Crosstalk between integrin/FAK and Crk/Vps25 governs invasion of bovine mammary epithelial cells by S. agalactiae

Abstract

Streptococcus agalactiae (S. agalactiae) is a contagious obligate parasite of the udder in dairy cows. Here, we examined S. agalactiae-host interactions in bovine mammary epithelial cells (BMECs) in vitro. We found that S. agalactiae infected BMECs through laminin β2 and integrin. Crk, Vps25, and RhoA were differentially expressed in S. agalactiae-infected cells. S. agalactiae infection activated FAK and Crk. FAK deficiency decreased the number of intracellular S. agalactiae and Crk activation. Knockdown of Crk or Vps25 increased the level of intracellular S. agalactiae, whereas its overexpression had the opposite effect. RhoA expression and actin cytoskeleton were altered in S. agalactiae-infected BMECs. Crk and Vps25 interact in cells, and invaded S. agalactiae also activates Crk, allowing it to cooperate with Vps25 to defend against intracellular infection by S. agalactiae. This study provides insights into the mechanism by which intracellular infection by S. agalactiae is regulated in BMECs.

Keywords: Bacteriology; Cell biology; Genetics.

Graphical abstract

Figure 1

S. agalactiae invades BMECs S. agalactiae was stained with CFSE (green) and used to infect BMECs at an MOI of 100 for 2 h, and the S. agalactiae-infected BMECs were maintained in culture medium with antibiotics and lysozyme to kill and lyse the extracellular bacteria. Nuclei were costained with DAPI (blue), and actin was stained with Alexa Fluor 594 Phalloidin (red), and cells were observed by laser scanning confocal microscopy. Scale bars represent 20 μm n = 3 independent experiments.

Figure 2

Laminin β2 mediates invasion of BMECs by S. agalactiae (A) Intracellular bacterial count in LAMBP2-silenced BMECs infected with S. agalactiae for 2 h at MOI 100. (B) Intracellular bacterial count in BMECs incubated with anti-laminin β2 and infected with S. agalactiae. ∗p < 0.05, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.

Figure 3

Integrin mediates invasion of BMECs by S. agalactiae and FAK activation (A) BMECs were pretreated with 5 μM RGD for 24 h and infected with S. agalactiae for 2 h at MOI 100. (B) Silencing of ITGB1 impairs S. agalactiae infection. (C) S. agalactiae infection enhances FAK phosphorylation (Tyr397), whereas RGD inhibits it. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.

Figure 4

Verification of differential expressed proteins in S. agalactiae-infected BMECs identified by comparative proteomic analysis Crk, Vps25, and RhoA were examined by western blot in S. agalactiae-infected BMECs. ∗p < 0.05, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.

Figure 5

TAE226 inhibits S. agalactiae internalization and enhances Crk phosphorylation in BMECs BMECs were pretreated with TAE226 for 24 h and infected with S. agalactiae for 2 h at MOI 100. (A) Colony count of S. agalactiae. (B) Western blot of phosphorylation of FAK at Tyr397 and Crk at Tyr221. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.

Figure 6

Knockdown of FAK decreases the number of intracellular S. agalactiae and enhances the phosphorylation of Crk in MCF-10-A cells FAK was silenced by a targeting shRNA in MCF-10A nonneoplastic breast epithelial cells, and the cells were infected with S. agalactiae for 2 h at MOI 100. (A) Knockdown of FAK decreases the number of S. agalactiae in MCF-10A cells compared with control. (B) Knockdown of FAK enhances the phosphorylation of Crk at Tyr221 and alters the expression of RhoA in MCF-10A cells. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.

Figure 7

Crk prevents S. agalactiae invasion and regulates the expression of Vps25 and RhoA in BMECs Crk was silenced or overexpressed in BMECs, and the cells were infected with S. agalactiae for 2 h at MOI 100. (A and B) (A) Knockdown of Crk increases the number of intracellular S. agalactiae in BMECs, whereas its overexpression decreases it compared with control (B). (C) Knockdown of Crk downregulates Vps25 and RhoA. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.

Figure 8

Vps25 protects against S. agalactiae invasion and cooperates with Crk in BMECs Vps25 was silenced or overexpressed in BMECs, and the cells were infected with S. agalactiae for 2 h at MOI 100. (A and B) (A) Knockdown of Vps25 increases the number of intracellular S. agalactiae in BMECs, whereas its overexpression decreases it, compared with control (B). (C) Knockdown of Vps25 decreases the expression of RhoA. (D) Coimmunoprecipitation of Vps25 and Crk with anti-Vps25. N1 denotes no antibody, and N2 is anti-his. Total protein lysates of BMECs were used as a positive control. ∗p < 0.05, ∗∗p < 0.01, one-way ANOVA followed by Tukey’s method. n = 3 independent experiments. Error bar indicates SD.