title: Factors required for activation of urease as a virulence determinant in Cryptococcus neoformans.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA.
authors: Arpita
authors: A
authors: Singh
authors: Robert J
authors: RJ
authors: Panting
authors: Ashok
authors: A
authors: Varma
authors: Tomomi
authors: T
authors: Saijo
authors: Kevin J
authors: KJ
authors: Waldron
authors: Ambrose
authors: A
authors: Jong
authors: Popchai
authors: P
authors: Ngamskulrungroj
authors: Yun C
authors: YC
authors: Chang
authors: Julian C
authors: JC
authors: Rutherford
authors: Kyung J
authors: KJ
authors: Kwon-Chung
mesh_terms: D000818:Animals; D001921:Brain / Q000382:microbiology / Q000473:pathology; D003453:Cryptococcosis / Q000382:microbiology / Q000473:pathology; D003455:Cryptococcus neoformans / Q000201:enzymology* / Q000472:pathogenicity*; D004195:Disease Models, Animal; D005260:Female; D005656:Fungal Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009532:Nickel / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D014510:Urease / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D037521:Virulence Factors; D009532:Nickel; D014510:Urease
references: J Clin Invest. 2010 May;120(5):1683-93
references: 20424328
references: BMC Evol Biol. 2011 Mar 29;11:80
references: 21447149
references: J Bacteriol. 1997 Jul;179(13):4081-6
references: 9209019
references: QJM. 2008 May;101(5):359-63
references: 18334496
references: Microbiology (Reading). 2006 Dec;152(Pt 12):3723-3731
references: 17159224
references: Protein Sci. 1993 Jun;2(6):1034-41
references: 8318888
references: EMBO J. 2006 Apr 19;25(8):1775-83
references: 16601688
references: Plant Mol Biol. 2001 Jan;45(2):169-79
references: 11289508
references: J Biol Chem. 2000 Jun 16;275(24):18029-33
references: 10748059
references: Anal Biochem. 2001 Mar 1;290(1):102-7
references: 11180943
references: Curr Infect Dis Rep. 2007 Nov;9(6):457-64
references: 17999881
references: J Bacteriol. 1990 Oct;172(10):5837-43
references: 2211515
references: Genetics. 1996 Mar;142(3):777-87
references: 8849887
references: J Biol Chem. 2012 May 4;287(19):15298-306
references: 22418440
references: Mol Genet Genomics. 2002 Dec;268(4):525-34
references: 12471450
references: Annu Rev Microbiol. 2006;60:281-303
references: 16753032
references: Plant Physiol. 1990 Oct;94(2):681-9
references: 16667765
references: Cell Microbiol. 2008 Jun;10(6):1313-26
references: 18248627
references: Gene. 1997 Oct 1;198(1-2):387-91
references: 9370306
references: Cell. 1994 Nov 4;79(3):389-92
references: 7954805
references: Infect Immun. 2009 Jan;77(1):120-7
references: 18936186
references: J Biol Chem. 1991 Feb 15;266(5):3222-7
references: 1847142
references: Microbiol Mol Biol Rev. 1997 Mar;61(1):17-32
references: 9106362
references: Infect Immun. 2004 Sep;72(9):4985-95
references: 15321990
references: Nature. 2008 Oct 23;455(7216):1138-42
references: 18948958
references: Am J Physiol. 1999 Oct;277(4):L719-26
references: 10516212
references: Mycoses. 2009 Mar;52(2):166-70
references: 18643920
references: Am J Pathol. 2004 May;164(5):1761-71
references: 15111322
references: Microbiology (Reading). 2002 Aug;148(Pt 8):2607-2615
references: 12177355
references: J Biol Chem. 1997 Jul 4;272(27):17139-44
references: 9202033
references: Respir Res. 2007 Nov 01;8:78
references: 17976234
references: J Cell Biol. 1988 Apr;106(4):1141-9
references: 2452168
references: Science. 1995 May 19;268(5213):998-1004
references: 7754395
references: J Bacteriol. 1993 Mar;175(5):1405-11
references: 8444802
references: Nature. 1989 Jul 20;340(6230):245-6
references: 2547163
references: J Bacteriol. 2002 Jul;184(13):3569-77
references: 12057951
references: Plant Physiol. 2005 Nov;139(3):1155-62
references: 16244137
references: J Mol Biol. 2004 Jul 23;340(5):1197-206
references: 15236977
references: Methods Mol Biol. 2005;310:227-40
references: 16350956
references: Biochemistry. 2010 Jul 20;49(28):5859-69
references: 20533838
references: J Biol Chem. 2001 Dec 28;276(52):49359-64
references: 11591723
references: Infect Immun. 2006 Jan;74(1):504-15
references: 16369007
references: Infect Immun. 2000 Feb;68(2):443-8
references: 10639402
references: Clin Microbiol Rev. 1999 Jul;12(3):394-404
references: 10398672
references: J Bacteriol. 1998 Apr;180(7):1962-4
references: 9537401
references: J Bacteriol. 1992 Jul;174(13):4324-30
references: 1624427
references: J Neurosurg. 2001 Mar;94(3):397-402
references: 11235942
references: Phytochemistry. 2008 Jan;69(1):18-28
references: 17706733
references: Microbiol Rev. 1995 Sep;59(3):451-80
references: 7565414
references: Am J Pathol. 2009 Mar;174(3):932-43
references: 19218345
references: Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):13095-9
references: 21788478
references: Environ Microbiol. 2008 Oct;10(10):2586-97
references: 18564183