Date of Award

Spring 8-1-2004

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Life Sciences

First Advisor

H. Kathleen Dannelly

Second Advisor

David Prentice

Third Advisor

Gary Stuart

Abstract

Tetracycline has been used in combination with other antibiotics in treatment of Helicobacter pylori infection. However, tetracycline resistance has developed in clinical isolates of H. pylori, rendering treatment failure. Mutations in the 16S rRNA gene have been reported to mediate tetracycline resistance in some patient isolates. Nevertheless, the diversity of tetracycline resistance cases suggests multiple genes are involved. HPl 165, a putative tetracycline resistance gene in the genome of H. pylori 26695, displays 49.8% identity to the tetracycline efflux gene tetA (P) from Clostridium perfringens. To determine the function of the HPl 165 gene and gain a better understanding of the mechanisms of tetracycline resistance in H. pylori, the tetracycline resistance phenotype was investigated, transcription of the HPl 165 gene in H. pylori was examined by R T-PCR, a .::\HP 1165 mutant of H. pylori was generated for gene function study by insertion of the pBCa3 plasmid, and a fusion protein ofHPl 165 was constructed. The results showed that H. pylori strains harboring HPl 165 were induced to intermediate level resistance in the laboratory (Minimum Inhibitory Concentration=4~6 μg/ml). The induced resistance was not due to 16S rRNA gene mutations since no mutation was found at or near the tetracycline binding sites. The gene was transcribed both in the induced tetracycline resistant strain and tetracycline susceptible wild type, indicating translational or posttranslational control of the gene function. Mutation ofHPl 165 gene in H. pylori resulted in increased tetracycline susceptibility and loss of inducible tetracycline resistance, suggesting that the HPl 165 gene is involved in the inducible tetracycline resistance in H. pylori. Sequencing verified in-frame cloning of the HPl 165 ORF into pET28 and successful construction of the fusion protein with a N-his tag. Transcription of the gene in E. coli BL21 (D E3) was detected by R T-PCR. However, the protein was not detected by SDS-P AGE and western blot.

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