Identification of Antimicrobial Peptide Variants From Lactobacillus spp. Against H. pylori-Mediated Gastric Cancer: An In-Silico Approach

Article information

Korean J Helicobacter Up Gastrointest Res. 2026;26(1):60-67

Publication date (electronic) : 2026 March 5

doi : https://doi.org/10.7704/kjhugr.2025.0049

Tammanna R. Sahrawat, Sushant

Center for Systems Biology and Bioinformatics, UIEAST, Panjab University, Chandigarh, India

Corresponding author Tammanna R. Sahrawat, PhD Center for Systems Biology and Bioinformatics, UIEAST, Panjab University, Chandigarh 160014, India E-mail: tammanna@pu.ac.in

Received 2025 July 22; Revised 2025 August 28; Accepted 2025 November 17.

Abstract

INTRODUCTION

Helicobacter pylori infection is strongly associated with gastric carcinogenesis, with chronic gastritis induced by H. pylori being the strongest known risk factor for gastric adenocarcinoma, and it has been classified as a Group I carcinogen by the WHO [1-4]. The association between H. pylori infection and the development of gastric cancer (GC) has been well established through epidemiological and molecular studies, and is reported to be responsible for nearly 90% of non-cardia GC cases [2,5-7]. GC ranks as the fifth most prevalent cancer and the fourth leading cause of cancer-related deaths as of 2022 [6]. Chronic infection induces both epigenetic modifications and genetic mutations within gastric epithelial cells, thereby promoting genomic instability [7].

Helicobacter pylori is a spiral, gram-negative bacterium that plays a pivotal role in promoting gastric tumorigenesis through key virulence factors such as cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA), which interfere with host cellular signaling, alter cell morphology, and compromise epithelial integrity, thereby inducing chronic inflammation and promoting the oncogenic transformation of gastric epithelial cells. It induces DNA damage through superoxides and promotes carcinogenesis via inflammation-derived cytokines and growth factors [8]. Additionally, its ability to persist in the highly acidic gastric environment through urease activity and biofilm formation further contributes to persistent infection and carcinogenesis [2,7].

H. pylori eradication therapy demonstrates anti-cancer effects by reducing gastric inflammation, inflammatory cell infiltration, and atrophy/intestinal metaplasia [8]. Animal studies have shown that antibiotic treatment significantly reduces gastric dysplasia and cancer development, with effectiveness dependent on the timing of intervention, being most effective before premalignant changes develop and showing better outcomes in younger patients [3,4].

The antibiotic resistance of H. pylori has become a significant global challenge, with the WHO designating clarithromycin-resistant H. pylori, which exceeds 15% in many regions, as a high-priority pathogen requiring urgent research and development [9-11]. This resistance is primarily caused by point mutations in the bacterial chromosome, resulting in decreased membrane permeability, altered oxidation-reduction potential, and enhanced efflux pump systems. Therefore, alternative treatment strategies have been developed, including sequential, concomitant quadruple, hybrid, and standard quadruple therapies [12].

Antimicrobial peptides (AMPs) represent a promising therapeutic substitute for antibiotics, particularly in combating multidrug-resistant infections [13]. These are short polypeptides comprising 10–50 amino acids with a molecular weight of less than 10 kDa, and they are present across a wide range of organisms, including animals, insects, plants, and humans [14]. AMPs are naturally expressed in the stomach and play key roles in innate immune responses; therefore, they can function synergistically with conventional drugs to enhance therapeutic efficacy against H. pylori [15]. AMP Tilapia piscidin 4 has been shown to effectively inhibit both antibiotic-sensitive and multidrug-resistant strains of H. pylori using a mechanism that involves membrane disruption and micelle formation, while also suppressing regulatory T cells and inflammatory cytokines to restore immune balance [16]. Similarly, supramolecular AMP hydrogels have demonstrated potent antimicrobial activity, offering advantages over conventional antibiotics, including rapid killing in acidic gastric environments and promotion of inflammation resolution [17].

In recent years, probiotics have gained significance because of their positive effects on the host through the maintenance of gut health and overall well-being. Probiotics are beneficial, non-pathogenic microbes, including Lactobacillus species, which are rod-shaped, gram-positive bacteria that predominantly synthesize lactic acid via fermentative metabolism [18,19]. Probiotics have been reported to exert protective effects against gastrointestinal and urogenital disorders owing to their notable antimicrobial activity and provide various health benefits, including reduced lactose intolerance, decreased cancer risk and progression, and improved immunity [20].

Lactobacillus species have emerged as important sources of bioactive compounds, including AMPs [21], and various studies have revealed that they can decrease the activity of H. pylori virulence factors [20,22]. The metabolites synthesized by L. casei have been reported to reduce the motility and internalization abilities of H. pylori [23]. Similarly, L. reueri has been shown to notably inhibit flaA and vacA gene expression [24], while L. paraplantarum cell-free supernatant decreased the adhesion of H. pylori to human gastric adenocarcinoma AGS cells [25]. These findings suggest that Lactobacilli can serve as excellent candidates for developing AMP-based therapies targeting H. pylori-mediated GC, an approach that has not yet been explored. Therefore, this in silico study was undertaken to identify AMP variants derived from Lactobacillus spp. that exhibit potential inhibitory effects against H. pylori infection by targeting virulence factors previously associated with GC development. The integration of computational methods has significantly advanced AMP research by enabling efficient prediction and characterization of novel AMP sequences, as conventional AMP discovery strategies are laborious, time-consuming, costly, and require extensive biological assays [26,27].

