Helicobacter pylori l-asparaginase: a study of immunogenicity from an in silico approach

Abstract

Helicobacter pylori has become the causal agent of multiple forms of gastric disease worldwide, including gastric cancer. The enzyme l-asparaginase (ASNase) has been studied as a virulence factor. In this work, we performed an in silico investigation to characterize the immunological profile of H. pylori ASNase (HpASNase) to ascertain the possible implication of HpASNase immunogenicity in the H. pylori virulence mechanism. We applied a workflow based on bioinformatics tools, which, by calculating the relative frequency of immunogenic T-cell and B-cell epitopes, allowed us to predict the immunogenicity and allergenicity of HpASNase in silico. We also visualized the epitopes by mapping them into the native structure of the enzyme. We report for the first time the T-cell and B-cell epitope composition that contributes to the immunogenicity of this HpASNase, as well as the regions that could generate a hypersensitivity response in humans. ASNase from H. pylori resulted in highly immunogenic and allergenic. The high immunogenicity of HpASNase could imply the pathogenic mechanisms of H. pylori. This knowledge could be important for the development of new drugs against H. pylori infections.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03359-0.

Main text

Helicobacter pylori is a bacterium found on the surface of the gastric epithelium (Roesler et al. 2014) that infects about 60% of the population worldwide (Sitarz et al. 2018). However, in patients with gastric cancer, these values can reach almost 90%, making the bacterium a causal agent of this carcinoma (Moss 2017). This type of cancer contributes to more than 1 million newly diagnosed people globally every year, which is why it constitutes an important health issue (Thrift and El-Serag 2020). Several proteins have been described as pathogenicity factors of this bacterium (Roesler et al. 2014). Among these, it has been suggested that l-asparaginase (ASNase) might act as a pathogenicity attribute for H. pylori (Vimal and Kumar 2017) with an important role in bacterial colonization through a mechanism that relates the enzyme to pH modulation for the bacterial adaptation in the acid environment of the stomach (Merrell et al. 2003; Leduc et al. 2010). l-Asparaginase (EC 3.5.1.1) belongs to the amidohydrolase family and catalyzes the conversion of l-asparagine into l-aspartate and ammonia, although this enzyme is also able to carry out l-glutamine hydrolysis (Maggi et al. 2015a). H. pylori ASNase (HpASNase) is a type II l-asparaginase, which, in contrast to other types II l-asparaginases, presents a lower affinity for l-glutamine (Stivala et al. 2010), suggesting different structural features in comparison to other asparaginases, thus affecting the substrate interaction (Maggi et al. 2015b). During infection with H. pylori, the immune system can respond through multiple mechanisms. For example, a strong production of IgA and IgG antibodies leads to complement activation (Moyat and Velin 2014) and has also been associated with increased chronic gastritis (Stubljar 2015). Regarding the association of ASNase with this humoral immune response, it is proposed that the interaction between the enzyme and the host requires the secretion of ASNase through membrane vesicles, which contribute to the stimulation of the immune system, and as a consequence, the production of specific IgG-type antibodies (Stivala et al. 2010). Although this enzyme has shown an immunogenic potential in mice and human serum studies performed in vitro by ELISA test (Maggi et al. 2018), the impact of HpASNase from an immunological point of view in vivo has not been deeply studied to date. These results, in addition to the fact that HpASNase could behave as a potential antigen in humans, make it interesting to research. In this work, we performed an immunoinformatic study of HpASNase showing the structural determinants involved in its immunogenicity and allergenicity, which could influence the immunological response in humans against this pathogen as well as in the colonization phenomenon.

Vaccination from an early age could provide immunity against H. pylori infection. Currently, some vaccine candidates are at different stages of pre-clinical or clinical development, mainly composed of purified or recombinant components of H. pylori antigens with an adjuvant (Sutton and Boag 2019). Knowledge of immunological mechanisms during natural H. pylori infection is necessary due to the complicated host response against the pathogen, and the factors that allow the persistence of bacteria (Stubljar et al. 2018). For these reasons, this paper may add important information for the development of effective epitope-based drugs against H. pylori, taking advantage of the information obtained through immunoinformatic tools about the composition of T-cell and B-cell epitopes present in the HpASNase structure.

The amino acid sequences of l-asparaginases from H. pylori (2WLT) (Dhavala and Papageorgiou 2009) and E. coli (3ECA) were retrieved from the Protein Data Bank (Burley et al. 2019) and are also presented in Supplementary Material 1. The NetMHCII 2.3 Server (Jensen et al. 2018) was used for the T-cell epitope prediction, selecting as immunogenic epitopes the peptides predicted as "strong binder" and "weak binder". The computation of epitope density was carried out using the relative frequency:$fi=ni/N=ni/\sum jnj$ where n_i is the number of immunogenic epitopes predicted and $N$ is the total number of epitopes (both immunogenic and non-immunogenic). For this, we used the HLA-DRB1*01:01, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*11:01, HLA-DRB1*13:01, and HLA-DRB1*15:01 alleles since these are reference alleles with a high global distribution (Gonzalez-Galarza et al. 2011).

