Clinical relevance and mechanism of natural competence in helicobacter pylori

• Main Author: 葉郁青
• Other Authors: 王錦堂
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/43978512238595593898

博士 === 國立臺灣大學 === 微生物學研究所 === 91 === Abstract To study whether the capability of horizontal DNA transfer is associated with metronidazole resistance in Helicobacter pylori, a total of 81 clinical isolates were tested for MICs of metronidazole (MTZ) and the capability of natural transformation. The MIC assays were performed by using E-testing and reconfirmed by the agar dilution method. Natural competence assays were performed by transferring a chloramphenicol acetyltransferase (CAT) cassette and a 23S rRNA gene from a clarithromycin-resistant strain (with A-to-G mutation at nucleotide 2143) using natural transformation. Of the 81 isolates, 65 (80.3 %) were naturally competent while 16 were not. Among the 65 naturally competent strains, 39 (60.0%) were highly resistant to MTZ (MICs >32 　µg/mL) while only 2 of 16 (12.5%) noncompetent strains were highly MTZ-resistant (p <0.1). Therefore, there is an association between natural competence and MTZ resistance. Next, we used bioinformatics database search to find genes involved in natural competence, and two transformation-related open reading frames were found: a comE3 homologue (HP1361) of Bacillus subtilis and a comL homologue (HP1378) of Neisseria gonorrhoea. However, we were unable to obtain HP1378 knock-out mutant. HP1361 knock-out mutant was obtained by transposon shuttle mutagenesis. For DNA transformation experiments, 3 donor DNAs were used: theCAT-cassette-inserted HP0691, the clarithromycin resistant 23S rRNA gene and an Escherichia coli-H. pylori shuttle vector pHel2. DNA transformation ability was severely impaired (frequency <1.0x10-9) in HP1361 knock-out mutant, both by natural transformation and electroporation. Complementation with a plasmid or chromosomal integration restored partial capabilities of natural competence (to frequencies 2.8x10-7-8.2x10-6) and electroporation (to frequencies 3.0x10-7-1.0x10-5). Isotope-labeled DNA assays revealed that DNA binding and uptake capabilities were impaired in HP1361 knock-out mutant and restored after complementation. The results suggest that HP1361, a comE3 homologue, is required for DNA binding and uptake during DNA transformation of H. pylori.