: The first aim of the proposal is to clone, express, purify, and characterize the DHPS from human derived Pc, similar to what was done for DHPS from rat derived Pc in earlier studies. The second aim will be to determine whether, and to what extent, sulfa resistance is appearing in human strains of Pc. This aim is based upon the fact that DHPS amino acid sequence changes have been found in several (five) isolates of human-derived Pc that are consistent with the evolution of sulfa resistance. The first component of this aim will be to sequence Pc DHPS genes from several more patients and to determine whether specific mutations are associated with poor drug responses of certain strains. Strain identification will be done by PCR of the ITS regions of mitochondrial ribosomal RNA by Dr. Lee at Indiana University School of Medicine. The second component of this aim will be to perform site-directed mutagenesis to determine whether the observed mutations in DHPS affect the enzymes sensitivity to SMX and dapsone, two of the most commonly used sulfa drugs. Finally, the third aim of this proposal is to evaluate and compare the efficacies of a series of sulfa drugs in vitro (using recombinant DHPS from human-derived Pc and possibly variants--drug resistant isolates) once the recombinant tools are in place. Sulfa drugs that are effective in vitro will then be tested in vivo in a mouse model.
NIH ExPorter
NIH ExPorter