Journal: Antimicrobial Agents and Chemotherapy
Article Title: Giardia, Entamoeba, and Trichomonas Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases) ▿ Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases) ▿ †
Figure Lengend Snippet: A nonsense mutation (in-frame stop codon) was present in the Eh ntr1 gene of genome project strain HM-1:IMSS of Entamoeba and three clinical isolates. (A) The nitroreductases of Entamoeba strains HM-1:IMSS and 200:NIH, as well as those Methanothermobacter thermautotrophicus (Mt) and Archaeoglobus fulgidus (Af), were aligned by using the single-letter code. In this alignment, identical amino acids are marked with an asterisk, while similar amino acids are marked with a colon or a period. Within a putative zero-frame 81-bp intron of EhNTR1 of genome project strain HM-1:IMSS, there was a stop codon (#) where there was an Arg (R) in the wild-type EhNTR1 of 200:NIH. The stop codon, which was also present in the Eh ntr1 genes of 3 of 22 clinical isolates examined (isolates BM1, CM2, and H22), was present in a region that is conserved in bacterial nitroreductases. (B) PCR and RT-PCR with Eh ntr1 gene primers from HM-1:IMSS DNA and RNA, respectively, produced products of the same size, arguing against the presence of an in-frame intron in the Eh ntr1 gene. In contrast, the product obtained by RT-PCR with Eh ntr3 gene primers from HM-1:IMSS RNA was smaller than the product obtained by PCR from DNA, consistent with the presence of an intron in the Eh ntr3 gene of Entamoeba . The sequence of the Eh ntr3 RT-PCR product confirmed the presence of the intron at the position predicted by the genome project (data not shown). (C) Western blotting with a rabbit polyclonal antibody to a recombinant EhNTR1 protein showed that strains 200:NIH and Rahman, which had the wild-type Eh ntr1 gene, both expressed the NTR1 protein. In contrast, strain HM-1:IMSS, which had a nonsense mutation in the Eh ntr1 gene, did not express the NTR1 protein.
Article Snippet: Total RNA was isolated from mid-log-phase Entamoeba and Trichomonas with the Trizol reagent (Invitrogen), and the RNA was treated with a DNA-free reagent (Ambion), according to the manufacturer's instructions.
Techniques: Mutagenesis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Produced, Sequencing, Western Blot, Recombinant