Multiplex-PCR based screening and computational modeling of virulence factors and T cell mediated immunity in Helicobacter pylori infections for accurate clinical diagnosis

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, spiral-shaped bacterium that colonizes the stomach of more than half of the world's population during their life span. The bacteria appear around 3μm in length and possess a unipolar bundle of two to six sheathed flagella, about 0,5μm in size, that enable the bacteria to move in the acidic environment of stomach. H. pylori infection is usually acquired in early childhood and persists throughout the life. While most infected individuals remain asymptomatic, H. pylori infection can cause peptic ulcer, chronic gastritis, mucosa-associated lymphoid tissue (MALT) lymphomas and gastric cancer in 20% of the infected ones. The prevalence of H. pylori is higher in developing countries and it may reach 70-80% of adults in Turkish population. While the exact route of transmission is unknown, acquisition may occur through fecal to oral or oral to oral contact. H. pylori infection can be diagnosed by the presence of dispeptic symptoms. Invasive and non-invasive methods are also used for the diagnosis of the H. pylori infection. Combination of antimicrobial agents and proton pump inhibitors has been used for the treatment of H. pylori infection; however, drug resistance may cause treatment failures, for which different drug combinations are used to overcome this problem. The outcome of H. pylori infection is closely related with bacteria's virulence factors, host genetic factors and host immune response. Bacterial virulence factors contribute to the inflammatory response towards H. pylori either by different mechanisms like host-signaling pathways, maintaining tissue homeostasis in epithelial cells or by stimulating the innate immune cells. Cytotoxin-associated gene A (cagA), vacuolating cytotoxin gene A (vacA), outer inflammatory protein A (OipA), blood group antigen-binding adhesion (BabA), putative neuraminyllactose-binding hemaggluttinin homolog A (HpaA), neutrophil activating protein A (NapA), duodenal ulcer promoting gene A (dupA), urease subunit A (UreA), urease subunit B (UreB) are the most important H. pylori virulence factors that play a role in the bacterial adhesion, colonization and infection. Besides the virulence factors, type of the immune response of the host against H. pylori is crucial for the outcome of the infection. T cells are types of lymphocytes that play a central role in cell-mediated immunity against H. pylori infection. T cells develop in the thymus, and then enter the circulatory system. T cells are classified as cytotoxic T cells (CD8+ T cell), helper T cells (CD4+ T cell) and regulatory T cells (CD4+CD25+ T cell). Helper T cells differentiate into five major subsets known as T helper 1 (Th1), T helper 2 (Th2), T helper 17 (Th17), T helper 9 (Th9) and T helper 22 (Th22). T helper 1-based immune responses are required for protection against intracellular pathogens. T helper 2 cells are responsible for the protection against extracellular parasites. Recently described T helper 17 (Th17) cells are responsible for mediating defensive mechanisms of the host against various infections including extracellular bacteria and fungi infections. In previous studies, the association between T cells and H. pylori infection has already been identified. Th1 and Th17 cells are involved in response to H. pylori infection and contribute to the development of H. pylori – related pathology. Moreover, colonization with H. pylori leads to the expansion of regulatory T cells (Tregs) which limits the inflammatory response and cause the establishment of chronic infection. Th1, Th17, and regulatory T-cells (Treg) are localized in both gastric mucosa and peripheral blood of H. pylori – infected patients. It is anticipated that there may be a close relationship between the H. pylori infection and bacterial virulence factors for the clinical outcomes of the infection. However, the effects of nine major Helicobacter pylori specific virulence factors; CagA, VacA, OipA, BabA, HpaA, NapA, DupA, UreA, UreB on Th1, Th17 and Treg cell response in H. pylori - infected patients with gastritis and ulcer have not been fully elucidated. Therefore in this study, we aimed to investigate the association between the nine major (CagA, VacA, OipA, BabA, HpaA, NapA, DupA, UreA, UreB) Helicobacter pylori specific virulence factors with three various T cell subsets (Th1, Th17 and Treg) at mRNA expression levels in H. pylori-infected gastritis and ulcer patients. The presence/absence of nine virulence factors were detected using multiplex-PCR and mRNA expression levels of T cell specific factors were determined using quantitative real time-PCR. In total of 80 adult patients, 18 with ulcer and 62 with gastritis were included in this study. In addition, 18 patients with reactive gastropathy were included as the negative control group. These patients were admitted to Acibadem hospitals with gastrointestinal symptoms and underwent endoscopy. During endoscopy, two gastric antral and corpus biopsies were taken from each patient. One antral and one corpus samples were used for DNA isolation and the other antral and corpus samples were used for RNA isolation. Isolated DNA samples were used to develop a multiplex-PCR assay to detect nine H. pylori-specific virulence factor genes. Moreover, RNA samples were used to detect expression levels of Th1, Th17 and Treg cells - specific cytokines and transcription factors using quantitative real- time PCR (RT-PCR) assays. Primers were designed using primer design software for the H. pylori – specific virulence genes, Th1, Th17 and Treg cell – specific cytokines and transcription factors. First step of the study was the development of multiplex-PCR to detect virulence factor genes. Optimization of multiplex-PCR assay was done by using H. pylori G27 strain that includes all the virulence genes. Also, a multiplex urease PCR assay was developed to detect ureA and ureB genes in a single PCR assay. In the second step, positive samples were detected by the conventional PCR for each cytokine and transcription factor. These positive samples were used to set up the standard curve in the quantitative real time-PCR for each cytokine and transcription factor. Detection of the nine virulence genes using a multiplex-PCR assay within a three polymerase chain reaction was achieved; ureA, ureB, cag A, hpaA, napA in a one reaction, oipA, dupA, vacA in another reaction and babA in a third reaction. All DNA isolated from biopsy samples were tested with the developed multiplex urease PCR assay. A comparison study was performed between the results of the multiplex urease PCR assay, rapid urease test and pathological evaluation that were applied to patient samples. According to the results, it was determined that multiplex urease PCR and pathological evaluation methods produced more similar and precise results than the rapid urease test method. Within all virulence factors that were tested, only the prevalence of NapA virulence factor was significantly higher in patients with ulcer than gastritis. 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 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) mRNA expression levels. Herein, 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 were reported. Furthermore, a novel expert - derived model has been developed to identify the set of factors and rules that may distinguish the ulcer patients from gastritis patients.