https://orcid.org/0000-0002-3078-9423
Figures
Abstract
Helicobacter pylori is a microorganism associated with serious gastric pathologies. This bacterium presents specific genes that encode for different virulence factors associated with the development of gastric disease. The VapD protein has rarely been studied, although it has been previously demonstrated its participation in the protection of Helicobacter pylori within gastric cells. In the present work, we document the protocols developed to generate the VapD recombinant protein and the subsequent production of polyclonal antibodies. Our research group faced several problems throughout the trials; however, all of them were successfully solved.
Citation: Flores-Alanis A, Delgado G, Santiago-Olivares C, Luna-Pineda VM, Cruz-Rangel A, Guerrero-Mejía D, et al. (2025) Effective polyclonal antibodies against the virulence-associated protein D (VapD) of Helicobacter pylori, obtained from recombinant VapD. PLoS ONE 20(4): e0321455. https://doi.org/10.1371/journal.pone.0321455
Editor: Daniela Flavia Hozbor, Universidad Nacional de la Plata, ARGENTINA
Received: July 4, 2024; Accepted: March 6, 2025; Published: April 16, 2025
Copyright: © 2025 Flores-Alanis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: “All relevant data are within the paper and its Supporting Information files.”
Funding: RME received: DGAPA-PAPIIT grant IN213921 and CONAHCYT CF-2023-G-919.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Virulence-associate protein D (VapD) from Helicobacter pylori was first structurally and biochemically characterized 12 years ago [1]. It is a small protein of 11.2 kDa with purine-specific endoribonuclease activity related to the Cas2 family from the CRISPR/Cas system-associate proteins. Nonetheless, there are no studies that documented the potential role of this protein in H. pylori pathogenesis. VapD, however, has been studied in other microorganisms, including Rhodococcus equi and Haemophilus influenzae, suggesting that vapD may protect these bacteria from respiratory bursts within macrophages, or facilitate persistence in the respiratory epithelial cells microenvironment, respectively [2–4]. On the other hand, the vapD gene has been found to be overexpressed during the biofilm formation of pathogenic strains of Xylella fastidiosa; suggesting a role in bacterial virulence [5].
We reported that the transcription of the vapD gene in H. pylori when observed in adenocarcinoma gastric cells (AGS) [6]. In this microenvironment, this bacterium adopted a coccoid form, but still metabolically active. This fact was demonstrated through real-time PCR [6]. Moreover, it has been also observed the expression of the vapD in gastric biopsies of patients with atrophic chronic gastritis, follicular gastritis, peptic ulcers, and gastric adenocarcinoma [6]. These findings suggest the expression of the VapD protein, and its possible significant in the protection and/or persistence of H. pylori within gastric epithelial cells. Consequently, we believe that studying the role of VapD in the pathogenesis of chronic H. pylori infection is crucial to the understanding of the development of chronic inflammation and oncogenesis.
In the present study, we developed reproducible protocols that enable the cloning, expression and purification of recombinant VapD (rVapD) from H. pylori, as well as the production of polyclonal antibodies in mice. These antibodies were effective for in vivo immunolocalization and in vitro immunoprecipitation of VapD. Throughout the various assays, we encountered and successfully solved several technical challenges.
Materials and methods
The protocol described in this peer-reviewed article has been previously published in the server protocols.io, (dx.doi.org/10.17504/protocols.io.dm6gpz251lzp/v1) which is included for printing purposes in the Supplementary Material (S1 File).
Expected results
The vapD gene from H. pylori strain 22695 was identified through end-point PCR, resulting in a 282 bp amplicon. This amplicon was subsequently cloned into the pLATE31 expression vector and transformed into E. coli Rosetta (DE3). The colonies were screened by PCR, and the plasmids from positive colonies were verified through Sanger sequencing. The vapD gene was located in an open-read frame with a six histidine tag at the 3’ end (Fig 1).
The vapD gene lacking a stop codon was cloned into a pLATE31 vector. This expression vector presents a six histidine coding sequence (6xHis), which allows the expression of the protein with a C-terminal 6xHis-tag. Additionally, the vector presents the following characteristics: a β-lactamase gene (bla) conferring resistance to ampicillin (APR); an origin of replication (rep [pMB1]); the rop protein gene, which regulates the number of copies of the plasmid; the lac repressor (lacl), which ensures a strict control of the basal expression of the T7 RNA polymerase promoter (PT7); the T rrnBT1-2 transcription terminator, which prevents basal gene expression from vector derived promoter-like elements; the lacO operator, which ensures a strict control of gene expression; a ribosome binding site (RBS); Ptet, a promoter reduces basal expression from the PT7; the T7 terminator (TT7), which terminates transcription from the PT7. The vector was created in BioRender (https://BioRender.com).
