A serine/threonine phosphatase 1 of Streptococcus suis type 2 is an important virulence factor

Abstract

Streptococcus suis is regarded as one of the major pathogens of pigs, and Streptococcus suis type 2 (SS2) is considered a zoonotic bacterium based on its ability to cause meningitis and streptococcal toxic shock-like syndrome in humans. Many bacterial species contain genes encoding serine/threonine protein phosphatases (STPs) responsible for dephosphorylation of their substrates in a single reaction step. This study investigated the role of stp1 in the pathogenesis of SS2. An isogenic stp1 mutant (Δstp1) was constructed from SS2 strain ZJ081101. The Δstp1 mutant exhibited a significant increase in adhesion to HEp-2 and bEnd.3 cells as well as increased survival in RAW264.7 cells, as compared to the parent strain. Increased survival in macrophage cells might be related to resistance to reactive oxygen species since the Δstp1 mutant was more resistant than its parent strain to paraquat-induced oxidative stress. However, compared to parent strain virulence, deletion of stp1 significantly attenuated virulence of SS2 in mice, as shown by the nearly double lethal dose 50 value and the lower bacterial load in organs and blood in the murine model. We conclude that Stp1 has an essential role in SS2 virulence.

Keywords: Streptococcus suis type 2; serine/threonine protein phosphatase; virulence.

Fig. 1. Construction of the stp1 deletion mutant of Streptococcus suis type 2 and its identification by polymerase chain reaction (PCR). (A) Schematic representation of the allelic exchange of chromosomal stp1 with vector-carried cat between pSET4s-Δstp1 and the chromosome of ZJ081101 by homologous recombination. (B) PCR confirmation of the mutant strain Δstp1. Lane 1, primers gadph and cps2J for Δstp1; Lane 2, gadph and cps2J for ZJ081101; Lanes 3, 5, and 7, primers In1/In2, Cat1/Cat2 and Cat1/Out2 for Δstp1; Lanes 4, 6, and 8, primers In1/In2, Cat1/Cat2 and Cat2/Out1 for ZJ081101; M, Marker DL2000. (C) Stp1 protein is absent in the stp1 mutant as determined by western blotting with anti-Stp1 polyclonal antibody (CPP, cytoplasmic proteins; CWP, cell-wall proteins).

Fig. 2. Cell morphology of Streptococcus suis type 2 wild-type (WT) and its isogenic Δstp1 mutant. Transmission electron micrographs of bacteria (A–C). Scale bars = 5 µm (A), 0.2 µm, 100 nm, 50 nm (B, from left to right), 0.2 µm, 100 nm, 100 nm (C, from left to right).

Fig. 3. Phosphatase activity of Stp1 of Streptococcus suis type 2. (A) Purification of protein Stp1. (B) Dephosphorylation of p-nitrophenyl phosphate (pNPP) with Stp1 in the Mn²⁺ buffer. (C) Kinetics of pNPP hydrolysis by Stp1 as measured at different substrate concentrations over 10 min. The K_m and V_max were determined to be 5.95 (± 1.265) mM and 1.586 (± 0.09) nmol/µg per min, respectively. OD, optical density.

Fig. 4. Comparison of Streptococcus suis type 2 wild-type (WT) and its isogenic Δstp1 mutant effects on adhesion to bEnd.3 and HEp-2 cell monolayers. ^*p < 0.05, ^**p < 0.01.

Fig. 5. Comparison of Streptococcus suis type 2 wild-type (WT) and its isogenic Δstp1 mutant effects on phagocytosis and survival in RAW 264.7 macrophage cells. ^**p < 0.01. n.s., no significant difference.

Fig. 6. Resistance of Streptococcus suis type 2 wild-type (WT) and its isogenic Δstp1 mutant in BHI broth containing gradient levels of H₂O₂ or paraquat incubated at 37℃. The optical density at 620 nm was measured at hours 4 and 8. ^*p < 0.05, ^**p < 0.01. n.s., no significant difference.

Fig. 7. Bacterial load in organs of mice infected with Streptococcus suis type 2 wild-type (WT) and its isogenic Δstp1 mutant. As the lower limit of detection was 100 colony-forming unit (CFU), we did not suggest a null result when no single colony was present in a 10 µL spot of the organ homogenates; in such a case, we assigned an arbitrary value of 50 CFU to that sample.