Extracellular release of antigenic proteins by Helicobacter pylori

Abstract

Screening a Helicobacter pylori genomic library with antisera raised against H. pylori broth culture supernatant resulted in the identification of six antigens: urease, HspB, Lpp20, DnaK, MsrA, and a cysteine-rich 28-kDa protein (designated HcpA). H. pylori antigens may be released into the extracellular space by multiple mechanisms, including specific secretion pathways, autolysis, and membrane vesicle formation.

FIG. 1

Analysis of antigenic proteins in broth culture supernatant from H. pylori 60190. Proteins in broth culture supernatant from H. pylori 60190 were precipitated with a 50%-saturated solution of ammonium sulfate, resuspended, passed through a 0.2-μm-cutoff filter, and then fractionated by passage over a Superose 6 HR 16/50 gel filtration column. (A) Tracing of absorbance at 280 nm. Fraction numbers are indicated underneath; each fraction represents 2 ml. (B) Immunoblot analysis of proteins in selected fractions. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% acrylamide gel and transfer to nitrocellulose paper, the proteins were immunoblotted with rabbit antiserum to supernatant proteins (1:300 dilution of antiserum). Fraction 19 corresponds to the void volume of the column.

FIG. 2

Immunoblot analysis of E. coli DH5αMCR expressing recombinant H. pylori antigens. E. coli containing the designated plasmids were grown in Luria-Bertani broth containing ampicillin (50 μg/ml), and the bacterial pellets were analyzed by immunoblotting with rabbit antiserum to supernatant proteins (1:1,000 dilution). Plasmid designations and the names of the expressed H. pylori antigens are as follows: pBluescript (a), pH102 (UreA and UreB) (b), pH109 (HspB) (c), pH113 (DnaK) (d), pH105 (MsrA) (e), pH103 (HcpA) (f), and pH120 (Lpp20) (g).

FIG. 3

Alignment of the deduced HcpA amino acid sequence from H. pylori 60190 with deduced amino acid sequences of seven members of a paralogous gene family from H. pylori 26695 (23). Amino acid residues that are conserved in all eight sequences are indicated by asterisks, and conserved cysteine residues are indicated by plus signs.