METHODS

Docking studies

Peptide-protein docking was performed using HADDOCK 2.4 (https://rascar.science.uu.nl/haddock2.4/), followed by determination of binding free energy using HawkDock webserver (https://cadd.zju.edu.cn/hawkdock/) and visualization with Discovery Studio Visualizer (https://discover.3ds.com/discovery-studio-visualizer-download). A graphical abstract illustrating the research methodology is presented in Fig. 1.

Fig. 1.

Graphical abstract summarizing the in silico workflow of this study. AMPs, antimicrobial peptides; ADMET, Absorption, Distribution, Metabolism, Excretion, and Toxicity.

RESULTS

AMP region, anti-biofilm peptide, and AMP properties prediction

A total of 109 AMPs derived from Lactobacillus spp. were retrieved from the four databases (Supplementary Table 1 in the online-only Data Supplement), from which 138 AMP regions, each representing the highest scoring segment with an AMP probability score ≥0.5, were selected for further analysis (Supplementary Table 2 in the online-only Data Supplement), while regions with scores <0.5 were excluded due to their low predicted antimicrobial potential. Among these, only 45 AMPs showed an AntiBFP score ≥0.5, indicating strong anti-biofilm activity, and were further analyzed for biological properties (Supplementary Table 2 in the online-only Data Supplement), whereas AMPs with scores <0.5 were excluded due to their low anti-biofilm potential. Nine AMPs were selected based on AIP prediction confidence ≥medium, being anti-cancer, nonallergens, and non-antigenic peptides (Table 1, Supplementary Table 3 in the online-only Data Supplement), while the remaining peptides were excluded due to low AIP prediction confidence, low anti-cancer, or were predicted to be potential allergens or antigenic peptides.

Table 1.

Biological and physicochemical properties of the selected nine peptides

The molecular weights of these 9 peptides ranged from approximately 1.15 kDa to 1.61 kDa, and their theoretical isoelectric points (pI) spanned a wide range from acidic (pI 3.37) to highly basic (pI 11.00). Only AMP seq30 demonstrated low stability, exhibiting an instability index above the threshold of 40, whereas all others were classified as stable. The GRAVY scores ranged from -1.425 to 1.091, with negative values indicating hydrophilic peptides and positive values indicating hydrophobic peptides (Table 1) [27]. ADMET property analysis revealed that the nine AMPs possessed low absorption, moderate distribution, minimal metabolic interaction, moderate excretion, low toxicity risks, and acceptable safety profiles (Supplementary Table 4 in the online-only Data Supplement) [31].

Structure prediction and docking

The structures of the AMPs were predicted using PEPFOLD4, which revealed that most exhibited a primarily alpha-helical conformation. However, seq78 exhibited a coiled structure, suggesting limited structural diversity among the predicted AMPs. The structures of the four target virulence proteins and their 14 corresponding interacting host proteins reported to be involved in GC were retrieved from the PDB (Table 2) for docking analysis.

Table 2.

H. pylori virulence factors and host protein targets in gastric cancer, showing their docked peptides and binding free energies

A total of 162 protein–protein docking analyses were performed between the nine selected AMPs and the four target virulence proteins, along with their 14 corresponding interacting host proteins involved in GC. The docking studies indicated strong interactions between the modeled AMPs and the target proteins, highlighting the strong binding affinity of several AMPs (Supplementary Table 5 in the online-only Data Supplement). Strong binding free energies were observed for the complexes with the most favorable HADDOCK scores (Table 2). Notably, peptide seq30 exhibited potent binding to CagA with a binding free energy of -75.28 kcal/mol, while seq55 interacted strongly with c-Met (-99.15 kcal/mol), seq28 with Ecadherin (-75.58 kcal/mol), and seq78 with both β-catenin and proteinase-activated receptor 1 (-85.52 kcal/mol and -84.74 kcal/mol, respectively) (Fig. 2).

Fig. 2.

Docking and interacting residues of the most favorable scoring complexes of a representative H. pylori virulence factor, CagA (PDB ID: 4DVY), and the host protein, c-Met (PDB ID: 7B43), with their respective AMPs. A: Docked complex of CagA with seq30, showing a binding free energy of -75.28 kcal/mol. B: CagA residues interacting with seq30. C: Docked complex of c-Met with seq55 with binding free energy of -99.15 kcal/mol. D: c-Met residues interacting with seq55. AMPs are shown in red, and the interacting residues of the target proteins are shown in cyan. AMPs, antimicrobial peptides; PDB, Protein Data Bank.

