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. 2022 Apr 19;10(5):932.
doi: 10.3390/biomedicines10050932.

BifA Triggers Phosphorylation of Ezrin to Benefit Streptococcus equi subsp. zooepidemicus Survival from Neutrophils Killing

Fei Pan  1   2 , Jie Peng  1   2   3 , Dandan Yu  1   2 , Lianyue Li  1   2 , Hongjie Fan  1   2   4 , Zhe Ma  1   2   4
Affiliations

BifA Triggers Phosphorylation of Ezrin to Benefit Streptococcus equi subsp. zooepidemicus Survival from Neutrophils Killing

Fei Pan et al. Biomedicines. .

Abstract

Streptococcus equi subsp. zooepidemicus (SEZ) ATCC35246 can invade the brain and cause severe neutrophils infiltration in brain tissue. This microorganism can survive and reproduce to an extremely high CFU burden (108-109/organ) under stressful neutrophils infiltration circumstances. The aim of this research is to explore the mechanism of the SEZ hypervirulent strain with its specific bifA gene which avoids being eliminated by neutrophils in the brain. We isolated the primary mouse neutrophils to treat SEZ WT and bifA gene defective (ΔBif) strains. The ΔBif strain had a weakened function of defending against neutrophils killing in vitro. The interaction between BifA and ezrin proteins in neutrophils were identified by co-IP and immunoblot. In neutrophils, the BifA interacts with ezrin and triggers the phosphorylation of ezrin at its Thr567 site in a PKC-dependent manner, then the excessive elevation of phosphorylated-ezrin recruits Dbl and activates Rac1. Since the Rac1 is closely relevant to several critical cellular functions, its abnormal activation will lead to neutrophils dysfunction and benefit to SEZ survival from neutrophils killing. Our findings reveal a novel consequence of BifA and ERM family protein (for ezrin, radixin, moesin) interaction, which happens between BifA and ezrin in neutrophils and contributes to SEZ survival in the brain. BifA should be considered as a potential target for drug development to prevent SEZ infection.

