Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori.
Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the beta-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.
Harris
A G
AG
School of Microbiology and Immunology, University of New South Wales, Sydney 2052, NSW.
Hazell
S L
SL
Netting
A G
AG
D000667:Ampicillin / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004076:Digoxigenin / Q000737:chemistry / Q000378:metabolism; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D000667:Ampicillin; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D004076:Digoxigenin