For the prediction of T-cell allergenic epitopes, the AllerTOP v.2.0 server (http://www.ddg-pharmfac.net/AllerTOP/) was used (Dimitrov et al. 2014). Epitopes are classified by the software as probable allergen or probable non-allergen. The relative frequency of the predicted allergenic epitopes was obtained by dividing the number of immunogenic epitopes for the HLA-DRB1*07:01 allele over the total immunogenic epitopes determined in the previous section (Belén et al. 2020).

For the B-cell epitope prediction (linear and discontinuous epitopes), we used the ElliPro server ((Ponomarenko et al. 2008) from the IEDB Analysis Resource database. The epitope visualization in the native structure of HpASNase was carried out using the PyMOL software (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC).

The comparative analysis of epitope density was performed with the GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego, California, USA) package, using Student’s t test (p ≤ 0.05).

For decades, bacteria have been an important source of obtaining ASNase for therapeutic purposes in the treatment of acute lymphoblastic leukemia (Beckett and Gervais 2019). Five formulations of bacterial ASNase are currently on the market (Lew 2020; Völler et al. 2018). Among them, the native E. coli ASNase is considered the most immunogenic, causing hypersensitivity reactions in 30–40% of patients (Beckett and Gervais 2019). Because the response of the human immune system to these ASNase formulations has been well studied, we consider it would be interesting to compare the HpASNase immunogenicity with ASNase from E. coli. The comparative analysis of epitope density was performed with the GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego, California, USA) package, using Student’s t test (p ≤ 0.05) by in silico analysis. This would allow us to predict if the HpASNase could have some immunological impact and act as a relevant antigen in vivo, contributing to H. pylori virulence. H. pylori is known to be responsible for one of the most chronic bacterial infections in humans (Rieder et al. 2005), and the antigens secreted by the bacteria are highly immunogenic (Khalilpour et al. 2013). We applied the relative frequency as a measure of the immunogenic T-cell epitope density. Epitope density has been used to evaluate the immunogenicity of proteins (Belén et al. 2019; Lissabet 2018; Groot and Martin 2009; Groot et al. 2014) since high relative frequencies can be linked with high protein immunogenicity. It is known that T-cell epitope density is an important measure for evaluating the antigenicity of proteins, since this content provides a degree of the binding capacity of the epitopes to the major histocompatibility complex (MHC) and therefore, the recognition by the immune system (Weber et al. 2009).

Hence, this evaluation is essential to the development of new drugs based on immunological stimulation. In Supplementary Material 2, we present the T-cell epitopes of HpASNase predicted by the software as immunogenic. As observed in Fig. 1, the relative frequency of T-cell epitopes was significantly higher for HpASNase (p = 0.0006), resulting in more immunogenic than the E. coli enzyme. This indicates that the immunogenic epitopes of HpASNase could behave as potential antigens in vivo and therefore generate a significant immune response. Maggi et al. suggested that HpASNase plays a role in the antibody response generation in humans infected by H. pylori (Maggi et al. 2018) and also has an influence on H. pylori acid acclimation and resistance (Sachs et al. 2011; Miller and Maier 2014). Although commercial E. coli ASNase formulations generate high immunogenicity, it is important to note that E. coli forms part of the normal intestinal microbiome (Clements et al. 2012); therefore, it is logical to expect that infection with H. pylori will produce this type of response. Additionally, we identified and mapped the B-cell epitopes present in the HpASNase, both linear (continuous) and conformational (discontinuous). In Figs. 2 and 3, we show linear and discontinuous B-cell epitopes, respectively, as well as the mapping of these epitopes in the HpASNase structure. As can be observed, in general, the B epitopes are extensive and located on the most exposed surface of the enzymatic structure. This increases the chances of presentation to the immune system, and therefore antigenic processing and the intensity of the immune response. Single and multi-epitope-based vaccines have shown promising results in the protection against tumors, bacterial and viral infections, stimulating both cellular and humoral responses (Zhang et al. 2013; Christodoulides and Heckels 1994; Liu and Chen 2004; Zhang 2018; Kaumaya 2020). The high immunogenicity predicted for HpASNase and the knowledge of the amino acid composition of immunogenic T-cell and B-cell epitopes could be exploited in the development of epitope-based vaccine candidates against H. pylori.

Fig. 1.

Relative frequency of immunogenic T-cell epitopes. Significant differences were observed between the immunogenicity of H. pylori and E.coli ASNase (0.0006)

Fig. 2.

