MOLECULAR CHARACTERIZATION OF A CONSERVED 20-KILODALTON MEMBRANE-ASSOCIATED LIPOPROTEIN ANTIGEN OF HELICOBACTER-PYLORI

Antisera raised in rabbits to whole cells of Helicobacter pylori recog nized as a major antigen a protein with an apparent molecular weight o f 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate foll owed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this pr otein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the nati ve H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragme nt as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,09 4 containing a 21-residue typical lipoprotein signal peptide and conse nsus prolipoprotein processing site. The mass of the deduced 154-resid ue mature protein was 16,865 Da. Growth of E. coli cells expressing th e cloned H. pylori lpp20 gene in the presence of [H-3] palmitic acid r esulted in radiolabelled Lpp20 while treatment of the E. coli cells wi th globomycin caused accumulation of unprocessed Lpp20, consistent wit h Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also foun d in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonesse ntial class of lipoproteins.