Active transport in L. donovani is dependent on a proton gradient across the plasma membrane that is established through the action of an ATPase that extrudes protons. This proton must also play a vital role in the survival of the parasite within the acidic environment of the vertebrate host's phagolysosomes. Our long term goal is to understand the structure, function and regulation of this enzyme and, through this understanding to provide avenues to the control of this important pathogen.
The aim of this proposal is to apply molecular cloning technology to produce molecular probes with which to analyze the macromolecules that mediate the proton pumping system. We have succeeded in the first phase of this endeavor and have cloned parts of two tandemly arrayed genes that are structurally capable of encoding plasma membrane cation pumps. We will clone this tandem pair of L. donovani genes in their entirety. We will determine the DNA sequences of the genes, and predict the amino acid sequences of their protein products. This sequence information will be used produce nucleic acid and antibody probes for the analysis of expression in the two life cycle stages of the parasite. We will compare the abundance of these gene products to proton pumping activity, and examine the enzymatic activities of the proteins encoded by the presumptive proton pump genes.
