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

Jan M Boitz,Buddy Ullman

doi:10.1074/jbc.m110.125393

Abstract

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Deltahgprt/Deltaxprt 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 Deltahgprt/Deltaxprt knock-out 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 Deltahgprt/Deltaxprt 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 Deltahgprt/Deltaxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Deltahgprt/Deltaxprt 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 Deltahgprt/Deltaxprt 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 Deltahgprt/Deltaxprt parasites displayed persistence and increased virulence in mice.

Highlights

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
Virulence Defect of ⌬hgprt/⌬xprt Parasites in Mice—Because ⌬hgprt/⌬xprt L. donovani are effectively noninfectious in murine peritoneal macrophages [10], the ability of the double knock-out to infect Balb/c mice, a well characterized rodent model for leishmaniasis [13,14,15,16], was evaluated
The parasite burdens in the livers and spleens of mice infected with wild type L. donovani were ϳ10,000- and ϳ100-fold higher, respectively, than those from mice infected with the ⌬hgprt/⌬xprt knock-out (Fig. 1A)

Summary

Introduction

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. Tent ⌬hgprt/⌬xprt parasites in the second round of infection and ⌬hgprt/⌬xprt[Ino/Hyp] cells lines were capable of suswere only 750-fold less than those of wild type parasites in liver, tained proliferation in inosine and could grow in adenine or whereas splenic parasitemias between wild type and knock-out adenosine without dCF supplementation (Fig. 2).

Results

Conclusion