1887

Abstract

A urease-negative serotype A strain of (B-4587) was isolated from the cerebrospinal fluid of an immunocompetent patient with a central nervous system infection. The gene encoding urease failed to complement the mutant phenotype. Urease-positive clones of B-4587 obtained by complementing with a genomic library of strain H99 harboured an episomal plasmid containing DNA inserts with homology to the gene of . The gene harboured by these plasmids was named since it enabled the transformants to grow on media containing urea as the sole nitrogen source while the transformants with an empty vector failed to grow. Transformation of strain B-4587 with a plasmid construct containing a truncated version of the gene failed to complement the urease-negative phenotype. Disruption of the native gene in a wild-type serotype A strain H99 and a serotype D strain LP1 of resulted in the inability of the strains to grow on media containing urea as the sole nitrogen source, suggesting that the gene product is involved in the utilization of urea by the organism. Virulence in mice of the urease-negative isolate B-4587, the urease-positive transformants containing the wild-type copy of the gene, and the urease-negative vector-only transformants was comparable to that of the H99 strain of regardless of the infection route. Virulence of the disruption stain of H99 was slightly reduced compared to the wild-type strain in the intravenous model but was significantly attenuated in the inhalation model. These results indicate that the importance of urease activity in pathogenicity varies depending on the strains of used and/or the route of infection. Furthermore, this study shows that complementation cloning can serve as a useful tool to functionally identify genes such as that have otherwise been annotated as hypothetical proteins in genomic databases.

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2006-12-01
2024-03-28
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References