Keywords: Streptococcus equi subsp. zooepidemicus; bifA; ezrin; neutrophils.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The bifA gene defective SEZ strain is more susceptible to neutrophils killing. (A) SPF BALB/c mice were challenged with 1 × 106 CFU wild-type SEZ (SEZ ATCC 35246) or PBS as a control. Brains were stained with immunohistochemistry after the paraffin section. Granulocytes, especially the neutrophils, were stained densely. Severe neutrophils infiltration happened in SEZ infected mouse brain. (B) The survival percentage of SEZ, ΔBif and CΔBif after engaging with primary mouse neutrophils (multiplicity of infection, MOI = 1:10) for 60 min in vitro (n = 3, p values were calculated with one-way ANOVA, * indicates p < 0.05; ns indicates no significant difference) (C) The survival percentage of SEZ, ΔBif and CΔBif in dHL60 cells at different time points (n = 6, p values were calculated with one-way ANOVA for each time point, * indicates p < 0.05; *** indicates p < 0.001).
Figure 2
Figure 2
The bifA gene defective SEZ strain is more susceptible to neutrophils killing. BifA interacts with ezrin and elevates p-ezrin levels in dHL60 cells. (A) Observation of dHL60 cells incubated with BifA or Negative Control (NC) by immunofluorescence microscopy. BifA and p-ezrin were detected by immunostaining with an anti-BifA polyclonal antibody (green) and an anti-p-ezrin antibody (red), respectively, and DAPI was used as a counterstain for nuclei (blue). (Bar = 20 μm) The yellow lines in the merged pictures were in accordance with the line length (X-axis) of graphs on the right. The Y-axis showed the signal intensity of the three colors channels along with the yellow line. The overlap of green and red curves indicated the colocalization of BifA and p-ezrin in merged pictures. (B) Immunoblot detection of ezrin phosphorylation in dHL60 cells under BifA or NC treatment at different time points with anti-p-ezrin antibody. The gray intensity was measured with Image J. Gray intensity ratio of p-ezrin/GAPDH shown on the below graph (n = 3, ** indicates p < 0.01; *** indicates p < 0.001). (C) As the phos-tag can bind to p-ezrin and shift its mobility in gel, ezrin and p-ezrin were separated in phos-tag acrylamide gel, and detected by immunoblot. The gray intensity ratio of p-ezrin/GAPDH shown on the below graph (n = 3, * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; ns indicates no significant difference). (D) The phosphorylation level of ezrin in dHL60 cell lysate (input) after BifA or BifA + staurosporine (BifA + Stau.) treatment for 2 h was detected by immunoblot; the blank group was untreated cells. The p-ezrin was concentrated by immunoprecipitation with anti-p-ezrin antibody-conjugated beads and detected by immunoblot to confirm ezrin phosphorylation abolishment due to staurosporine. (E) The ezrin-HA-pCMV/BifA-pAcGFP or HA-pCMV/pAcGFP plasmids were co-transfected into 293T cells for GFP-BifA and HA-ezrin expression. The cell lysate was immunoprecipitated with an anti-GFP antibody. Elution 1 and 2 were detected by immunoblot with anti-HA or anti-GFP antibodies to identify the co-precipitation of GFP-BifA and HA-ezrin. (F) The truncated ezrin proteins with different domains were co-expressed with BifA protein in 293T cells and immunoprecipitated by anti-HA or anti-GPF antibody. Western blot was used to detect the precipitated proteins and verify their interaction.
Figure 3
Figure 3
Rac1-GTP formation increased in BifA-treated dHL60 cells due to p-ezrin recruited Dbl. (A) Western blots were performed on lysates of dHL60 cells after BifA treatment. Total Rac1 and rhotekin protein precipitated GTP-bound Rac1 were detected with anti-Rac1 antibody. The graph shown below is normalized grayscale intensity analyses (measured with ImageJ software). (B) BifA-treated dHL60 cell lysate (input) was used to CO-IP with p-ezrin conjugated beads. Western blots were performed to detect the existence of Dbl and p-ezrin in the elution. (C) The dHL60 cell lysate was used as reaction system 1 (Rxn 1). Reaction system 2 (Rxn 2) was Dbl depleted dHL60 cell lysate generated with anti-Dbl antibody-conjugated beads for depletion. Ezrin, p-ezrin, Dbl in Rxn 1 and 2 were detected with immunoblot directly. The amount of Rho-GTP was measured by a pull-down method based on its specific binding to Rhotekin-RBD followed by immunoblot with an anti-Rac1 antibody. (D) Immunofluorescent staining of F-actin in dHL60 cells was observed by confocal microscopy. (Bar = 20 μm). The optical density of the scribed line in the picture was measured with Image J.
Figure 4
Figure 4
Appropriate concentration of Y27632 restored the dHL60 cells killing capability to SEZ. (A) The survival percentage of wild-type SEZ in dHL60 cells at different time points. With 1 nM Y27632, dHL60 killed more bacteria within 60 min than control group (n = 3, p values were calculated with Student’s t-test for each time point, * indicates p < 0.05; ** indicates p < 0.01; ns indicates no significant difference compare to control.). (B) In contrast, high concentration (10 nM, 50 nM and 100 nM) of Y27632 impaired dHL60 killing capability to wild-type SEZ. (n = 3, p values were calculated with one-way ANOVA for each time point, * indicates p < 0.05; **** indicates p < 0.0001; ns indicates no significant difference compare to control).
Figure 5
Figure 5
Schematic model of molecular mechanism of BifA decreases neutrophils killing capability to SEZ. BifA increases and keeps the ezrin aberrant phosphorylation level in a PKC-dependent manner. The p-ezrin recruits Dbl (guanine nucleotide-exchange factor) and promotes inactive GDP-bound Rac1 to transfer to active GTP-bound Rac1. The extraordinary activation of Rac1 may lead to phagocytosis dysfunction and impair other actin-dependent processes of dHL60 cells. The appropriate concentration of ROCK inhibitor Y27632 restores neutrophils function by maintaining Rho-ROCK pathway stability.
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