Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Adenine and adenosine salvage in Leishmania donovani

“…As expected, wild type, Δ aprt , and Δ aah promastigotes displayed robust growth in adenine, while Δ aah /Δ aprt promastigotes failed to proliferate (Table I). As shown previously, neither Δ adss nor Δ hgprt /Δ xprt promastigotes could grow in adenine [9,17], although when a Δ aah mutation was inserted into each of these genetic backgrounds, the cells grew robustly [10,17]. Furthermore, pharmacologic simulation of the AAH deficiency with dCF enabled Δ adss [17] and Δ hgprt /Δ xprt (Table 1) promastigotes to proliferate with adenine as the sole purine nutrient but prevented growth of Δ aprt promastigotes by blocking adenine conversion to hypoxanthine via AAH.…”

“…Included in this nutritional analysis were previously isolated gene deletion mutants with known 6-aminopurine growth deficits. These mutants include L. donovani harboring genetic lesions in adenylosuccinate synthetase (ADSS), AAH and ADSS (Δ aah /Δ adss ), HGPRT and XPRT (Δ hgprt /Δ xprt ), and the triple null mutant Δ aah /Δ hgprt /Δ xprt (Table I) [9,10,17]. Nutritional assessments were performed in purine-deficient Dulbecco’s modified Eagle medium- Leishmania (DME-L) growth medium supplemented with 5% dialyzed fetal bovine serum to which either 100 μM adenine or adenosine was added.…”

GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites

“…GMPR was localized in L. donovani promastigotes by indirect immunofluorescence as described [21,22,25,26] using a 1:500 dilution of anti-LmGMPR antibody and a 1:10,000 dilution of secondary goat anti-rabbit Oregon Green-conjugated antibody (Thermo Fisher Scientific). Parasites were costained with guinea pig antibodies to the L. donovani IMPDH protein (1:200) and goat anti-guinea pig Rhodamine Red-conjugated (1:10,000) secondary antibody (Thermo Fisher Scientific) to stain glycosomes.…”

“…1) [5,7,17–21]. The importance of the nucleotide interconversion pathways in growth and infectivity was validated by the isolation and characterization of Δ adss and Δ asl and Δ impdh null mutants [22,23]. The Δ adss and Δ asl parasites exhibited growth phenotypes essentially indistinguishable from that of the previously characterized Δ hgprt /Δ xprt double knockout, whereas the Δ impdh cells could utilize guanine, guanosine, xanthine or xanthosine, all guanylate precursors, to fulfill their purine requirements [14,22,23].…”

“…The importance of the nucleotide interconversion pathways in growth and infectivity was validated by the isolation and characterization of Δ adss and Δ asl and Δ impdh null mutants [22,23]. The Δ adss and Δ asl parasites exhibited growth phenotypes essentially indistinguishable from that of the previously characterized Δ hgprt /Δ xprt double knockout, whereas the Δ impdh cells could utilize guanine, guanosine, xanthine or xanthosine, all guanylate precursors, to fulfill their purine requirements [14,22,23]. All three null mutants exhibited reduced infectivity phenotypes in macrophages; however, only Δ asl parasites were severely incapacitated in their ability to establish a visceral infection in the mouse model, whereas parasite burdens in mice inoculated with Δ adss or Δ impdh cells were robust [22,23].…”