Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA.
mBio. 2013 May 7;4(3):e00220-13. doi: 10.1128/mBio.00220-13.
Urease in Cryptococcus neoformans plays an important role in fungal dissemination to the brain and causing meningoencephalitis. Although urea is not required for synthesis of apourease encoded by URE1, the available nitrogen source affected the expression of URE1 as well as the level of the enzyme activity. Activation of the apoenzyme requires three accessory proteins, Ure4, Ure6, and Ure7, which are homologs of the bacterial urease accessory proteins UreD, UreF, and UreG, respectively. A yeast two-hybrid assay showed positive interaction of Ure1 with the three accessory proteins encoded by URE4, URE6, and URE7. Metalloproteomic analysis of cryptococcal lysates using inductively coupled plasma mass spectrometry (ICP-MS) and a biochemical assay of urease activity showed that, as in many other organisms, urease is a metallocentric enzyme that requires nickel transported by Nic1 for its catalytic activity. The Ure7 accessory protein (bacterial UreG homolog) binds nickel likely via its conserved histidine-rich domain and appears to be responsible for the incorporation of Ni(2+) into the apourease. Although the cryptococcal genome lacks the bacterial UreE homolog, Ure7 appears to combine the functions of bacterial UreE and UreG, thus making this pathogen more similar to that seen with the plant system. Brain invasion by the ure1, ure7, and nic1 mutant strains that lack urease activity was significantly less effective in a mouse model. This indicated that an activated urease and not the Ure1 protein was responsible for enhancement of brain invasion and that the factors required for urease activation in C. neoformans resemble those of plants more than those of bacteria.
Cryptococcus neoformans is the major fungal agent of meningoencephalitis in humans. Although urease is an important factor for cryptococcal brain invasion, the enzyme activation system has not been studied. We show that urease is a nickel-requiring enzyme whose activity level is influenced by the type of available nitrogen source. C. neoformans contains all the bacterial urease accessory protein homologs and nickel transporters except UreE, a nickel chaperone. Cryptococcal Ure7 (a homolog of UreG) apparently functions as both the bacterial UreG and UreE in activating the Ure1 apoenzyme. The cryptococcal urease accessory proteins Ure4, Ure6, and Ure7 interacted with Ure1 in a yeast two-hybrid assay, and deletion of any one of these not only inactivated the enzyme but also reduced the efficacy of brain invasion. This is the first study showing a holistic picture of urease in fungi, clarifying that urease activity, and not Ure1 protein, contributes to pathogenesis in C. neoformans.
新型隐球菌中的脲酶在真菌向大脑传播并导致脑膜脑炎方面起着重要作用。尽管尿素不是由 URE1 编码的脱氨酶合成所必需的，但可用的氮源会影响 URE1 的表达以及酶活性水平。脱辅基酶的激活需要三种辅助蛋白，Ure4、Ure6 和 Ure7，它们分别是细菌脲酶辅助蛋白 UreD、UreF 和 UreG 的同源物。酵母双杂交测定显示 Ure1 与 URE4、URE6 和 URE7 编码的三种辅助蛋白之间存在阳性相互作用。使用电感耦合等离子体质谱法 (ICP-MS) 对隐球菌裂解物进行金属蛋白质组学分析和脲酶活性的生化测定表明，与许多其他生物体一样，脲酶是一种金属中心酶，其催化活性需要由 Nic1 转运的镍。Ure7 辅助蛋白（细菌 UreG 同源物）可能通过其保守的富含组氨酸的结构域结合镍，并且似乎负责将 Ni(2+) 掺入脱辅基酶中。尽管新型隐球菌基因组缺乏细菌 UreE 同源物，但 Ure7 似乎结合了细菌 UreE 和 UreG 的功能，因此使这种病原体更类似于植物系统。在缺乏脲酶活性的 ure1、ure7 和 nic1 突变株的小鼠模型中，对大脑的侵袭明显减少。这表明，激活的脲酶而不是 Ure1 蛋白负责增强大脑侵袭，并且新型隐球菌中脲酶激活所需的因素与植物更相似，而不是与细菌更相似。
新型隐球菌是人类脑膜脑炎的主要真菌病原体。尽管脲酶是新型隐球菌大脑入侵的重要因素，但尚未研究该酶的激活系统。我们表明，脲酶是一种需要镍的酶，其活性水平受可用氮源类型的影响。除了镍伴侣 UreE 之外，新型隐球菌还包含所有细菌脲酶辅助蛋白同源物和镍转运蛋白。新型隐球菌的 Ure7（UreG 的同源物）显然在激活 Ure1 脱辅基酶方面既充当细菌 UreG 又充当 UreE。新型隐球菌的脲酶辅助蛋白 Ure4、Ure6 和 Ure7 在酵母双杂交测定中与 Ure1 相互作用，并且缺失这些中的任何一个不仅使酶失活，而且还降低了大脑入侵的功效。这是第一项研究真菌脲酶的整体情况的研究，阐明了脲酶活性而不是 Ure1 蛋白有助于新型隐球菌的发病机制。