E. coli Rosetta (DE3) carrying the pLATE31-vapD-6xHis construct was induced with 0.1 mM IPTG.The rVapD protein was observed at approximately 12 kDa (Fig 2). No difference in protein expression was observed with different IPTG concentrations (0.5 and 1.0 mM). The His-tagged rVapD was purified through immobilized metal affinity chromatography (IMAC) (Fig 3A). The protein was then concentrated, and its identity was confirmed by Western blotting with an anti-His antibody (Fig 3B).
Aliquots of 20 µl of culture were collected at 0, 1, 2, 3, and 4 h after the addition of 0.1mM IPTG to verify the correct expression of rVapD by SDS-PAGE. MW, molecular weight.
A. Recuperation of rVapD. Aliquots of 20 µl from each elution were separated by SDS-PAGE. Lane 1: flow-through of the E. coli Rosetta (DE3) lysate after the His-tag column; lanes: 2-3, washes with buffer containing 50 mM imidazole; lanes: 4-8, rVapD eluted from the His-tag column with buffer containing 325 mM imidazole. B. rVapD was concentrated after IMAC and confirmed by Western blotting anti-His using 1 µg of rVapD. MW, molecular weight.
We observed that when bacterial lysis was performed with lysozyme, rVapD was located in the insoluble fraction (Fig 4A). However, non-denaturing methods are available for recovering soluble proteins from inclusion bodies by addition a mix of detergents such as CHAPS, Triton X-100 or sarkosyl. Studies have shown that combining these detergents yields better results than using them individually [7,8]. In these studies, optimal results were achieved with 1–2% sarkosyl, 2–4% Triton X-100 and 20–40 mM CHAPS. The authors initially solubilized the insoluble pellet with sarkosyl for 6–12 h after centrifugation; subsequently, to increase the efficiency of binding to chromatography, Triton X-100 and CHAPS were added as well.
A. Recovery of rVapD. Lanes 1 and 2: soluble (20 µg) and insoluble (2 µl) fractions, respectively, from E. coli Rosetta (DE3) lysate overexpressing rVapD with lysozyme method. Lanes 3 and 4: soluble (20 µg) and insoluble (2 µl) fractions from E. coli Rosetta (DE3) lysate overexpressing rVapD with lysozyme and non-denaturing method, respectively. B. Purification of rVapD by IMAC after non-denaturing method. Aliquots of 20 µl from each elution were separated by SDS-PAGE. Lane 1: flow-through of the E. coli Rosetta (DE3) lysate after the His-tag column; lanes: 2-3, washes with buffer containing 50 mM imidazole; lanes: 4-8, rVapD eluted from the His-tag column with buffer containing 325 mM imidazole. MW, molecular weight.
In this work, we reduced the concentration of sarkosyl to 0.7% to prevent the protein extract from becoming too viscous. We employed a mix of the three detergents simultaneously after the bacteria pellet was resuspended, as previously reported [9]. The results were similar: the purification ratio between our method and the previously reported detergent concentrations [7] was 1:0.96, suggesting that the modifications did not alter the purification. Furthermore, we observed that the yield of rVapD was lower when it was purified without the use of detergents (0.66 mg/ml) than when it was purified with non-denaturing detergents (1.7 mg/ml). Our method did not affect the purification efficiency of rVapD (Fig 4B).
A previous study reported complications when obtaining stable H. pylori rVapD protein fused to a His-tag, as it precipitated at concentrations above 25 µM. The authors addressed this issue by fusing VapD with the streptococcal protein G (GB1) [1]. In the present work, we observed that after IMAC purification, rVapD remained stable at concentrations up to 150 µM for one month at 4°C. This fact did not represent a problem for our objectives; however, it should be considered for further studies.
Although VapD is a small protein (11.2 kDa), we found that the combination of rVapD with Freund’s adjuvant was sufficient to induce an immune response, as depicted in Fig 5A. The result of the indirect ELISA demonstrated that immunization and booster doses increased the immune response over time; at the end of the immunization schedule, the highest titers were observed at dilutions between 1:500 and 1:4,000 (Fig 5B). The polyclonal serum from the final bleeding of the mice was tested against various concentrations of rVapD; dot blotting revealed that the serum recognized up to 30 ng of rVapD at dilutions of 1:500 and 1:1,000, whereas at a dilution of 1:2,000, rVapD was detected at a concentration of 60 ng (Fig 5C). Finally, the serum was tested by Western blot against the same rVapD concentrations used in the dot blot, with a 1:2,000 dilution (Fig 5D).
