Porollo Aleksey, Sesterhenn Thomas M, Collins Margaret S, Welge Jeffrey A, Cushion Melanie T
Center for Autoimmune Genomics and Etiology, Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA
Department of Psychiatry and Behavioral Neurosciences, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
mBio. 2014 Nov 4;5(6):e01834. doi: 10.1128/mBio.01834-14.
In the context of deciphering the metabolic strategies of the obligate pathogenic fungi in the genus Pneumocystis, the genomes of three species (P. carinii, P. murina, and P. jirovecii) were compared among themselves and with the free-living, phylogenetically related fission yeast (Schizosaccharomyces pombe). The underrepresentation of amino acid metabolism pathways compared to those in S. pombe, as well as the incomplete steroid biosynthesis pathway, were confirmed for P. carinii and P. jirovecii and extended to P. murina. All three Pneumocystis species showed overrepresentation of the inositol phosphate metabolism pathway compared to that in the fission yeast. In addition to those known in S. pombe, four genes, encoding inositol-polyphosphate multikinase (EC 2.7.1.151), inositol-pentakisphosphate 2-kinase (EC 2.7.1.158), phosphoinositide 5-phosphatase (EC 3.1.3.36), and inositol-1,4-bisphosphate 1-phosphatase (EC 3.1.3.57), were identified in the two rodent Pneumocystis genomes, P. carinii and P. murina. The P. jirovecii genome appeared to contain three of these genes but lacked phosphoinositide 5-phosphatase. Notably, two genes encoding enzymes essential for myo-inositol synthesis, inositol-1-phosphate synthase (INO1) and inositol monophosphatase (INM1), were absent from all three genomes, suggesting that Pneumocystis species are inositol auxotrophs. In keeping with the need to acquire exogenous inositol, two genes with products homologous to fungal inositol transporters, ITR1 and ITR2, were identified in P. carinii and P. murina, while P. jirovecii contained only the ITR1 homolog. The ITR and inositol metabolism genes in P. murina and P. carinii were expressed during fulminant infection as determined by reverse transcriptase real-time PCR of cDNA from infected lung tissue. Supplementation of in vitro culture with inositol yielded significant improvement of the viability of P. carinii for days 7 through 14.
Microbes in the genus Pneumocystis are obligate pathogenic fungi that reside in mammalian lungs and cause Pneumocystis pneumonia in hosts with weakened immune systems. These fungal infections are not responsive to standard antifungal therapy. A long-term in vitro culture system is not available for these fungi, impeding the study of their biology and genetics and new drug development. Given that all genomes of the Pneumocystis species analyzed lack the genes for inositol synthesis and contain inositol transporters, Pneumocystis fungi, like S. pombe, appear to be inositol auxotrophs. Inositol is important for the pathogenesis, virulence, and mating processes in Candida albicans and Cryptococcus neoformans, suggesting similar importance within the Pneumocystis species as well. This is the first report to (i) characterize genes in the inositol phosphate metabolism and transport pathways in Pneumocystis species and (ii) identify inositol as a supplement that improved the viability of P. carinii in in vitro culture.
在解读肺孢子菌属专性致病真菌的代谢策略背景下，比较了三种肺孢子菌（卡氏肺孢子菌、鼠肺孢子菌和耶氏肺孢子菌）的基因组，并与自由生活的、系统发育相关的裂殖酵母（粟酒裂殖酵母）进行了比较。与粟酒裂殖酵母相比，卡氏肺孢子菌和耶氏肺孢子菌氨基酸代谢途径表达不足以及类固醇生物合成途径不完整得到了证实，并扩展到鼠肺孢子菌。与裂殖酵母相比，所有三种肺孢子菌的肌醇磷酸代谢途径表达均过量。除了粟酒裂殖酵母中已知的那些基因外，在两种啮齿动物肺孢子菌（卡氏肺孢子菌和鼠肺孢子菌）基因组中鉴定出四个基因，分别编码肌醇多磷酸多激酶（EC 2.7.1.151）、肌醇五磷酸2激酶（EC 2.7.1.158）、磷脂酰肌醇5磷酸酶（EC 3.1.3.36）和肌醇-1,4-二磷酸1磷酸酶（EC 3.1.3.57）。耶氏肺孢子菌基因组似乎包含其中三个基因，但缺少磷脂酰肌醇5磷酸酶。值得注意的是，所有三个基因组均缺少编码肌醇合成必需酶的两个基因，即肌醇-1-磷酸合酶（INO1）和肌醇单磷酸酶（INM1），这表明肺孢子菌为肌醇营养缺陷型。为满足获取外源性肌醇的需求，在卡氏肺孢子菌和鼠肺孢子菌中鉴定出两个与真菌肌醇转运蛋白具有同源产物的基因ITR1和ITR2，而耶氏肺孢子菌仅含有ITR1同源物。通过对感染肺组织cDNA进行逆转录酶实时PCR测定，发现鼠肺孢子菌和卡氏肺孢子菌中的ITR和肌醇代谢基因在暴发性感染期间表达。在体外培养中添加肌醇可使卡氏肺孢子菌在第7至14天的活力显著提高。
肺孢子菌属微生物是专性致病真菌，寄居于哺乳动物肺部，可在免疫系统受损的宿主中引起肺孢子菌肺炎。这些真菌感染对标准抗真菌治疗无反应。目前尚无针对这些真菌的长期体外培养系统，这阻碍了对其生物学、遗传学及新药研发的研究。鉴于所分析的所有肺孢子菌物种基因组均缺乏肌醇合成基因且含有肌醇转运蛋白，肺孢子菌似乎与粟酒裂殖酵母一样为肌醇营养缺陷型。肌醇对白色念珠菌和新型隐球菌中的致病机制、毒力及交配过程很重要，这表明在肺孢子菌中也具有类似的重要性。这是首份（i）描述肺孢子菌物种中肌醇磷酸代谢和转运途径中的基因以及（ii）鉴定出肌醇作为一种可提高卡氏肺孢子菌体外培养活力的补充剂的报告。
