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Genetic characterization of trimethoprim resistance in Haemophilus influenzae
R de Groot1, M Sluijter, A de Bruyn
1Department of Pediatrics, Sophia Children's Hospital, Erasmus University Rotterdam, The Netherlands.
Abstract:
We previously demonstrated that trimethoprim (Tmp) resistance in Haemophilus influenzae is mediated by chromosomally encoded dihydrofolate reductase (DHFR) with a modified primary structure and distinct kinetic properties. To gain insight into the relationship of the DHFR structure and the level of Tmp resistance that it confers on the host bacterium, we cloned and characterized the folH genes of one Tmp-susceptible and two Tmp-resistant H. influenzae strains. Differences were observed between Tmp-susceptible and Tmp-resistant isolates both in the promoter region and in the coding sequences. The effect of differences between H. influenzae folH genes on Tmp susceptibility was investigated in Escherichia coli. Various folH gene hybrids were constructed, and their influence on Tmp susceptibility was determined. Resistance in E. coli mediated by folH from H. influenzae strain R1047 was associated with alterations in the promoter and the central part of folH. In contrast, the E. coli Tmp resistance phenotype associated with the folH gene of H. influenzae R1042 was characterized by alterations in one or more of three amino acid residues at the C-terminal part of the protein. These data indicate that Tmp resistance is not only related to alterations in the promoter region of the folH gene and the Tmp binding domains at the N-terminal and central part of DHFR. Alterations in the C-terminal part may also cause Tmp resistance, probably as a result of a change in secondary structure and the subsequent loss of Tmp binding affinity.
Insights
Trimethoprim resistance in Haemophilus influenzae is linked to changes in dihydrofolate reductase (DHFR). Alterations in the DHFR gene
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Trimethoprim (Tmp) resistance in Haemophilus influenzae is primarily mediated by chromosomally encoded dihydrofolate reductase (DHFR) with altered structure and kinetics.
- Understanding the structure-function relationship of DHFR is crucial for comprehending Tmp resistance mechanisms.
Purpose of the Study:
- To investigate the relationship between dihydrofolate reductase (DHFR) gene structure and the level of trimethoprim (Tmp) resistance in Haemophilus influenzae.
- To characterize the folH genes from Tmp-susceptible and Tmp-resistant H. influenzae strains.
Main Methods:
- Cloning and characterization of folH genes from susceptible and resistant H. influenzae strains.
- Construction and analysis of folH gene hybrids in Escherichia coli to assess Tmp susceptibility.
- Investigating the impact of promoter and coding sequence variations on Tmp resistance.
Main Results:
- Differences in promoter regions and coding sequences of folH genes were observed between susceptible and resistant H. influenzae isolates.
- In E. coli, resistance mediated by H. influenzae folH (R1047) involved promoter and central folH alterations.
- Resistance mediated by H. influenzae folH (R1042) in E. coli was linked to alterations in C-terminal amino acid residues.
Conclusions:
- Trimethoprim resistance is associated with modifications beyond the promoter and N-terminal/central Tmp binding domains of DHFR.
- Alterations in the C-terminal region of DHFR can contribute to Tmp resistance, likely by affecting secondary structure and Tmp binding affinity.
- These findings expand the understanding of molecular mechanisms underlying trimethoprim resistance in H. influenzae.