DISCUSSION

Lactobacilli are well-known probiotics that exert protective effects against gastrointestinal disorders and have been reported to be associated with reduced cancer risk. However, despite improved host immunity and their rich source of bioactive compounds capable of inhibiting H. pylori [20-22], AMPs remain largely unexplored against H. pylori-mediated GC. Therefore, the present study was undertaken to explore AMP variants sourced from Lactobacillus spp. to assess their potential inhibitory effects on H. pylori infection, which could suppress cell invasion and disrupt signaling cascades that lead to oncogenic transformation and GC.

A total of 109 AMPs were retrieved from AMP databases, of which 138 AMP regions were identified; only nine AMPs were shortlisted based on ADMET analysis, and their physicochemical characteristics were safe and pharmacologically stable. Tertiary structure prediction of these AMPs indicated that they adopt an alpha-helical form, a common secondary structural motif associated with antimicrobial activity, which is frequently observed in many naturally occurring AMPs [32]. Docking analysis revealed that all nine selected AMPs had significant binding affinities with the target proteins, including H. pylori virulence factors and their human host-interacting proteins (Table 2). Stable peptide–protein interactions of seq28, seq30, seq55, and seq78, indicated by their strong binding free energies, highlight their potential as therapeutic candidates for preventing H. pylori infection.

In the present study, seq30 (derived from plantaricin-S-β produced by L. plantarum strain LPCO10), seq78 (derived from plantaricin JY22 produced by L. plantarum strain JY22), and seq55 (originating from plantaricin ZJ5 produced by L. plantarum strain ZJ5) have been previously reported to possess high antimicrobial activity against Enterococcus faecalis [33], Bacillus cereus [34], and Staphylococcus aureus [35], respectively. Peptide seq28, derived from plantaricin NC8 β peptide and produced by L. plantarum strain NC8, has been reported to show significant antimicrobial effects against H. pylori strain ZJC03 by disrupting the capacity of the strain to modify the host microenvironment, thereby offering a novel approach for the prevention and control of H. pylori infection [36].

H. pylori CagA is a major virulence factor strongly associated with GC development. CagA functions as an oncoprotein delivered to gastric epithelial cells via the type IV secretion system, where it undergoes tyrosine phosphorylation. This is followed by the initiation of multiple cellular signaling pathways that lead to alterations in cell morphology, increased inflammation, and enhanced cell proliferation, contributing to various gastric diseases, including GC [7,37]. CagA also disrupts E-cadherin and β-catenin by interacting with E-cadherin independently of its tyrosine phosphorylation, leading to β-catenin accumulation in both the cytoplasm and nucleus, which contributes to the onset of intestinal metaplasia, a precursor lesion in the transition from normal to neoplastic gastric epithelium [7]. Through direct interaction, CagA inhibits proteinase-activated receptor 1, a pivotal protein involved in maintaining cell polarity and microtubule stability, and this interaction disrupts cell structure and function, potentially contributing to tissue damage and inflammation, as well as the development of GC [38]. Furthermore, non-phosphorylated CagA can interact with the hepatocyte growth factor receptor c-Met, triggering downstream signaling cascades such as MAPK and PI3K/Akt, which in turn promote cellular proliferation, migration, and survival, which are hallmarks of oncogenic transformation [7,39].

Peptides seq30, seq28, seq55, and seq78 showed strong binding affinities (ranging from -99.15 kcal/mol to -75.58 kcal/mol) with targets namely CagA virulence factor (PDB ID: 4DVY) of H. pylori and human host proteins i.e., E-cadherin (PDB ID: 4ZTE), c-Met (PDB ID: 7B43), β-catenin (PDB ID: 1JDH), and proteinase-activated receptor 1 (PDB ID: 3VW7). This indicates their ability to effectively bind these targets, potentially preventing activation of downstream signaling mechanisms and thereby curtailing oncogenic transformation of gastric epithelial cells following H. pylori infection. The proposed mechanism of action of these AMPs is illustrated in Fig. 3.

Fig. 3.

Proposed mechanism of action of the designed AMPs. AMPs, antimicrobial peptides; GC, gastric cancer.

A key limitation of our study was the absence of experimental validation. Therefore, further in vitro and in vivo studies are needed to evaluate the antimicrobial activities of the designed AMPs against H. pylori infection to reduce the incidence of H. pylori-mediated GC. In vitro assays, such as H. pylori growth inhibition, biofilm disruption, and cytotoxicity analyses, along with in vivo studies on animal models of H. pylori-induced gastric pathology, will aid in assessing the stability and bioavailability of AMPs for effective delivery into target cells without loss of activity or increased toxicity, thereby supporting the successful translation of computational predictions into efficacious therapeutic interventions.

In conclusion, the designed AMPs seq28, seq30, seq55, and seq78 offer a targeted approach with the dual benefits of inhibiting bacterial colonization and interfering with H. pylori-mediated oncogenic signaling, while exhibiting low toxicity and a low likelihood of antimicrobial resistance. The AMPs can therefore be further explored as therapeutic candidates for the prevention of H. pylori infection and the development of GC.

Supplementary Materials

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