Studies of the Genetic Heterogeneity of Helicobacter pylori Vacuolating Toxin and Relationship with Its Biological Functions

• Main Authors: Hung-Jung Wang, 王鴻俊
• Other Authors: Wen-Ching Wang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/44243597511089421431

博士 === 國立清華大學 === 生命科學系 === 89 === The vacuolating cytotoxin, VacA, secreted by Helicobacter pylori (H. pylori) is a major virulence factor in the pathogenesis of gastroduodenal diseases. Analysis of vacA alleles shows two divergent regions: signal sequence (s1a, s1b, s2) and middle region (m1 and m2). The s1a and m1 type isolates are indicated to associate with higher virulence in Western countries. In order to investigate the relationship between vacA gene diversity and pathogenesis in Taiwan, we first investigated vacA heterogeneity among 119 clinical isolates obtained from National Taiwan University Hospital. Of 119 isolates, 104 strains had the genotype of s1a/m2 (87%), 13 strains (11%) were characterized as the s1a/m1T, that was more homologous to the s1a/m1, and 2 (2%) were the s1a/m1Tm2 chimeric type. Production of high-grade VacA among 11 strains with s1a/m1T (72.7%) was higher than those of 66 strains with s1a/m2 (21.2%). The second part was aimed to investigate the relationship between genetic diversity of vacA C-terminal domain and the level of VacA secretion by utilizing these isolates. Sau3AI-HeaIII RFLP patterns of the 1.1-kb PCR-amplified vacA C-terminal fragment revealed four popular but distinct RFLP patterns (denoted as A-a, A-b, A-f, and B-a) among 87 isolates. The A-a type strains produced a higher level of VacA than A-b and A-f strains. Despite this, there was high conservation (96.7%) among these alleles. Sequence analysis and secondary structure prediction suggested a β-barrel structure that may act as a selective export channel like Igaβ-core of IgA proteases. To facilitate the study of the binding mechanism of VacA acting on cell surface, we have established an expression system to produce a soluble form of VacA fused with GST in Escherichia coli. Although the GST-VacA lacked vacuolating activity, it had a binding affinity similar to that of native VacA. Moreover, GST-VacA can block the vacuolating activity induced by the native VacA. To investigate the role of VacA C-terminal region in its cell binding activity, we constructed different GST-VacAs with truncated C-terminal and analyzed their cell binding activity. Our results suggested that the m1-type VacA P58 domain contributed to a full cell-binding activity in HeLa cells, particularly about 100 residues in the C-terminal region. To investigate the role of the m1/m2 midle region sequences in cell binding activity, we constructed vacA alleles including 60190 (m1), v226 (m2), and v225 (m1m2) and assayed their cell binding activity using the same expression system. Kinetic analysis indicated that binding equilibrium could be reached after 10 hour incubation. Further kinetic, Scatchard, and inhibition analyses revealed the existence of at least two populations of distinct affinity receptors (m1R and m2R) in RK13 cells: one m1-specific receptor interacts with m1 VacA with a Kd ≈ 5 nM and the other common receptor interacts similarly with m1, m2, or m1/m2 with a lower affinity (Kd ≈ 46-60 nM)