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

All Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel-dependent urease of H, pylori. This protein was recently recognized as a homologue of the heat-shock protein of the GroEL class, The gene encoding the GroEL-like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes, In Escherichia coli, the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs), IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process, hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively, Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured 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 homologues so far characterized, When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed, This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in the chelation of nickel ions, The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltase-binding protein (MBP) and purified in large scale, The MBP-HspA and MBP-HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori-infected patients, Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.