A major goal of our studies is to develop therapeutic agents for the treatment of P. carinii pneumonia. One of the targets for therapy is dihydropteroate synthase (DHPS), the enzyme that is inhibited by sulfonamides and sulfones. P. carinii pneumonia can be treated with sulfonamides in combination with inhibitors of dihydrofolic reductase such as trimetheprim or trimetrexate. To investigate the possibility of finding better inhibitors of DHPS of P. carinii, we have undertaken to isolate the gene coding for P. carinii DHPS with the ultimate goal being to express it in large quantities which will allow drug screening studies with a wide variety of sulfonamides. The approaches that we have taken have been two- fold. First we have obtained a DHPS-containing plasmid that codes for the DHPS of streptococcus pneumonia. This has been used to see if it would crosshybridize with P. carinii DHPS. In preliminary screening, including screening of the chromosomes of P. carinii and southern blots of crude P. carinii that has been treated with restriction enzymes, we have not been able to find cross-reactivity. The second approach that we have been using is to attempt to identify a clone coding for P. carinii DHPS by utilizing sulfonamide resistance for selection. E. coli are sensitive to sulfanomides such as sulfa methoxazole and sulfadoxine. Our approach is to use E. coli containing plasmids from a CDNA library of P. carinii and to evaluate sulfonamide resistance among these E. coli. If one of the plasmids is producing DHPS, the E. coli would potentially be resistant to certain sulfonamides based on either overproduction of the DHPS or on different levels of inhibition of DHPS derived from either E. coli or P. carinii with individual sulfonamides. Using this approach to date we have identified a number of clones that appear to be sulfonamide resistant. These clones are currently being characterized to see if in fact they do contain DHPS activity and to ensure that they are obtained from P. carinii and not from another source.
