Abstract
Abstract Helicobacter pylori (H. pylori) is a spiral microaerophilic Gram-negative bacterium which colonizes the gastric mucosa of the human stomach. Infection with H. pylori is epidemiologically associated with development of gastrointestinal diseases. Vacuolating cytotoxin (VacA) is the major disease-related virulence factor of H. pylori, whose molecular mechanisms in cellular damages and gastric diseases are obscure. In Chapter 1, the background regarding H. pylori in aspects of microbiology, epidemiology, and clinical investigations are described, followed by introduction of major virulence factors of H. pylori with emphasis on VacA. In Chapter 2, H. pylori strain diversity was investigated in 55 couples in Taiwan. Only one of 25 couples had the same strain in both partners as characterized by two DNA typing methods. Comparison of isolates from the antrum and corpus in each of 40 patients showed that 9 pairs were distinct but might be related. Peptic ulcer occurred in 77.8% of these 9 patients compared with 29% of 31 patients with the same predominant strain in two biopsies (P = 0.025). RAPD and sequence analysis of two very closely related isolates from one patient supports the hypothesis that development of genetic diversity results from horizontal genetic exchange during long-term colonization of mixed bacterial populations.In Chapter 3, we determined and analyzed cagA status, urease activities, and VacA genetic-functional properties from 189 clinical isolates to investigate the roles of virulence factors in H. pylori-involved carcinogenesis. Undifferentiated urease activities were detected among gastric cancer (GC), peptic ulcer (PU), and non-ulcer dyspepsia (NUD) groups. The presences of cagA gene and s1a genotype of vacA gene were regardless of clinical outcomes due to their extremely high prevalence (~ 98%), whereas GC isolates were significantly associated with higher frequency of vacA m1T genotype (44.7 %) as compared with 71.8 % of m2 strains in PU (P < 0.01). Extensive screening of VacA functional properties showed that m1T isolates displayed comparable production of exotoxins but apparently higher specific vacuolating activities as compared with m2 strains. Flow cytometric examination of VacA-binding on human gastric epithelial cells showed high and moderate-to-high binding competence for m1 and m2 form, respectively. Functional analyses of VacA in different clinical groups revealed that GC was associated with higher cytotoxicities as compared with PU; m1T isolates in GC group possessed significantly higher vacuolating activities and VacA productions than did those in PU (P < 0.05). We thus collectively verified the association of VacA with gastric carcinoma and suggested that high VacA activities might propel the pathogenic progress toward GC by worsening degenerative damages. Infection with either m1 strains or strains carrying high VacA cytotoxicity could both be regarded as the high-risk indicator for developing gastric malignancy.Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) and the lipid microdomain (lipid raft) are speculated to play roles in the process of VacA intoxication, despite that comprehension of the relevant mechanism is largely in absence. In Chapter 4, the interactions among VacA, GPI-APs, and the lipid raft were investigated with an aim to understand the molecular action and biological denotation of VacA. Transfected CHO cells expressing fasciclin I, a GPI-AP, displayed higher VacA sensitivity as compared with parental cells. PI-PLC removal of GPI-APs impaired vacuolating activities on AGS, MDCK, and both CHO cell lines. Flow cytometric binding analysis showed independence of VacA cell-binding from the presence of fasciclin I, whereas internalization of VacA was increased by fasciclin I-expression as detected by protease protection assay and confocal microscope. Internalized VacA was transiently delivered to fasciclin I-enriched perinuclear compartments identified as Golgi complex and/or recycling endosome (RE), followed by redistribution to the membrane of large vacuoles. Perinuclear fasciclin I dispersed from Golgi/RE and subsequently re-clustered around the vacuoles at 2-4 h after VacA intoxication. Colocalization of VacA with fasciclin I could be detected as internalization of VacA began. Cholesterol-depletion led to partial loss of fasciclin I and to significantly reduced VacA binding as well as impaired vacuolating activities. Differential detergent extraction and fractionation in sucrose density gradient showed both VacA and fasciclin I were mainly co-resided in the lipid microdomain and more fasciclin I was recruited to the raft after VacA treatment. We proposed that GPI-APs are not primary receptors of VacA and that VacA is internalized via the endocytosis pathway of GPI-APs. Lipid rafts serve as the platform for VacA binding and for further intracellular conveyance of GPI-APs and VacA. VacA may coordinate with lipid rafts to exert cellular functions for assisting H. pylori survivability.