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.

The Nudix hydrolase superfamily

“…Direct evidence for this was provided by the demonstration that deletion of the S. typhimurium ygdP and apaH genes leads to synergistic increases in bacterial Ap 4 N levels while decreasing cellular invasion [51]. Furthermore, pnhA -mutants of P. multocida are 1000-fold less virulent in a chick embryo animal model [53]. Disruption of E. coli ygdP also relieves lethal hybrid jamming, the blocking of the general protein secretory pathway caused by the expression of normally cytoplasmic proteins when fused to an N-terminal Sec signal sequence.…”

“…They hydrolyse Np n N where n>3, always producing an NTP product. E. coli YgdP and the orthologues from Salmonella typhimurium [51], Rickettsia prowazekii (InvA) [52] and Pasteurella multocida (PnhA) [53] prefer Ap 5 A over Ap 4 A in vitro, whereas Ap 4 A is preferred by the enzymes from Bartonella bacilliformis (IalA) [54,55] and Helicobacter pylori (NudA) [56]. E. coli K1 YgdP and B. bacilliformis IalA have both been implicated in the ability of these pathogens to invade brain microvascular endothelial cells [57] and erythrocytes [58], respectively, suggesting that proper regulation of bacterial Np n N is required for expression of the invasive phenotype.…”

The evolution of function within the Nudix homology clan

“…Diadenosine polyphosphates (Ap n As) are structurally related hydrolase substrates implicated in modulating an alarmone response upon pathogen infection or other cellular stress events . Hydrolyzing these metabolites potentially diminishes the effect of a host stress‐response, a feature for which pathogenic bacteria use Nudix hydrolases to their advantage . On the other hand, some plants employ Nudix hydrolases to mitigate pathogen infection and boost host immunity via activity on a variety of substrates .…”

Intruders below the Radar: Molecular Pathogenesis of Bartonella spp

“…These enzymes are involved in the catabolism of alarmones, and homologs of IalA in other pathogens were shown to be crucial for infectivity (38,128,200,422). It is thus reasonable to assume that IalA may contribute indirectly to invasion, and a homolog in Rickettsia prowazekii was indeed suggested to "buffer" the bacterial homeostasis against stressful conditions during growth in a hostile, intracellular environment (154).…”