The pnhA gene of Pasteurella multocida encodes a dinucleoside oligophosphate pyrophosphatase member of the Nudix hydrolase superfamily

Abstract

The pnhA gene of Pasteurella multocida encodes PnhA, which is a member of the Nudix hydrolase subfamily of dinucleoside oligophosphate pyrophosphatases. PnhA hydrolyzes diadenosine tetra-, penta-, and hexaphosphates with a preference for diadenosine pentaphosphate, from which it forms ATP and ADP. PnhA requires a divalent metal cation, Mg(2+) or Mn(2+), and prefers an alkaline pH of 8 for optimal activity. A P. multocida strain that lacked a functional pnhA gene, ACP13, was constructed to further characterize the function of PnhA. The cellular size of ACP13 was found to be 60% less than that of wild-type P. multocida, but the growth rate of ACP13 and its sensitivity to heat shock conditions were similar to those of the wild type, and the wild-type cell size was restored in the presence of a functional pnhA gene. Wild-type and ACP13 strains were tested for virulence by using the chicken embryo lethality model, and ACP13 was found to be up to 1,000-fold less virulent than the wild-type strain. This is the first study to use an animal model in assessing the virulence of a bacterial strain that lacked a dinucleoside oligophosphate pyrophosphatase and suggests that the pyrophosphatase PnhA, catalyzing the hydrolysis of diadenosine pentaphosphates, may also play a role in facilitating P. multocida pathogenicity in the host.

FIG. 1.

Alignment of dinucleoside oligophosphate pyrophosphatases from various bacterial pathogens. Only the Nudix motif (shown in bold) and the highly conserved sequence (shaded) are shown for each of the dinucleoside oligophosphate pyrophosphatases. Asterisks indicate identical amino acid residues, and ≫ indicates proteins that have been identified as dinucleoside oligophosphate pyrophosphatases. All of the above sequences were obtained via BLASTP (2) and by using the PnhA amino acid sequence as the query sequence.

FIG. 2.

Expression and purification of the PnhA Nudix hydrolase protein. Shown is a gradient polyacrylamide gel (4 to 20%) containing 1% sodium dodecyl sulfate, stained with Coomassie brilliant blue. Lane 1 is protein standards with the indicated molecular masses; lane 2 contains approximately 10 μg of crude extract of uninduced cells of ACP1009; lane 3 contains approximately 20 μg of crude extract of isopropyl-β-d-thiogalactopyranoside-induced ACP1009; lane 4 contains approximately the same number of enzyme units of the purified PnhA protein (from fraction V) as in lane 3.

FIG. 3.

Reaction products from hydrolysis of Ap₅A by PnhA. High-performance liquid chromatography was used to identify the reaction products as described previously (16). Samples were collected at 0, 10, and 40 min of reaction, respectively.

FIG. 4.

Morphological characterization of wild-type P. multocida, ACP13, and ACP17. Electron micrographs of negatively stained (A) wild-type P. multocida, (B) ACP13 mutant, (C) ACP13 cell undergoing replication, and (D) ACP17 complemented mutant. The arrows (D) indicate small cells that are assumed to be those that have lost the pACP1007 plasmid with the functional pnhA and are thus exhibiting the mutant phenotype (B, C). The TEM micrographs were taken at magnification ×20,000.