Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Abstract

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal ⌬hgprt/⌬xprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084 -16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the ⌬hgprt/⌬xprt knockout cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of ⌬hgprt/⌬xprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which ⌬hgprt/⌬xprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured ⌬hgprt/⌬xprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of ⌬hgprt/⌬xprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which ⌬hgprt/⌬xprt parasites displayed persistence and increased virulence in mice.Leishmania donovani is a protozoan parasite that is the causative agent of visceral leishmaniasis, a debilitating and often fatal disease in humans. Leishmania spp. are digenetic protozoan parasites that exist as flagellated, motile promastigotes within the alimentary tract and salivary glands of their insect vector, members of the Phlebotomine sandfly family and as nonflagellated, amotile amastigotes within macrophages and other reticuloendothelial cells of the mammalian host. No effective vaccines are available for visceral leishmaniasis-or for that matter any disease caused by protozoan parasites, and therefore chemotherapy offers the only means of defense for the treatment and prevention of leishmaniasis and other diseases of parasitic origin. Unfortunately, the current armamentarium of drugs employed against visceral and other forms of leishmaniasis is far from ideal and is adversely affected by toxicity, protracted and invasive routes of administration, and therapeutic unresponsiveness. As a result, there is an acute need for better and more efficacious drugs to combat the disease.The establishment of an efficacious, parasite-specific ...

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

“…The relative percentages of each product are quantified in Table 1. Mouse Infections-Because both ⌬adss and ⌬asl parasites exhibited markedly reduced parasite loads in macrophages, the ability of the null mutants and their respective complements to infect BALB/c mice, a well characterized rodent model for L. donovani infections (48,49,(62)(63)(64), was evaluated. Unexpectedly, despite the reduced parasite numbers observed in peritoneal macrophages infected with ⌬adss parasites (Fig.…”

“…Groups of five 7-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA) were inoculated by tail vein injection with 5 ϫ 10 6 late log or stationary phase wild type, ⌬adss, ⌬adss[pADSS], ⌬aah/ ⌬adss, ⌬asl, or ⌬asl[pASL] promastigotes (48,49). Four weeks post-infection, mice were sacrificed and their livers and spleens harvested as reported (48,49).…”

“…Macrophage Infections-Macrophage infectivity experiments with wild type, ⌬adss, ⌬adss[pADSS], ⌬asl, and ⌬asl[pASL] promastigotes were performed using either peritoneal macrophages harvested from BALB/c mice or cultured J774 murine macrophages (ATCC, Manassas, VA) (DMEM supplemented with 4 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, and 10% FBS but without added purine) and incubated at 37°C in a humidified 5% CO 2 incubator as described (16,21,48,49). 72 h post-infection, macrophages were washed, stained using the Diff-Quik kit (International Medical Equipment Inc., San Marcos, CA), and amastigotes were enumerated (16,21,49).…”

“…Groups of five 7-week-old female BALB/c mice (Charles River Laboratories, Wilmington, MA) were inoculated by tail vein injection with 5 ϫ 10 6 late log or stationary phase wild type, ⌬adss, ⌬adss[pADSS], ⌬aah/ ⌬adss, ⌬asl, or ⌬asl[pASL] promastigotes (48,49). Four weeks post-infection, mice were sacrificed and their livers and spleens harvested as reported (48,49). Single-cell suspensions from mouse organs were prepared by passage through a 70-m cell strainer (BD Falcon), and parasite burdens were then determined in 96-well microtiter plates using a standardized limiting dilution assay (50).…”

“…The immunofluorescence assay was performed on L. donovani promastigotes as described (11,12,27,48) using a 1:1000 dilution of recombinant anti-ASL antibody and a 1:10,000 dilution of secondary goat anti-rabbit Oregon Green-conjugated antibody (Invitrogen).…”