Proteomics Reveals the Response Mechanism of Embryonic Bovine Lung Cells to Mycoplasma bovis Infection

Abstract

Mycoplasma bovis (M. bovis) has caused huge economic losses to the cattle industry. The interaction between M. bovis and host cells is elucidated by screening and identifying the target protein of M. bovis adhesin on the surface of the host cell membrane. However, the response mechanism of embryonic bovine lung (EBL) cells to M. bovis infection is not yet fully understood. Additionally, it is necessary to further explore whether infection with M. bovis induces oxidative stress and mitochondrial damage in EBL cells. In this study, oxidation reaction, mitochondrial membrane potential, mitochondrial structure, and apoptosis ability of EBL cells infected with M. bovis were assessed at different times (12, 24, 48 h post-infection; hpi). Then, the differential proteomic analysis of M. bovis-infected EBL cells at 12 h and 24 h was performed with uninfected cells as the control. The results showed that M. bovis infection reduced the antioxidant capacity of EBL cells, increased ROS levels, and decreased mitochondrial membrane potential. The mitochondrial membrane of EBL cells was damaged, and the ridge arrangement was disordered after infection by transmission electron microscopy. With the increase in infection time, the mitochondrial matrix partially dissolved and spilled. The apoptosis rate of EBL cells increased with the increase in infection time of M. bovis. Furthermore, proteomic analysis identified 268 and 2061 differentially expressed proteins (DEPs) at 12 hpi and 24 hpi, respectively, compared with the uninfected cells. According to GO analysis, these DEPs were involved in the mitosis and negative regulation of cell growth. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to mitochondrial damage or cell growth regulation, including glycolysis/gluconeogenesis, pentose phosphate pathway, oxidative phosphorylation, AMPK, cGMP-PKG, cAMP, calcium, Wnt, Phospholipase D, apoptosis, MAPK, cell cycle, Ras, PI3K-Akt, mTOR, HIF-1. PPI results indicated that YWHAZ, PIK3CA, HSP90AB1, RAP1A, TXN, RAF1, MAPK1, PKM, PGK1, and GAPDH might be involved in mitochondrial pathway apoptosis induced by M. bovis infection. This study offers helpful data toward understanding the response of mitochondria of EBL cells to M. bovis infection.

Keywords: Mycoplasma bovis; embryonic bovine lung cells; mitochondrial damage; proteomics; signaling pathway.

Figure 1

The survival rate in EBL cells were infected with different MOI of M. bovis at different times (24 h, 48 h, 72 h). Data are presented as mean ± standard deviation (n = 3). The figure contains different lowercase letters, indicating significant differences under different concentration treatment levels at the same time (p < 0.05). The figure contains different uppercase letters indicating significant differences at different times under the same concentration treatment level (p < 0.05).

Figure 2

The related indexes of oxidative stress in EBL cells after 12 h, 24 h, and 48 h infection with M. bovis. (A) CAT levels at different time periods (12 hpi, 24 hpi, 48 hpi). (B) SOD levels at different time periods (12 hpi, 24 hpi, 48 hpi). (C) LDH levels at different time periods (12 hpi, 24 hpi, 48 hpi). (D) ROS level was detected by DCFH-DA staining fluorescence microscopy at different time periods (12 hpi, 24 hpi, 48 hpi). (E) Green fluorescence intensity was analyzed by image J. Data are presented as mean ± standard deviation (n = 3). Infection group compared with control group, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Effects of M. bovis on mitochondrial membrane potential in EBL cells. (A) EBL cells infected with M. bovis were collected, and the mitochondrial membrane potential was evaluated by JC-1 at different time periods (12 hpi, 24 hpi, 48 hpi). Aggregate: polymer (red light). Monomer; JC-1 monomer (green light). (B) Fluorescence intensity was analyzed by image J. Data are presented as mean ± standard deviation (n = 3). Infection group compared with control group, ns p > 0.05, * p < 0.05, *** p < 0.001.

Figure 4

Effect of M. bovis on mitochondrial morphology in EBL cells. EBL cells infected with M. bovis at different time periods were collected and mitochondrial morphology was observed by transmission electron microscopy. (A,E) Mitochondrial morphology of uninfected group. (B,F) Mitochondrial morphology at 12 hpi. (C,G) Mitochondrial morphology at 24 hpi. (D,H) Mitochondrial morphology at 48 hpi (A–D) scale bar = 2 µm. (E–H) scale bar = 500 nm.

Figure 5

The apoptosis rate in EBL cells after 12 h, 24 h, and 48 h infection with M. bovis. (A) Apoptosis rate was detected by flow cytometry at 12 hpi, 24 hpi, and 48 hpi. (a) The apoptosis rate of uninfected EBL cells. (b–d) The apoptosis rates of M. bovis-infected cells at different time periods (12, 24, and 48 h). (B) The percentage of M. bovis-induced apoptosis in EBL cells with different times (12 hpi, 24 hpi, 48 hpi). (C) Apoptosis-related proteins were detected by Western Blotting at 12 hpi, 24 hpi, and 48 hpi. Data are the mean ± standard deviation of three independent experiments. Infection group compared with control group, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Proteomic data analysis. (A) Volcano map shows the DEPs at 12 hpi group. (B) Volcano map shows the DEPs at 24 hpi group. Blue-colored plots illustrate upregulated proteins, red-colored plots illustrate down-regulated proteins and green plots reflect proteins that did not show changes in expression. (C) Venn map shows the common up-down-regularized DEPs at 12 hpi and 24 hpi. (D) GO functional enrichment pathway of 12 hpi. (E) GO functional enrichment pathway of 24 hpi.

Figure 7

The enrichment analysis of selected DEPs in EBL cells infected with M. bovis. (A) The KEGG enrichment analysis of selected DEPs at 12 hpi. (B) The KEGG enrichment analysis of selected DEPs at 24 hpi. (C) The interaction network map of important DEPs. (D) The heatmap of common DEPs cluster analysis.