: Toxoplasma gondii is an opportunistic pathogen that causes disastrous consequences for the vulnerable and immunocompromised, like AIDS patients. Its activity is controlled through extensive treatment regimens, which often yield allergic side effects for the patient. In an effort to search for novel alternative chemotherapeutic agents, this research will examine herbicide action and resistance in T. gondii. Dinitroaniline herbicides have been shown to kill Toxoplasma as well as other related parasites, without harming mammalian cells. These agents are thought to affect tubulin proteins and thus, hamper microtubule polymerization. Studies on herbicide action in plants and green algae identified specific point mutations in tubulin genes that correlated with dinitroaniline resistance. To assess the nature of herbicide resistance in T. gondii, dinitroaniline-resistant mutants will be generated through chemical mutagenesis, and the single copy a- and B- tubulin genes from mutant parasites will be amplified by PCR (Polymerase Chain Reaction). The tubulin DNA sequences from these mutant parasites will be compared to the wild-type and the effect of the point mutations on tubulin protein structure will be predicted using crystallographic data from other species. To confirm that the mutation(s) is responsible for drug resistance, the mutant tubulin gene will be cloned into a T. gondii expression vector and transformed into wild-type parasites. Those parasite clones that received the mutant allele should exhibit drug resistance upon repeated selection. The existence of the mutant allele in these resistant parasites will be verified using southern hybridization. This protocol will be repeated using cDNA (complementary DNA) constructs prepared by RT-PCR (reverse transcription PCR). The expression of mutant tubulin protein will also be confirmed via western blot analysis and immunofluorescence microscopy.
