A Riemerella anatipestifer Metallophosphoesterase That Displays Phosphatase Activity and Is Associated with Virulence

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that bacterial virulence and secretion proteins of the type IX secretion system (T9SS) mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, in comparison to those of wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which is absent from the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of gene mutation and complementation strains. The virulence assessment showed >1,000-fold attenuated virulence and significantly reduced bacterial loads in the blood of ducks infected with Yb2Δ00980, the AS87_RS00980 gene deletion mutant strain. Bacterial virulence was recovered in complementation strain cYb2Δ00980 Further study indicated that the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase (MPPE), which displayed phosphatase activity and was cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined to be 7.0 and 60°C, respectively, and the K and V_max were determined to be 3.53 mM and 198.1 U/mg. The rMPPE activity was activated by Zn²⁺ and Cu²⁺ but inhibited by Fe³⁺, Fe²⁺, and EDTA. There are five conserved sites, namely, N267, H268 H351, H389, and H391, in the metallophosphatase domain. Mutant proteins Y267-rMPPE and Y268-rMPPE retained 29.30% and 19.81% relative activity, respectively, and mutant proteins Y351-rMPPE, Y389-rMPPE, and Y391-rMPPE lost almost all MPPE activity. Taken together, these results indicate that the R. anatipestiferAS87_RS00980 gene encodes an MPPE that is a secretion protein of T9SS that plays an important role in bacterial virulence.IMPORTANCERiemerella anatipestifer T9SS was recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes have been identified, but no effector has been reported in R. anatipestifer to date. In this study, we identified the T9SS secretion protein AS87_RS00980 as an MPPE that displays phosphatase activity and is associated with bacterial virulence. The enzymatic activity of the rMPPE was determined, and the K and V_max were 3.53 mM and 198.1 U/mg, respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated >1,000-fold, indicating that MPPE is an important virulence factor. In summary, we identified that the R. anatipestiferAS87_RS00980 gene encodes an important T9SS effector, MPPE, which plays an important role in bacterial virulence.