DESCRIPTION (provided by the applicant): Leishmaniasis is a protozoan parasitic infection affecting almost 12 million people worldwide. The pentavalent antimony containing drugs, Pentostam and Glucantime are the first line of treatment against this disease. We hypothesized that the drugs are accumulated by macrophages, which reduce the Sb(V) to Sb(lll), the active form of the drug. Sb(lll) is then taken up by the intracellular amastigote form of the parasite, where it exerts its action. A complete understanding of their action requires knowledge of the pathway for these drugs. In bacteria, yeast and mammals aquaglyceroporin channels have been shown to transport As(lll) and Sb(lll). We have recently identified the genes for an aquaglyceroporin homologue, LtAQPI and LmAQPI, in Leishmania tarentolae and Leishmania major, respectively. Drug resistance has frequently been reported in field isolates, and clinical resistance is a major impediment to the treatment of this disease. Drug resistance can arise through mutation or down regulation of drug uptake system(s). When over expressed, LmAQPI sensitizes both wild type and drug resistant Leishmania promastigotes to both Sb(lll) and As(lll) in vitro. LmAQPI also re-sensitized a field resistant isolate from an Indian patient unresponsive to pentavalent antimonials. High intracellular accumulation of both Sb(lll) and As(lll) was demonstrated to be responsible for sensitivity. Specific aims include: 1) Characterization of aquaglyceroporin and Identification of additional transporters: LmAQPI will be characterized by determining its substrate specificity in oocyte or Leishmania whole cells. The channel will also be localized and residues involved in metalloid uptake will be identified. Additional homologues of LmAQPI will be identified by RT-PCR and functional complementation. 2) Generation of aquaglyceroporin knockouts: The knockouts will be created in L. major, L infantum and L. tarentolae. 3) Differential regulation ofAQPI and drug sensitivity of amastigotes in macrophages: Differential regulation of LmAQPI will be studied in vitro. Drug sensitivity of amastigotes over expressing LmAQPI will be examined in vitro and in vivo in susceptible mice. 4) Expression of AQPI in laboratory mutants and field isolates and correlation with drug uptake and resistance. Expression of LmAQPI will be monitored by western blot analysis and correlated with resistance. Drug uptake will be determined by using ICP-MS. The proposed research will greatly improve human health and advance basic research in the field of drug action and resistance in parasitic protozoa.
