[Detection of point mutation in an in vitro-selected amoxicillin-resistant strain of Helicobacter pylori].
OBJECTIVE
To investigate the relationship between point mutation of penicillin-binding protein gene (pbp) and amoxicillin resistance (AMOgamma) of Helicobacter pylori (H. pylori) as well as to compare the protein profiles under proteomic technology to get the candidate resistance-related proteins.


METHODS
(1) AMOgamma strains were selected from the sensitive H. pylori strain 26695 by serial passage technique in vitro. (2) Point mutations of five putative resistance genes (HP0597, HP1565, HP1542, HP1556, and HP0160) were analyzed by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing. (3) Proteins samples were separated by two-dimensional electrophoresis (2-DE). Protein profiles were compared between the AMOgamma strain obtained in vitro and its sensitive parent strain 26695. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed to identify the proteins of interest. The proteins were searched by software MASCOT and identified by peptide fingerprint map using the program MS-FIT of Protein Prospect.


RESULTS
(1) An AMOgamma strain (MIC 8 microg/ml) was obtained. Complete loss of the resistant phenotype was observed after cultivation in the absence of AMO or storage at - 80 degrees C. (2) DHPLC and Sequencing result showed no point mutations in five pbp genes in the AMOgamma strain when compared with the corresponding PCR products from its parent strain 26695. (3) Protein profiling showed that eleven protein spots were differently expressed between 26695 and the AMOgamma strain. Of these protein spots in the AMOgamma strain, two new spots (Spot 1 and Spot 2) were observed with one (Spot 3) was up-regulated three-fold and the remained ones (Spot 4-11) were down-regulated.


CONCLUSIONS
AMO resistance of H. pylori might be resulted from, unstable phenotype change rather than point mutations of pbp genes. These differentially regulated proteins in AMOgamma strain might play a role in development of resistance to AMO in H. pylori.
Shen
Jing
J
Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing 100036, China.
Deng
Da-Jun
DJ
Ke
Yang
Y
Zhang
Jian-Zhong
JZ
D000658:Amoxicillin / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D000658:Amoxicillin