Multiplex-PCR-Based Screening and Computational Modeling of Virulence Factors and T-Cell Mediated Immunity in Helicobacter pylori Infections for Accurate Clinical Diagnosis.

Sinem Oktem-Okullu,Tanil Kocagoz,Ugur Sezerman,Ayca Sayi-Yazgan,Nurdan Tozun,Arzu Tiftikci,Murat Saruc,Eser Vardareli,Bahattin Cicek,Ahmet Sinan Yavuz,Jian Zhang

doi:10.1371/journal.pone.0136212

Sinem Oktem-Okullu, Tanil Kocagoz + Show 9 more

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https://doi.org/10.1371/journal.pone.0136212

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Journal: PloS one	Publication Date: Aug 19, 2015
Citations: 9	License type: CC BY 4.0

Affiliation: Istanbul Technical University, Acıbadem University

Abstract

The outcome of H. pylori infection is closely related with bacteria's virulence factors and host immune response. The association between T cells and H. pylori infection has been identified, but the effects of the nine major H. pylori specific virulence factors; cagA, vacA, oipA, babA, hpaA, napA, dupA, ureA, ureB on T cell response in H. pylori infected patients have not been fully elucidated. We developed a multiplex- PCR assay to detect nine H. pylori virulence genes with in a three PCR reactions. Also, the expression levels of Th1, Th17 and Treg cell specific cytokines and transcription factors were detected by using qRT-PCR assays. Furthermore, a novel expert derived model is developed to identify set of factors and rules that can distinguish the ulcer patients from gastritis patients. Within all virulence factors that we tested, we identified a correlation between the presence of napA virulence gene and ulcer disease as a first data. Additionally, a positive correlation between the H. pylori dupA virulence factor and IFN-γ, and H. pylori babA virulence factor and IL-17 was detected in gastritis and ulcer patients respectively. By using computer-based models, clinical outcomes of a patients infected with H. pylori can be predicted by screening the patient's H. pylori vacA m1/m2, ureA and cagA status and IFN-γ (Th1), IL-17 (Th17), and FOXP3 (Treg) expression levels. Herein, we report, for the first time, the relationship between H. pylori virulence factors and host immune responses for diagnostic prediction of gastric diseases using computer—based models.

Highlights

Helicobacter pylori (H. pylori) is a gram negative, microaerophilic, spiral-shaped bacteria that colonize in human gastric mucosa and if not treated, it persists through lifespan
It is well established that virulence factors with potential value for specific pathologies are the cytotoxin associated gene A, vacuolating cytotoxin gene A, outer inflammatory protein A, the blood group antigen binding adhesin gene A, the putative neuraminyllactose-binding hemagglutinin homolog, neutrophil-activating protein A, duodenal ulcer promoting gene A, urease A, and urease B [7, 8]
We optimized multiplex-PCR of genomic DNA derived from H. pylori G27 strain to detect nine H. pylori virulence genes within a three PCR reaction; urease B (ureB), hpaA, cytotoxin associated gene A (cagA), neutrophil-activating protein A (napA), urease A (ureA) in a single reaction, duodenal ulcer promoting gene A (dupA), outer inflammatory protein A (oipA), vacuolating cytotoxin gene A (vacA) in a single reaction, and blood group antigen binding adhesin gene A (babA) in a single reaction (Fig 1(a) and 1(b))

Summary

Introduction

Helicobacter pylori (H. pylori) is a gram negative, microaerophilic, spiral-shaped bacteria that colonize in human gastric mucosa and if not treated, it persists through lifespan. The clinical outcome of H. pylori infection is determined by multiple factors including H. pylori related virulence factors, host genetic predisposition and immune response [5, 6]. Many putative virulence genes have been described to determine the clinical outcome of H. pylori infection. Virulence factors are important in conditioning the clinical outcome of the H. pylori–driven infection, the local immune response mechanisms have been claimed to play a role in the pathogenesis of the disease [9]. CD4+ T cells including T- helper cells (Th) and regulatory T-cells (Tregs) are shown to play critical roles in the pathogenesis of H. pylori infection. Described T helper 17 (Th17) cells are shown to play a role in host defense against H. pylori infection by mediating the recruitment of neutrophils and macrophages into infected tissues. Tregs have been identified as the major regulatory component of the adaptive immune response and they have been involved in Helicobacter pylori-related inflammation and bacterial persistence [10,11,12,13,14]

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