Biochemical and genetic analyses of the oomycetePythium insidiosumprovide new insights into clinical identification and urease-based evolution of metabolism-related traits

The oomycete microorganism,Pythium insidiosum, causes the life-threatening infectious condition, pythiosis, in humans and animals worldwide. Affected individuals typically endure surgical removal of the infected organ(s). Detection ofP. insidiosumby the established microbiological, immunological, or molecular methods is not feasible in non-reference laboratories, resulting in delayed diagnosis. Biochemical assays have been used to characterizeP. insidiosum, some of which could aid in the clinical identification of this organism. Although hydrolysis of maltose and sucrose has been proposed as the key biochemical feature useful in discriminatingP. insidiosumfrom other oomycetes and fungi, this technique requires a more rigorous evaluation involving a wider selection ofP. insidiosumstrains. Here, we evaluated 10 routinely available biochemical assays for characterization of 26P. insidiosumstrains, isolated from different hosts and geographic origins. Initial assessment revealed diverse biochemical characteristics across theP. insidiosumstrains tested. Failure to hydrolyze sugars is observed, especially in slow-growing strains. Because hydrolysis of maltose and sucrose varied among different strains, use of the biochemical assays for identification ofP. insidiosumshould be cautioned. The ability ofP. insidiosumto hydrolyze urea is our focus, because this metabolic process relies on the enzyme urease, an important virulence factor of other pathogens. The ability to hydrolyze urea varied amongP. insidiosumstrains and was not associated with growth rates. Genome analyses demonstrated that urease- and urease accessory protein-encoding genes are present in both urea-hydrolyzing and non-urea-hydrolyzing strains ofP. insidiosum. Urease genes are phylogenetically conserved inP. insidiosumand related oomycetes, while the presence of urease accessory protein-encoding genes is markedly diverse in these organisms. In summary, we dissected biochemical characteristics and drew new insights into clinical identification and urease-related evolution ofP. insidiosum.

The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein

ABSTRACT Four accessory proteins (UreD, UreE, UreF, and UreG) are typically required to form the nickel-containing active site in the urease apoprotein (UreABC). Among the accessory proteins, UreD and UreF have been elusive targets for biochemical and structural characterization because they are not overproduced as soluble proteins. Using the best-studied urease system, in which the Klebsiella aerogenes genes are expressed in Escherichia coli, a translational fusion of ureE and ureF was generated. The UreEF fusion protein was overproduced as a soluble protein with a convenient tag involving the His-rich region of UreE. The fusion protein was able to form a UreD(EF)G-UreABC complex and to activate urease in vivo, and it interacted with UreD-UreABC in vitro to form a UreD(EF)-UreABC complex. While the UreF portion of UreEF is fully functional, the fusion significantly affected the role of the UreE portion by interrupting its dimerization and altering its metal binding properties compared to those of the wild-type UreE. Analysis of a series of UreEF deletion mutants revealed that the C terminus of UreF is required to form the UreD(EF)G-UreABC complex, while the N terminus of UreF is essential for activation of urease.