Current studies were undertaken to characterize P. carinii ornithine decarboxylase (ODC) with the ultimate goal being to investigate inhibitors of ODC as potential therapeutic agents for the treatment of P. carinii pneumonia. Uncontrolled clinical studies have shown that difluoromethyl ornithine (DFMO) may be effective in treating P. carinii pneumonia. In addition to DFMO a number of other ODC inhibitors exist but currently evaluating such drugs for their activity against P. carnii is very cumbersome. In preliminary studies utilizing intact organisms of P. carinii obtained from an animal model both DFMO and analogs have been shown to be able to inhibit P. carinii ODC. Because of the lack of availability of large quantities of enzymes we have undertaken to identify the gene coding for P. carinii ODC with ultimate goal being to characterize ODC and express it in large quantities. A number of approaches to identify the gene for cDNA library of P. carinii have been tried but have been unsuccessful. These approaches have included using a plasmid which contains the ODC of S. cerevisiae as a probe; using degenerate oligonucleotides (that are based on DNA sequences conserved by all ODC genes sequenced to date) as probes; utilizing the polymerase chain reaction (PCR) to identify a fragment of ODC from the P. carinii genome; using an ODC deficient bacteria as the host for plasmids obtained from the library to try to complement ODC activity. Currently the approach being utilized is to use polyclonal antibodies raised against conserved oligopeptides, that are based on the ODC of a number of organisms, as an immunological probe to screen the P. carinii CDNA library. Screening of clones reactive with the polyclonal antibodies is currently underway,. A number of reactive clones have been identified and are being subcloned for further characterization. The ultimate goal of these studies would be to express recombinant ODC derived from P. carinii in large quantities to be able to use it to screen currently available inhibitors of ODC.
