Virulence (Dec 2024)

Serine protease RAYM_01812 (SspA) inhibits complement-mediated killing and monocyte chemotaxis and contributes to virulence of Riemerella anatipestifer in ducks

  • Rongkun Yang,
  • Sen Li,
  • Jie Guo,
  • Yanhua Wang,
  • Zeyuan Dong,
  • Qing Wang,
  • Hongying Bai,
  • Congran Ning,
  • Xiaotong Zhu,
  • Jiao Bai,
  • Sishun Hu,
  • Yuncai Xiao,
  • Zili Li,
  • Zutao Zhou

DOI
https://doi.org/10.1080/21505594.2024.2421219
Journal volume & issue
Vol. 15, no. 1

Abstract

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Riemerella anatipestifer (RA) is a significant poultry pathogen causing acute septicemia and inflammation. The function of protease RAYM_01812, responsible for gelatin degradation, is unexplored in RA pathogenesis. To elucidate its role, we generated a deletion mutant ΔRAYM_01812 (ΔRAYM) and complementary CΔRAYM_01812 (CΔRAYM) strain and revealed the protease’s role in extracellular gelatinase activity. By expressing full-length 76 kDa RAYM_01812 protein without signal peptide as well as seven partial structural domains fragments, we evidence that the N-terminal propeptide acts as an enzymatic activity inhibitor and it gets cleaved at A112. Also, we show that the β-fold sheet domain is necessary for enhancing the enzymatic protease activity. Sequential auto-proteolysis forms a stable 40 kDa enzyme. Then, testing the strains in duck sera indicated that the absence or presence of RAYM_01812 results in reduced or enhanced bacterial survival, respectively. Furthermore, we found that the protease is able to cleave IgY antibodies as well as the complement factors C3a and C5a, that the protease reduces C3a- or C5a-mediated monocyte chemotaxis, and results in enhanced membrane attack complex (MAC) formation on the surface of ΔRAYM compared to CΔRAYM. This suggests that RAYM_01812 plays a crucial role in protecting against the serum complement-mediated bactericidal effect through inhibiting MAC formation and monocyte chemotaxis. Animal infection assays showed a 1090-fold reduced virulence of ΔRAYM compared to RA-YM, evidenced by decreased tissue loading and weaker histopathological changes. In conclusion, RAYM_01812 acts as a vital virulence factor, enabling host innate immune defence escape through complement killing evasion and monocyte chemotaxis inhibition.

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