LAUSR

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrate that bacterial virulence and secretion proteins of the T9SS mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, compare to the wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which absent in the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of the gene mutation and complementation strains. The virulence assessment showed a more than 1,000-folds attenuated virulence, and significantly reduced bacterial loads in blood of infected ducks for the mutant strain Yb2Δ00980 The bacterial virulence was recovered in the complementation strain cYb2Δ00980 Further study indicated that the AS87_RS00980 gene encodes a secretion protein metallophosphatase (MPPE), which displayed phosphatase activity and cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined as 7.0 and 60°C, respectively; and the Km and Vmax were determined as 3.53 mM and 198.1U/mg. The MPPE activity was activated by Zn2+, Cu2+, but inhibited by Fe3+, Fe2+, and EDTA. There are five conserved sites of N267, H268 H351, H389, and H391 in the MEEP domain. The mutant proteins of 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 the MPPE activity. Taken together, these results indicated that R. anatipestifer AS87_RS00980 gene encodes a MPPE, which is a secretion protein of T9SS and plays an important role in bacterial virulence.Importance Riemerella anatipestifer T9SS is recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes are identified, but no effector is reported in the R. anatipestifer up to date. In this study, we identified the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase that displays phosphatases activity and associated with bacterial virulence. Enzymatic activity of the metallophosphoesterase was determined, and the Km and Vmax were 3.53 mM and 198.1U/mg respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated over 1000-fold, indicating it is an important virulence factor. In summary, we identified that R. anatipestifer AS87_RS00980 gene encodes an important T9SS effector MPPE, which plays an important role in bacterial virulence.