A. Representation of the immunization schedule. B. Absorbance values of the indirect ELISA with serum tested at 30, 45 and 60 days (gray arrows in A), rVapD was used at a final concentration of 100 ng. C. Dot blotting test of the final bleeding (red arrow in A) using three dilutions of the serum (1:500, 1:1,000, and 1:2,000) and a serially two-fold diluted concentration of rVapD (1 to 0.03 µg). D. Western blotting of the serum at a dilution of 1:2,000 against a serially two-fold diluted concentration of rVapD (1 to 0.12 µg). Fig 5A was created in BioRender (https://BioRender.com).
To demonstrate the in vivo expression of the VapD protein, we performed an immunofluorescence assay in co-cultures of AGS cells and H. pylori strain 26695 at 48 h post-inoculation. The co-cultures were incubated with anti-vapD (green) and anti-H. pylori (red) antibodies. The presence of VapD corresponded with the localization of intracellular bacteria (Fig 6). Furthermore, we observed multiple vesicles, some of which contained bacteria (arrows in Fig 6).
H pylori was observed in AGC cells at 48 h post-inoculation. The expression of VapD coincided with the presence of the bacterium. The arrows point to vesicles where H. pylori is internalized.
Anti-VapD polyclonal antibodies were also tested through an immunoprecipitation (IP) assay. IP efficiency was evaluated using lysates from Rosetta (DE3) cells overexpressing rVapD, and the antibodies successfully immunoprecipitated rVapD (Fig 7).
Lane 1: mouse serum (5 µl) + bacterial lysate (20 µl); lane 2: mouse pre-immune serum (5 µl) + bacterial lysate (20 µl); lane 3: lysate of Rosetta (DE3) cells overexpressing rVapD (20 µl). MW, molecular weight.
This work describes the protocols for the production, expression, and purification of H. pylori rVapD, as well as the production of its antibodies. The results indicate that the polyclonal antibodies successfully recognized both the quaternary and linear structures of VapD. The immunolocalization and IP results revealed that the polyclonal serum recognized the structural epitopes, whereas the Western blot result demonstrated the recognition of linear epitopes. These findings open the possibility of selecting monoclonal antibodies using the hybridoma method to identify antibodies on the basis of specific applications for further investigation. This is the first study to report the production of specific antibodies against VapD.
Supporting information
S1 File. Step-by-step protocol, also available on protocols.io.
https://doi.org/10.1371/journal.pone.0321455.s001
(PDF)
S3 File. Values used to build the graph in Fig 5C.
https://doi.org/10.1371/journal.pone.0321455.s003
(XLSX)
References
- 1. Kwon A-R, Kim J-H, Park SJ, Lee K-Y, Min Y-H, Im H, et al. Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity. Nucleic Acids Res. 2012;40(9):4216–28. pmid:22241770
- 2. Benoit S, Benachour A, Taouji S, Auffray Y, Hartke A. Induction of vap genes encoded by the virulence plasmid of Rhodococcus equi during acid tolerance response. Res Microbiol. 2001;152(5):439–49. pmid:11446512
- 3. Benoit S, Benachour A, Taouji S, Auffray Y, Hartke A. H(2)O(2), which causes macrophage-related stress, triggers induction of expression of virulence-associated plasmid determinants in Rhodococcus equi. Infect Immun. 2002;70(7):3768–76. pmid:12065520
- 4. Ren D, Walker AN, Daines DA. Toxin-antitoxin loci vapBC-1 and vapXD contribute to survival and virulence in nontypeable Haemophilus influenzae. BMC Microbiol. 2012;12:263. pmid:23157645
- 5. Mendes JS, da Santiago AS, Toledo MAS, Rosselli-Murai LK, Favaro MTP, Santos CA, et al. VapD in Xylella fastidiosa is a thermostable protein with ribonuclease activity. PLoS One. 2015;10(12):e0145765. pmid:26694028
- 6. Morales-Espinosa R, Delgado G, Serrano L-R, Castillo E, Santiago CA, Hernández-Castro R, et al. High expression of Helicobacter pylori VapD in both the intracellular environment and biopsies from gastric patients with severity. PLoS One. 2020;15(3):e0230220. pmid:32163505
- 7. Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques. 2010;48(1):61–4. pmid:20078429
- 8. Massiah MA, Wright KM, Du H. Obtaining soluble folded proteins from inclusion bodies using Sarkosyl, Triton X-100, and CHAPS: application to LB and M9 minimal media. Curr Protoc Protein Sci. 2016;84:6.13.1–6.13.24. pmid:27038270
- 9. Flores-Alanis A. Análisis de complejos ribonucleoprotéicos formados por la proteína de unión a Cap, nCBP de Arabidopsis thaliana. Universidad Nacional Autónoma de México. 2013. Available from: https://tesiunam.dgb.unam.mx/F/NAKCES967VVMJHCA1RX6I3AIUCRK39Q9FVDRM9L36Q7YFHN6T4-14370?func=full-set-set&set_number=018939&set_entry=000004&format=999