Functional and Phylogenetic Analysis of ureD in Shiga Toxin-Producing Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG, the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD, encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro. We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

Contribution of Urease to Colonization by Shiga Toxin-Producing Escherichia coli

“…Since other bacterial ureases have been found to exhibit optimal enzyme activity at low pH (55), we examined whether this is a mechanism of regulation for the STEC urease enzyme by determining the pH for optimal STEC enzyme activity. Cultures of two different phylogenetically distinct urease-positive STEC strains, STEC Mo28 and STEC 88-0643, were used for these experiments (46). Lysates of these cultures were used to quantify the urease activity at pH values 3 through 9.…”

“…Urease expression is constitutive for some bacterial species; however, most species have a method of regulating expression of the ure genes based on an environmental stimulus (29). In our previous work, we have demonstrated that SNPs in ureD affect the urease activity but that there must also be some trans-acting factor yet to be identified that is affecting the level of urease activity (46). Elucidation of the method of regulation of urease expression in STEC could help in determining the role urease plays in STEC pathogenesis.…”

“…Most STEC strains exhibit a urease-negative phenotype in vitro, and it was later discovered that the STEC Sakai strain carries a single nucleotide substitution in ureD that results in early truncation of the UreD chaperone protein due to replacement of a glutamine codon with an amber codon (34). The UreD protein has been demonstrated to be essential for activation of the urease apoprotein (29), and we have previously demonstrated that the single nucleotide polymorphism (SNP) in ureD abrogates urease activity (46). An identical SNP was found in other STEC O157:H7 isolates and was believed to be the basis for the urease-negative phenotype in all phenotypically urease-negative STEC strains (34).…”

“…However, our previous work in which a more phylogenetically diverse set of STEC strains was examined has demonstrated that this SNP is not as widespread as was thought and appears to be restricted to a branch of the STEC O157:H7 phylogenetic cluster, along with a subset of STEC O111 isolates. Furthermore, an additional SNP that we identified in the Sakai strain, resulting in the substitution of a leucine for a proline at position 38 in the UreD protein and contributing to the lack of urease activity, was determined to be phylogenetically restricted to a branch of the STEC O157:H7 isolates (46). These results indicate that many STEC strains carry genes encoding a urease enzyme that should function appropriately under inducing conditions, possibly in vivo in the human or animal host, and may provide a significant survival advantage.…”

“…In the first two STEC genomes sequenced, those of the O157:H7 EDL933 strain and O157:H7 Sakai strain, a genomic island containing, among other genes, ureDABCEFG, a cluster of genes coding for the enzyme urease and its accessory proteins, was identified (12, 39). Subsequently, many other STEC strains, including a wide variety of serotypes, have been found to carry the ure gene cluster (9,35,36,46). Urease, which hydrolyzes the substrate urea to form CO 2 and two molecules of NH 3 (29), has been demonstrated to contribute to virulence in several other bacterial species (1, 5-8, 25, 43).…”