Genomics and systems-level insights into Helicobacter pylori mediated virulence

Abstract

Helicobacter pylori (H. pylori) is associated with diverse gastric disorders leading to gastric carcinoma, the third leading cause of gastric mortality. Being a genetically highly diverse bacterium, H. pylori displays high variation among virulence factors associated with clinical outcomes of infection. Most important virulent factors include VacA and CagA encoded by almost 70% of the strains. These virulent proteins can interfere with multiple cellular activities and alter various host signaling pathways leading to cell proliferation, cytoskeletal rearrangements, and disruption of cell-cell junctions. The adapted virulence mechanism by the organism results into a high antibiotic resistance and hence contributing towards an increased global burden of gastric infections and carcinogenesis. Thus far, none of the conventional treatment regimens results in complete pathogen eradication, gastroduodenal ulcer relapse and bacterial resistance. The organism adopts various routes to enter the host cells for persistent colonization and pathogenicity. Besides the type IV secretion system, it also targets the epithelial barriers, tight junction proteins and the potent barrier adapter proteins including zonula occludens, claudins, and connexins. Dysregulation of zonula occluden-1 (Zo-1), Claudin-2 (CLDN2) and Connexin32(CX32) has already been reported during H. pylori infection but the actual mechanism is still unclear. This study aimed to unravel the mechanisms of modifying cell adhesion and dysregulation of ZO-1, CLDN2 and CX32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk ABSTRACT 2 between phosphorylation, O-glycosylation, palmitoylation and methylation within these junction proteins are explored which may compromise their integrity. Various novel PTM sites have been identified within junction proteins which can be further targeted to infer their potential impact in animal models. A qualitative logic-based model is designed highlighting the situation-dependent dynamic behaviors of the host and pathogenic proteins before and after infection, verified by the available experimental data. The model effectively illustrates the key regulatory mechanisms of tight junctions and how they respond to H. pylori infection. Model reflects the sequence of events and captures the logical interactions among entities and clearly depicts that, as a result of specific kinases, expression of CX32 and ZO-1 decreases up to significant levels whereas CLDN2 gets overexpressed to promote paracellular cation leak. The study also attempted to propose vaccines as a promising strategy to combat H. pylori mediated infections, effectively. Thus, a reverse vaccinology approach has been successfully employed to predict the potential vaccine candidates against H. pylori. The predicted potential vaccine candidates include VacA, BabA, SabA, FecA, and Omp16. Multivalent subunit vaccine constructs are designed with aim to induce better antigenic responses than a univalent subunit vaccine. Thus, surface-exposed, conserved and antigenic epitopes from the predicted candidate proteins are screened to design broad-spectrum poly-epitope based peptide vaccines. Seven novel poly-epitope proteins are designed along with suitable adjuvant (Cholera Toxin Subunit B adjuvant at 5’ end) and linkers (GPGPG and EAAAK) against H. pylori by predicting the best possible combinations of predicted epitopes. The proposed poly-epitope vaccines candidates can bind efficiently with A2, A3, B7 ABSTRACT 3 and DR1 superfamilies of HLA alleles, as checked in silico. They also form stable and significant interactions with Toll-like receptor 2 and Toll-like receptor 4. Keeping in mind the laborious and time-consuming process of vaccine candidate predictions, a highly scalable, multi-mode, and configurable pipeline has also been designed, term as VacSol. The pipeline efficiently integrates well-known and robust algorithms/tools for bacterial proteome analysis. The utility of VacSol is tested using the H. pylori reference strain (26695) as a benchmark. The study provided insights into H. pylori mediated virulence and infection and improved our understanding of the mechanism of bacterial pathogenesis. The described methodology can be easily reproduced, and can be extended to other bacterial infectionS