Contribution of specific amino acid changes in penicillin binding protein 1 to amoxicillin resistance in clinical Helicobacter pylori isolates.
Amoxicillin is commonly used to treat Helicobacter pylori, a major cause of peptic ulcers, stomach cancer, and B-cell mucosa-associated lymphoid tissue lymphoma. Amoxicillin resistance in H. pylori is increasing steadily, especially in developing countries, leading to treatment failures. In this study, we characterize the mechanism of amoxicillin resistance in the U.S. clinical isolate B258. Transformation of amoxicillin-susceptible strain 26695 with the penicillin binding protein 1 gene (pbp1) from B258 increased the amoxicillin resistance of 26695 to equal that of B258, while studies using biotinylated amoxicillin showed a decrease in the binding of amoxicillin to the PBP1 of B258. Transformation with 4 pbp1 fragments, each encompassing several amino acid substitutions, combined with site-directed mutagenesis studies, identified 3 amino acid substitutions in PBP1 of B258 which affected amoxicillin susceptibility (Val 469 Met, Phe 473 Leu, and Ser 543 Arg). Homology modeling showed the spatial orientation of these specific amino acid changes in PBP1 from 26695 and B258. The results of these studies demonstrate that amoxicillin resistance in the clinical U.S. isolate B258 is due solely to an altered PBP1 protein with a lower binding affinity for amoxicillin. Homology modeling analyses using previously identified amino acid substitutions of amoxicillin-resistant PBP1s demonstrate the importance of specific amino acid substitutions in PBP1 that affect the binding of amoxicillin in the putative binding cleft, defining those substitutions deemed most important in amoxicillin resistance.
Division of Biomedical Sciences, University of California, Riverside, Riverside, CA 92521-0121, USA.
Nadia N
NN
Qureshi
Dimitrios
D
Morikis
Neal L
NL
Schiller
D000595:Amino Acid Sequence; D000658:Amoxicillin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D046915:Penicillin-Binding Proteins; D000658:Amoxicillin