Linear immunogenic B-cell epitopes and mapping of these epitopes in the HpASNase structure

Fig. 3.

Conformational immunogenic B-cell epitopes and epitope mapping in the structure of HpASNase. BDE (B-cell discontinuous epitopes)

On the other hand, when comparing the relative frequencies for HLA-DRB1*07:01 alleles, we observed that HpASNase presents a higher number of allergenic epitopes (Fig. 4), which are listed in Table 1.

Fig. 4.

Relative frequency of allergenic T-cell epitopes for the HLA-DRB1*07:01 alleles. A higher epitope density was observed for H. pylori ASNase

Table 1.

Peptide sequence of allergenic T-cell epitopes of H. pylori ASNase determined with the HLA-DRB1*07:01 allele

Predicted T-cell allergenic epitopes
LGVKELLKAIPSLNK GVKELLKAIPSLNKI ELLKAIPSLNKIARI AYFLNLVLHSTKPVV FLNLVLHSTKPVVLV VLHSTKPVVLVGAMR LHSTKPVVLVGAMRN ADGALNLYEAVSVAV GALNLYEAVSVAVNE	ALNLYEAVSVAVNEK SVREVVKTHTTHVST REVVKTHTTHVSTFK EVVKTHTTHVSTFKA TTHVSTFKALNSGAI THVSTFKALNSGAIG HVSTFKALNSGAIGS VSTFKALNSGAIGSV STFKALNSGAIGSVY	TFKALNSGAIGSVYY KVDIIYTHAGMTPDL VDIIYTHAGMTPDLF DIIYTHAGMTPDLFQ LFQASLNSHAKGVVI EASQMGVVIVRSSRV ASQMGVVIVRSSRVG SQMGVVIVRSSRVGS QMGVVIVRSSRVGSG

The relationship between H. pylori infection and the incidence of allergic reactions remains a controversial topic. Some works support the hypothesis that there is a negative correlation between the occurrence of allergic reactions and the presence of H. pylori in the gastric mucosa (Sitaraman 2015; Imamura et al. 2010; Chen and Blaser 2007, 2008). However, other authors maintain that the presence of virulence factors such as the CagA⁺ protein in patients infected with H. pylori is positively correlated with high levels of IgE in serum. This type of association has been mainly related to food allergies, since the inflammation caused by the bacteria can affect the integrity of the intestinal mucosa and therefore stimulate the transmission of antigens from food exacerbating the inflammatory cytokines release and IgE-mediated allergic reactions (Censini et al. 1996; Figura et al. 1999; Rasmi et al. 2015). Nevertheless, the H. pylori and allergy connection have not been thoroughly clarified. Although the contribution of HpASNase in the previously suggested association between H. pylori and allergic reactions has not been studied or demonstrated, the allergenic determinants present in the ASNase structure of some bacteria and fungi have been determined by our group with in silico tools (Belén et al. 2019, 2020). In the present study, we compared the allergenicity of HpASNase with ASNase from E. coli. The higher relative frequency of allergenic epitopes for HpASNase compared to that of E. coli (Fig. 4) could point to a possible hypersensitivity response to the enzyme in humans, since the HLA-DRB1*07:01 alleles have been associated with the occurrence of hypersensitivity reactions in patients treated with bacterial ASNase, and it is well known that ASNase from E. coli frequently causes allergic reactions in patients (Fernandez et al. 2014; Kutszegi et al. 2017). Several proteins have been associated with the antigenicity and immunogenicity of H. pylori, as well as its influence on bacterium colonization and invasion ability (Zarei et al. 2017; Raoufi et al. 2015). However, the HpASNase contribution to pathogenicity and immunological response associated with bacterial infection remains poorly understood.

Conclusions

Helicobacter pylori is a widely studied bacterium, especially its pathogenicity mechanisms and virulence factors. L-asparaginase enzyme has been reported as one of these attributes, and in this work, we report, from an in silico approach, the antigenic determinants that could be contributing to the generation of immunological responses in humans. This information has considerable value since we reported the sequence of the antigenic determinants that are probably responsible for the antibodies formed in response to this enzyme. Asparaginases from some bacteria have proven to be immunogenic in humans; however, and unlike other virulence factors of H. pylori, little information is available about the enzyme's immunological potential, nor the information that this could contribute to bacterial infective mechanisms. Hence, these results shed new light on the possibility of considering HpASNase a possible therapeutic target in the treatment of H. pylori infections from an immunological point of view.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

LH and JFBL: conception and design of the work, acquisition, analysis, and interpretation of data. AP, COR-Y, RC, and JGF: revised the work critically for important intellectual content and made language suggestions.

Declarations

Coflict of interest

All the authors declare that they have no conflict of interest.