Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases
Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases
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DOI：
10.1074/jbc.m110.125393
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发表时间：
2010-06-11
影响因子：
4.8
通讯作者：
Ullman, Buddy
中科院分区：
文献类型：
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作者：
Boitz, Jan M.;Ullman, Buddy
多诺瓦利什曼原虫不能从头合成嘌呤，并强制清除宿主的嘌呤。在此之前，我们描述了L. donovani的条件致死性Delta hgprt/Delta xprt突变体(Boitz， J. M., and Ullman， B. (2006) J. Biol。该研究建立了L. donovani主要通过次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT）和黄嘌呤磷酸核糖转移酶（XPRT）来回收嘌呤。与野生型L. donovani不同，Delta hgprt/Delta xprt敲除不能在6-氧嘌呤上生长，并且绝对需要腺嘌呤或腺苷和2'-脱氧仿甲霉素（一种寄生虫腺嘌呤氨基水解酶活性抑制剂）。在这里，我们证明了德尔塔hgprt/德尔塔xprt寄生虫感染小鼠的能力被严重损害。令人惊讶的是，在小鼠体内存活下来的突变寄生虫部分恢复了它们的毒力特性，在随后的小鼠感染中表现出10倍的寄生虫负荷增加。为了剖析Delta hgprt/Delta xprt寄生虫在体内持续生长的机制，从培养的Delta hgprt/Delta xprt寄生虫中克隆出在限制条件下恢复生长能力的抑制菌株。这些抑制克隆在6-氧嘌呤中生长和代谢的能力可能归因于腺嘌呤磷酸核糖基转移酶（APRT）基因的显著扩增和过表达。此外，用APRT片段转染Delta hgprt/Delta xprt细胞在体外重现了抑制表型，并使其能够在6-氧嘌呤上生长。生化研究进一步表明，出乎意料的是，次黄嘌呤是APRT的低效底物，这可以解释抑制因子代谢次黄嘌呤的能力。随后的分析表明，APRT扩增也是Delta hgprt/Delta xprt寄生虫在小鼠中表现持久性和增强毒力的潜在机制。
Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Delta hgprt/Delta 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 Delta hgprt/Delta 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 Delta hgprt/Delta 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 Delta hgprt/Delta xprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Delta hgprt/Delta 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 Delta hgprt/Delta 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 Delta hgprt/Delta xprt parasites displayed persistence and increased virulence in mice.