HELICOBACTER-PYLORI HSPA-HSPB HEAT-SHOCK GENE-CLUSTER - NUCLEOTIDE-SEQUENCE, EXPRESSION, PUTATIVE FUNCTION AND IMMUNOGENICITY

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant pr otein that was reported to be associated with the nickel-dependent ure ase of H, pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class, The gene encoding the Gr oEL-like protein of H. pylori (HspB) was cloned (plLL689) and was show n to belong to a bicistronic operon including the hspA and hspB genes, In Escherichia coil, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion e lement that mapped upstream to the two open reading frames (ORFs), IS5 was absent from the H. pylori genome, and was thus acquired during th e cosmid cloning process, hspA and hspB encoded polypeptides of 118 an d 545 amino acid residues, corresponding to calculated molecular masse s of 13.0 and 58.2 kDa, respectively, Amino acid sequence comparison s tudies revealed that, although H. pylori HspA and HspB proteins were h ighly similar to their bacterial homologues, the H. pylori HspA featur ed a striking motif at the C-terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel-binding domain, which is not present in any of the other bacterial GroES homo logues so far characterized, When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) i nto an E. coil host strain, an increase of urease activity was observe d, This suggested a close interaction between the HspA and HspB protei ns and the urease enzyme, and a possible role for HspA in the chelatio n of nickel ions, The genes encoding each of the HspA and HspB polypep tides were cloned, expressed independently as proteins fused to the ma ltase-binding protein (MBP) and purified in large scale, The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic prop erties. Both HspA and HspB represent antigens that are specifically re cognized by the sera from H. pylori-infected patients, Whereas HspB wa s known to be immunogenic in humans, this is the first demonstration t hat HspA per se is also immunogenic.