  1. Bacanamwo M, Witte C. P, Lubbers M. W, Polacco J. C. 2002; Activation of the urease of Schizosaccharomyces pombe by the UreF accessory protein from soybean. Mol Genet Genomics 268:525–534 [CrossRef]
    [Google Scholar]
  2. Bava A. J, Negroni R, Bianchi M. 1993; Cryptococcosis produced by a urease negative strain of Cryptococcus neoformans . J Med Vet Mycol 31:87–89 [CrossRef]
    [Google Scholar]
  3. Canteros C. E, Rodero L, Rivas M. C, Davel G. 1996; A rapid urease test for presumptive identification of Cryptococcus neoformans . Mycopathologia 136:21–23 [CrossRef]
    [Google Scholar]
  4. Chang Y. C, Kwon-Chung K. J. 1994; Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14:4912–4919
    [Google Scholar]
  5. Chang Y. C, Kwon-Chung K. J. 1998; Isolation of the third capsule-associated gene, CAP60 , required for virulence in Cryptococcus neoformans . Infect Immun 66:2230–2236
    [Google Scholar]
  6. Chang Y. C, Kwon-Chung K. J. 1999; Isolation, characterization, and localization of a capsule-associated gene, CAP10 , of Cryptococcus neoformans . J Bacteriol 181:5636–5643
    [Google Scholar]
  7. Chang Y. C, Penoyer L. A, Kwon-Chung K. J. 1996; The second capsule gene of Cryptococcus neoformans , CAP64 , is essential for virulence. Infect Immun 64:1977–1983
    [Google Scholar]
  8. Chen G, Fournier R. L, Varanasi S, Mahama-Relue P. A. 1997; Helicobacter pylori survival in gastric mucosa by generation of a pH gradient. Biophys J 73:1081–1088 [CrossRef]
    [Google Scholar]
  9. Cox G. M, Mukherjee J, Cole G. T, Casadevall A, Perfect J. R. 2000; Urease as a virulence factor in experimental cryptococcosis. Infect Immun 68:443–448 [CrossRef]
    [Google Scholar]
  10. Curtis J. L, Huffnagle G. B, Chen G. H, Warnock M. L, Gyetko M. R, McDonald R. A, Scott P. J, Toews G. B. 1994; Experimental murine pulmonary cryptococcosis. Differences in pulmonary inflammation and lymphocyte recruitment induced by two encapsulated strains of Cryptococcus neoformans . Lab Invest 71:113–126
    [Google Scholar]
  11. Edman J. C, Kwon-Chung K. J. 1990; Isolation of the URA5 gene from Cryptococcus neoformans var. neoformans and its use as a selective marker for transformation. Mol Cell Biol 10:4538–4544
    [Google Scholar]
  12. Eitinger T, Degen O, Bohnke U, Muller M. 2000; Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe , provides nickel ion for urease biosynthesis. J Biol Chem 275:33184
    [Google Scholar]
  13. Holt C. L, May G. S. 1996; An extragenic suppressor of the mitosis-defective bimD6 mutation of Aspergillus nidulans codes for a chromosome scaffold protein. Genetics 142:777–787
    [Google Scholar]
  14. Kwon-Chung K. J, Bennett J. E. 1992 Medical Mycology Philadelphia: Lea & Febiger;
  15. Kwon-Chung K. J, Polacheck I, Popkin T. J. 1982; Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol 150:1414–1421
    [Google Scholar]
  16. Kwon-Chung K. J, Wickes B. L, Booth J. L, Vishniac H. S, Bennett J. E. 1987; Urease inhibition by EDTA in the two varieties of Cryptococcus neoformans . Infect Immun 55:1751–1754
    [Google Scholar]
  17. Kwon-Chung K. J, Varma A, Edman J. C, Bennett J. E. 1992; Selection of ura5 and ura3 mutants from the two varieties of Cryptococcus neoformans on 5-fluoroorotic acid medium. J Med Vet Mycol 30:61–69 [CrossRef]
    [Google Scholar]
  18. Lee M. H, Mulrooney S. B, Renner M. J, Markowicz Y, Hausinger R. P. 1992; Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes ( ureD , ureE , ureF , and ureG ) are involved in nickel metallocenter biosynthesis. J Bacteriol 174:4324–4330
    [Google Scholar]
  19. Lee H, Chang Y. C, Kwon-Chung K. J. 2005; TUP1 disruption reveals biological differences between MATa and MATα strains of Cryptococcus neoformans . Mol Microbiol 55:1222–1232
    [Google Scholar]
  20. Li A, Wu S. 1992; An urease negative Cryptococcus neoformans . Wei Sheng Wu Xue Bao 32:68–71
    [Google Scholar]
  21. Li A, Guo N, Wu S. 1993; A strain of urease negative Cryptococcus neoformans isolated from the environment in China. Chin Med Sci J 8:52–54
    [Google Scholar]
  22. Liu L, Wakamatsu K, Ito S, Williamson P. R. 1999; Catecholamine oxidative products, but not melanin, are produced by Cryptococcus neoformans during neuropathogenesis in mice. Infect Immun 67:108–112
    [Google Scholar]
  23. Mobley H. L. 1996; The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration. Aliment Pharmacol Ther 10:57–64 [CrossRef]
    [Google Scholar]
  24. Mobley H. L, Hu L. T, Foxal P. A. 1991; Helicobacter pylori urease: properties and role in pathogenesis. Scand J Gastroenterol 187:39–46
    [Google Scholar]
  25. Mondon P, Chang Y. C, Varma A, Kwon-Chung K. J. 2000; A novel episomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive selection marker in bacteria. FEMS Microbiol Lett 187:41–45 [CrossRef]
    [Google Scholar]
  26. Olszewski M. A, Noverr M. C, Chen G. H, Toews G. B, Cox G. M, Perfect J. R, Huffnagle G. B. 2004; Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion. Am J Pathol 164:1761–1771 [CrossRef]
    [Google Scholar]
  27. Perfect J. R, Wong B, Chang Y. C, Kwon-Chung K. J, Williamson P. R. 1998; Cryptococcus neoformans : virulence and host defences. Med Mycol 36:79–86
    [Google Scholar]
  28. Peterson C. L. 1994; The SMC family: novel motor proteins for chromosome condensation?. Cell 79:389–392 [CrossRef]
    [Google Scholar]
  29. Ruane P. J, Walker L. J, George W. L. 1988; Disseminated infection caused by urease-negative Cryptococcus neoformans . J Clin Microbiol 26:2224–2225
    [Google Scholar]
  30. Saitoh N, Goldberg I. G, Wood E. R, Earnshaw W. C. 1994; ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure. J Cell Biol 127:303–318 [CrossRef]
    [Google Scholar]
  31. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, Yanagida M. 1994; Fission yeast cut3 and cut14 , members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J 13:4938–4952
    [Google Scholar]
  32. Soriano A, Hausinger R. P. 1999; GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins. Proc Natl Acad Sci U S A 96:11140–11144 [CrossRef]
    [Google Scholar]
  33. Soriano A, Colpas G. J, Hausinger R. P. 2000; UreE stimulation of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein complex. Biochemistry 39:12435–12440 [CrossRef]
    [Google Scholar]
  34. Tanaka R, Nishimura K, Imanishi Y, Takahashi I, Hata Y, Miyaji M. 2003; Analysis of serotype AD strains from F1 progenies between urease-positive- and negative-strains of Cryptococcus neoformans . Nippon Ishinkin Gakkai Zasshi 44:293–297 [CrossRef]
    [Google Scholar]
  35. Toffaletti D. L, Rude T. H, Johnston S. A, Durack D. T, Perfect J. R. 1993; Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175:1405–1411
    [Google Scholar]
  36. Varma A, Kwon-Chung K. J. 2000; Characterization of the L41 gene in Cryptococcus neoformans : its application as a selectable transformation marker for cycloheximide resistance. Yeast 16:1397–1403 [CrossRef]
    [Google Scholar]
  37. Williamson P. R. 1997; Laccase and melanin in the pathogenesis of Cryptococcus neoformans . Front Biosci 2:e99–e107
    [Google Scholar]
  38. Zimmer B. L, Roberts G. D. 1979; Rapid selective urease test for presumptive identification of Cryptococcus neoformans . J Clin Microbiol 10:380–381
    [Google Scholar]
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