Trimethoprim-resistant DHFR

Figure 5.10. Protein complementation assay using murine DHFR. The F[l,2] and F[3] fragments are each fused to the homodimerizing GCN4 leucine zipper protein. A. Transformation of both Z-F[l,2] and Z-F[3] constructs results in reconstituted DHFR and growth of E. coh on agar plates containing trimethoprim. B. Transformation of Z-F[l,2] or Z-F[3] alone does not result in trimethoprim resistant E. coli cells. Figure adapted from Pelletier et al. (1998).

Plasmid- and transposon-mediated resistance to trimethoprim involves a by-pass of the sensitive step by duplication of the chromosomally-encoded dihydrofolate reductase (DHFR) target enzyme [203]. Several trimethoprim-resistant bacterial DHFRs have been identified, resistance ensuing because of altered enzyme target sites [204], Low-level resistance to tetracyclines arises in E. coli as a result of chromosomal mutations leading to loss of the outer membrane porin OmpF through which these drugs normally pass [6, 193],... 

Trimethoprim competitively inhibits dihydrofolate reductase (DHFR) and resistance can be caused by overproduction of host DHFR, mutation in the structural gene for DHFR and acquisition of the dfr gene encoding a resistant form. There are at least 15 DHFR enzyme types based on sequence homology and acquisition of dfr genes encoding alternative DHFR of type I, II or V is the most common mechanism of trimethoprim resistance among the Enterobacteriaceae. 

Overproduction of the chromosomal genes for the dihydrofolate reductase (DHFR) and the dihydroptero-ate synthase (DHPS) leads to a decreased susceptibility to trimethoprim and sulfamethoxazol, respectively. This is thought to be the effect of titrating out the antibiotics. However, clinically significant resistance is always associated with amino acid changes within the target enzymes leading to a decreased affinity of the antibiotics. 

Resistance to trimethoprim can be due to the acquisition of plasmid encoded non-allelic variants of the chromosomal DHFR enzyme that are antibiotic unsusceptible. The genes may be part of transposons that then insert into the chromosome. For instance, in gram-negative bacteria the most widespread gene is dhfrl on transposon Tn7. 

Chromosomal mutations in E. coli result in overproduction of dihydrofolate reductase (DHFR). Higher concentrations of trimethoprim, which may not be therapeutically achievable, are therefore required to inhibit nucleotide metabolism. Other mutations lower the affinity of DHFR for trimethoprim. These two mechanisms of resistance may coexist in a single strain, effectively increasing the level of resistance to the antibiotic. 

The isolation of the gene coding for H. volcanii DHFR (Rosenshine et al, 1987) deserves special mention. As mentioned above, H. volcanii is very sensitive to the antifolate drug trimethoprim. Spontaneous resistant colonies arise at frequencies of 10 10-10 9. The molecular basis for the resistance in all the resistant mutants studied so far is... 

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