Analysis of a [beta]-helical region in the p55 domain of Helicobacter pylori vacuolating toxin

• Published in: BMC microbiology Vol. 10; pp. 60 - 119
• Main Authors: Ivie, Susan E, McClain, Mark S, Algood, Holly M Scott, Lacy, D Borden, Cover, Timothy L
• Format: Journal Article
• Language: English
• Published: BioMed Central Ltd 23.02.2010
• Subjects: Analysis; Bacterial toxins; Carrier proteins; Genetic aspects; Health aspects; Helicobacter pylori; Physiological aspects; Structure; United States
• DOI: 10.1186/1471-2180-10-60
• ISSN: 1471-2180

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. VacA, a toxin secreted by H. pylori, is comprised of two domains, designated p33 and p55. Analysis of the crystal structure of the p55 domain indicated that its structure is predominantly a right-handed parallel [beta]-helix, which is a characteristic of autotransporter passenger domains. Substitution mutations of specific amino acids within the p33 domain abrogate VacA activity, but thus far, it has been difficult to identify small inactivating mutations within the p55 domain. Therefore, we hypothesized that large portions of the p55 domain might be non-essential for vacuolating toxin activity. To test this hypothesis, we introduced eight deletion mutations (each corresponding to a single coil within a [beta]-helical segment spanning VacA amino acids 433-628) into the H. pylori chromosomal vacA gene. All eight of the mutant VacA proteins were expressed by the corresponding H. pylori mutant strains and underwent proteolytic processing to yield ~85 kDa passenger domains. Three mutant proteins (VacA [DELTA]484-504, [DELTA]511-536, and [DELTA]517-544) were secreted and induced vacuolation of mammalian cells, which indicated that these [beta]-helical coils were dispensable for vacuolating toxin activity. One mutant protein (VacA [DELTA]433-461) exhibited reduced vacuolating toxin activity compared to wild-type VacA. Other mutant proteins, including those containing deletions near the carboxy-terminal end of the [beta]-helical region (amino acids Val.sup.559.sup.-Asn.sup.628.sup.), exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded. These results indicate that within the [beta]-helical segment of the VacA p55 domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. We propose that non-essential [beta]-helical coils and a carboxy-terminal [beta]-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains.