Abstract
Extracellular transport of Vacuolating cytotoxin (VacA) across Helicobacter pylori outer membrane is hypothesized to be facilitated by the C-terminal domain of VacA precursor. The genetic properties of this domain were studied by restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of a 1.1-kb PCR-amplified vacA fragment. Sau3AI-HaeIII digestions of this fragment yielded 14 distinct combinations of RFLP types. Of the 4 popular groups (A-a, A-b, A-f, and B-a), secreted VacA levels detected by Western blotting were statistically related to the RFLP types: A-a strains produced a higher level of VacA than A-b strains and than A-f strains. Despite the genetic heterogeneity revealed by PCR-RFLP analysis, amino acid sequences of 4 strains (1 A-a, 1 A-b, 1 A-f, and 1 B-a strain) were rather conserved. Secondary structure prediction of the C-terminal region supported a b-barrel structure that might act as an export channel like Igab-core of IgA proteases. To facilitate the study of the proposed translocation function of the C-terminal domain, a recombinant MBP-VacA C-terminal domain fusion protein was expressed in E. coli. However, the expressed MBP-VacA C-terminal fusion protein was found in the inclusion body fraction and was not suitable for functional study. Western blotting using antiserum against a recombinant C-terminal fragment was performed to analyze the membrane fraction proteins of Helicobacter. pylori. Four polypeptides with distinct molecular weight (~60 kDa, ~50 kDa, ~30 kDa, and ~20 kDa) were detected. Since the C-terminal domain is proposed to be ~50 kDa, the ~50-kDa detected polypeptide might be the C-terminal domain. Based on their molecular weights and immuno-reactivity with the anti-C-terminal antiserum, one possibility for the observed immuno reactive ~30-kDa and ~20-kDa polypeptides was that these two bands were the cleaved fragments of the C-terminal domain. In an effort to test this hypothesis, the ~50-kDa and ~20-kDa polypeptides were purified by Q-Sepharose binding or isoelectric focusing, followed by N-terminal amino acid sequencing. Sequence analysis showed that these two purified peptides were not part of the C-terminal domain of VacA precursor. One possibility is that the immuno-reactivity was non-specific; another possibility is that the immuno-detected C-terminal polypeptides were small proportion of the purified peptides and cannot be accurately sequenced. Production of antisera against two other C-terminal fragments is in progress, aiming to identify the ~50 kDa, ~30 kDa, and ~20 kDa peptides and possible cleavage sites of the C-terminal domain.