Identification of a novel gene, URE2, that functionally complements a urease-negative clinical strain of Cryptococcus neoformans

Abstract

A urease-negative serotype A strain ofCryptococcus neoformans(B-4587) was isolated from the cerebrospinal fluid of an immunocompetent patient with a central nervous system infection. TheURE1gene 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 thesudAgene ofAspergillus nidulans. The gene harboured by these plasmids was namedURE2since 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 theURE2gene failed to complement the urease-negative phenotype. Disruption of the nativeURE2gene in a wild-type serotype A strain H99 and a serotype D strain LP1 ofC. neoformansresulted in the inability of the strains to grow on media containing urea as the sole nitrogen source, suggesting that theURE2gene 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 theURE2gene, and the urease-negative vector-only transformants was comparable to that of the H99 strain ofC. neoformansregardless of the infection route. Virulence of theURE2disruption 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 ofC. neoformansused and/or the route of infection. Furthermore, this study shows that complementation cloning can serve as a useful tool to functionally identify genes such asURE2that have otherwise been annotated as hypothetical proteins in genomic databases.