Dihydropteroate synthase of Mycobacterium leprae and dapsone resistance

Two Mycobacterium leprae genes, folP1 and folP2, encoding putative dihydrop teroate synthases (DHPS), were studied for enzymatic activity and for the p resence of mutations associated with dapsone resistance. Each gene was clon ed and expressed in a folP knockout mutant of Escherichia coli (C600 Delta folP::Km(r)). Expression of di. leprae folP1 in C600 Delta folP::Km(r) conf erred growth on a folate-deficient medium, and bacterial lysates exhibited DHPS activity. This recombinant displayed a 256-fold-greater sensitivity to dapsone (measured by the MIG) than wild-type E. coli C600, and 50-fold les s dapsone was required to block (expressed as the 50% inhibitory concentrat ion [IC50]) the DHPS activity of this recombinant. When the folP1 genes of several dapsone-resistant M. leprae clinical isolates were sequenced, two m issense mutations were identified. One mutation occurred at codon 53, subst ituting an isoleucine for a threonine residue (T53I) in the DHPS-1, and a s econd mutation occurred in codon 55, substituting an arginine for a proline residue (P55R), Transformation of the C600 Delta folP::Km(r) knockout with plasmids carrying either the T53I or the P55R mutant allele did not substa ntially alter the DHPS activity compared to levels produced by recombinants containing wild-type M. leprae folP1, However, both mutations increased da psone resistance, with P55R having the greatest affect on dapsone resistanc e by increasing the MIC 64-fold and the IC50 68-fold. These results prove t hat the folP1 of M. leprae encodes a functional DHPS and that mutations wit hin this gene are associated with the development of dapsone resistance in clinical isolates of il M. leprae. Transformants created with M. leprae fol P2 did not confer growth on the C600 Delta folP::Km(r) knockout strain, and DNA sequences of folP2 from dapsone-susceptible and -resistant M leprae st rains were identical, indicating that this gene does not encode a functiona l DHPS and is not involved in dapsone resistance in M. leprae.