Interaction between M-like protein and macrophage thioredoxin facilitates antiphagocytosis for Streptococcus equi ssp. zooepidemicus

Abstract

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis.

Figure 1. The interaction between SzP and TRX.

A: The split-ubiquitin yeast two hybrid assay. SzP was cloned in frame into the yeast expression vector (pDHB1), fused at its N-terminus to a small membrane anchor (the yeast ER protein Ost4) and at its C-terminus to a reporter cassette composed of the C-terminal half of ubiquitin (Cub) and a transcription factor (LexA-VP16). The recombinant TRX-pPR3-N (fusion to the mutated N-terminal half of ubiquitin) and SzP-pDHB1 were co-transformed into the NMY51 yeast strain. The co-transformed cells were selected on Trp-/Ade-/His-/Leu- 80 mM Aminotriazole dropout plates. The control+ represented co-transformation of pDSL-Delta-p53 and pDHB1-largeT (Dualsystems Biotech, Switzerland). Tubes showed the results of Liquid β-galactosidase assay. B: Luminescence value of the substrate corresponding to the pProLabel tag fused to TRX. Histogram showed max luminescence value through 1 h monitoring (n = 3, mean±SD, ** indicates that a value was significantly different (p<0.01)from the control- group). C: SzP was immunoprecipitated from HEK293 cells expressing TRX. HEK293 lysates were immunoprecipitated with rabbit polyclonal antibodies against TRX. The immunoprecipitates were subjected to the western blotting analysis with GFP monoclonal antibodies against GFP -SzP. D: pDHB1-SzP and pPR3-N-mut-TRX were co-transformed into the yeast strain NMY51. SzP interacted with mutant TRX, which had mutations Cys³² and Cys³⁵ to Ser (C32S/C35S) in its active site. Tubes showed the results of Liquid β-galactosidase assay.

Figure 2. Oxidized and reduced forms of TRX can interact with SzP.

A: TRX bound to protein G beads was treated with either 1 mM H₂O₂ or 100 mM DTT for 15 min at room temperature. The proteins were separated by native PAGE and visualized by the western blot analysis for His-tag fused TRX. B: Oxidized and reduced forms of TRX were able to interact with SzP. His-TRX bound to protein G beads were incubated with Nonidet P-40 buffer or with buffer supplemented with 1 mM H₂O₂ for 15 min at room temperature. After extensive washing in the same buffer, the proteins were submitted to the binding assay. As a control to revert oxidation, a part of the H₂O₂-treated fusion protein was incubated with buffer containing 100 mM DTT for 15 min prior to SzP incubation. C: SzP did not inhibit TRX activity. The insulin disulfide reduction assay was performed in the lysates obtained from confluent cultures described under “Materials and Methods.” The activity of TRX bound to SzP in vitro: TRX activity was detected after incubating 10 µg TRX with 100 µg/ml or 500 µg/ml SzP at 37°C for 2 h. Control samples were treated with PBS. (n = 3, mean±SD).

Figure 3. Recruitment of TRX to the surface of viable S. zooepidemicus.

Flow cytometry results showed that TRX could bind to the surface SzP of S. zooepidemicus. A: S. zooepidemicus wild strain incubated with PBS (negative control); B: SzP-knockout strain incubated with TRX; C: S. zooepidemicus wild strain incubated with TRX. The SzP of S. zooepidemicus interacted with TRX and recruited TRX to the bacterial surface.

Figure 4. TRX knockdown.

A: The relative level of TRX mRNA was detected by RT-PCR. β-actin was used as the reference control. The mRNA level of control was considered as 100%. The relative level of TRX mRNA was calculated using equation 2^−ΔΔCT (n = 3, mean±SD, the symbols, * and #, indicate that a value differed significantly (p<0.05) from the control and nt-TRX RNAi treated groups, respectively.); B: Reduction in TRX protein levels was confirmed by the western blot analysis in Raw264.7 cell. Lane 1 and 3 were nt-TRX RNAi plasmid treated cell lysis, lane 2 and 4 were TRX RNAi plasmid treated cell lysis. All samples were extracted at 24 h after transfection.

Figure 5. The SzP/TRX interaction facilitated S. zooepidemicus to avoid being phagocytized.

The phagocytosis percentage of Raw264.7 wild strain and TRXi strain did not differ significantly. The S. zooepidemicus SzP-knockout strain were phagocytized effectually. In the presence of the serum, the antiphagocytosis of S. zooepidemicus was more pronounced with the SzP/TRX interaction, as there was significantly more macrophage containing ingested S. zooepidemicus in TRXi Raw264.7 cells than the wild type Raw264.7 cells. Thus, the SzP/TRX interaction reduced the phagocytosis of S. zooepidemicus by the macrophages in the host immune system. The phagocytosis percentage of the TRX pretreated S. zooepidemicus wild strain was significantly lower than the SzP-knockout strain in the TRXi Raw264.7 cells. In the absence of the serum, the phagocytosis of the macrophages was independent of TRX, and only SzP was able affect phagocytosis (n = 3, mean±SD, ** p<0.01).

Figure 6. SzP/TRX interaction contributed to the FH recruitment and reduced the C3 deposition on the bacterial surface, beneficial for S. zooepidemicus to evade phagocytosis of the host immune system.

A: The binding of FH to SzP (lane 1), TRX (lane 2) and the SzP/TRX complex (Lane 3) was demonstrated by using polyclonal goat antiserum to FH in an immunoblot analysis. B: TRX inhibited C3 convertase activity in the fluid phase. Fluid phase alternative pathway C3 convertase was generated by the addition of purified C3, C3i, factor B, and 0.1 M MgCl₂. 10 µg of TRX or FH were added followed by the addition of factor D to a final volume of 125 µl. Purified components only (control+); purified complement components without factor D (control-). The inhibition of C3 convertase was determined by C3a generation after 30 min of incubation and measured by a C3a ELISA. The effect of dosage increase of FH and TRX on C3a generation (ng/ml) was shown here. Reduction in C3a generation was correlated with the decreased C3 convertase activity. (Symbol * indicates that a value significantly differs from the control+ group). C: Immunoblot showing C3 components eluted from the surface of the S. zooepidemicus wild strains and the SzP knockout strains following treatment of TRX, FH or TRX and FH in porcine plasma. The blot was developed with the affinity purified antiserum to C3. C3 components eluted from the S. zooepidemicus wild strains and the SzP knockout strains after incubation in porcine plasma were used as the negative control. D: Flow cytometry analysis of S. zooepidemicus surface-bound C3b, a total of 10,000 events were collected per sample and a single gate was used to exclude debris. E: Flow cytometry analysis of gradient concentration TRX pretreated S. zooepidemicus surface-bound C3b. The antiphagocytic effectiveness of the TRX was dose-dependent until saturation. These results were the mean±SD for n = 3, *p<0.05, **p<0.01, ***p<0.001.