not belong to either CRISPR-Cas or TA system and may therefore be considered an ‘orphan’ endoribonuclease. However, the specific conditions under which VapD exerts its activity in H. pylori remain to be determined.

Based on these observations, we propose that VapD expression is associated with the intracellular physiological state of H. pylori and may reflect adaptive response to the intracellular stress, potentially involving the modulation of mRNAs and/or sRNA pools. While this study does not establish a causal role for VapD in persistence, it identifies VapD as a protein marker linked to intracellular state of H. pylori and provides a framework for future functional studies.

For future studies, the antibodies generated could be conjugated to gold particles for use in electronic microscopy, allowing more precise localization of the protein. Additionally, these antibodies could be applied in immunoprecipitation (IP) assays to identify protein‒protein interactions involving VapD, and in RNA IP assays to identify RNA targets, which would help elucidate the function of VapD in the intracellular environment.

Conclusions

In this study, we provide the first direct evidence that the VapD protein is detectably expressed when H. pylori is inside a eukaryotic cell. This finding complements previous transcriptional and genetic analysis and identifies VapD as a protein associated with the intracellular state of H. pylori. Moreover, this raises the question of the role of VapD in the intracellular environment and why its expression occurs under these conditions.

Supplementary material

10.1099/mic.0.001683Uncited Supplementary Material 1.