Neospora caninum infection induced mitochondrial dysfunction in caprine endometrial epithelial cells via downregulating SIRT1

Abstract

Background: Infection of Neospora caninum, an important obligate intracellular protozoan parasite, causes reproductive dysfunctions (e.g. abortions) in ruminants (e.g. cattle, sheep and goats), leading to serious economic losses of livestock worldwide, but the pathogenic mechanisms of N. caninum are poorly understood. Mitochondrial dysfunction has been reported to be closely associated with pathogenesis of many infectious diseases. However, the effect of N. caninum infection on the mitochondrial function of hosts remains unclear.

Methods: The effects of N. caninum infection on mitochondrial dysfunction in caprine endometrial epithelial cells (EECs), including intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) contents, mitochondrial DNA (mtDNA) copy numbers and ultrastructure of mitochondria, were studied by using JC-1, DCFH-DA, ATP assay kits, quantitative real-time polymerase chain reaction (RT-qPCR) and transmission electron microscopy, respectively, and the regulatory roles of sirtuin 1 (SIRT1) on mitochondrial dysfunction, autophagy and N. caninum propagation in caprine EECs were investigated by using two drugs, namely resveratrol (an activator of SIRT1) and Ex 527 (an inhibitor of SIRT1).

Results: The current study found that N. caninum infection induced mitochondrial dysfunction of caprine EECs, including accumulation of intracellular ROS, significant reductions of MMP, ATP contents, mtDNA copy numbers and damaged ultrastructure of mitochondria. Downregulated expression of SIRT1 was also detected in caprine EECs infected with N. caninum. Treatments using resveratrol and Ex 527 to caprine EECs showed that dysregulation of SIRT1 significantly reversed mitochondrial dysfunction of cells caused by N. caninum infection. Furthermore, using resveratrol and Ex 527, SIRT1 expression was found to be negatively associated with autophagy induced by N. caninum infection in caprine EECs, and the intracellular propagation of N. caninum tachyzoites in caprine EECs was negatively affected by SIRT1 expression.

Conclusions: These results indicated that N. caninum infection induced mitochondrial dysfunction by downregulating SIRT1, and downregulation of SIRT1 promoted cell autophagy and intracellular proliferation of N. caninum tachyzoites in caprine EECs. The findings suggested a potential role of SIRT1 as a target to develop control strategies against N. caninum infection.

Keywords: Autophagy; Caprine endometrial epithelial cells; Mitochondrial dysfunction; Neospora caninum; Propagation of parasite; Sirtuin 1.

Fig. 1

Mitochondrial dysfunction in caprine endometrial epithelial cells (EECs) induced by Neospora caninum infection. a, b ROS levels investigated by a DCFH-DA fluorescent probe in caprine EECs infected with N. caninum tachyzoites at a multiplicity of infection (MOI) of 3:1 (parasite:cell) for 48 h. Scale bar, 50 µm. c, d The mitochondrial membrane potential (MMP) detected by JC-1 in caprine EECs at 48 h post infection. Scale bar, 50 µm. e ATP contents detected by an ATP Determination Kit in caprine EECs at 48 h. f Mitochondrial DNA (mtDNA) copy numbers detected by qPCR in caprine EECs at 48 h. g Mitochondrial ultrastructure morphology in caprine EECs detected at 48 h by using transmission electron microscopy (TEM). Scale bar, 1 µm. Data are shown as mean ± standard deviation (SD) of three independent experiments. P-values were calculated using Student’s t test. **P < 0.01; ***P < 0.001

Fig. 2

Expression analysis of Sirtuin 1 (SIRT1) in caprine endometrial epithelial cells (EECs) infected with Neospora caninum. a The mRNA levels of SIRT1 determined by RT-qPCR. The protein levels of SIRT1 determined by western blotting. RT-qPCR (a) and western blotting (b–e) were performed to detect the expression of SIRT1 in caprine EECs infected with N. caninum tachyzoites at a multiplicity of infection (MOI) of 3:1 (parasite:cell) for 48 h. Additionally, the protein levels were determined in caprine EECs pre-treated with 50 μM resveratrol (RSV) (b, c) or 20 μM Ex 527 (d, e) for 1 h and then infected with N. caninum tachyzoites at MOI of 3:1 (parasite:cell) for 48 h. Data are shown as mean ± standard deviation (SD) of three independent experiments. P-values were calculated using Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

Effect of Sirtuin 1 (SIRT1) on Neospora caninum-induced mitochondrial dysfunction in caprine endometrial epithelial cells (EECs). a, b ROS levels detected by a DCFH-DA fluorescent probe. Scale bar, 50 µm. c, d The mitochondrial membrane potential (MMP) detected by JC-1. Scale bar, 50 µm. e ATP contents detected by an ATP Determination Kit. f Mitochondrial DNA (mtDNA) copy numbers detected by qPCR. The ROS levels, mitochondrial membrane potential, ATP levels and mtDNA copy numbers were investigated in caprine EECs pre-treated with 50 μM resveratrol (RSV) or 20 μM Ex 527 for 1 h and then infected with N. caninum tachyzoites at a multiplicity of infection (MOI) of 3:1 (parasite:cell) for 48 h. Data are shown as mean ± standard deviation (SD) of three independent experiments. P-values were calculated using Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001. NS no significant difference was observed

Fig. 4

Effect of Sirtuin 1 (SIRT1) on Neospora caninum-induced autophagy in caprine endometrial epithelial cells (EECs). a–d The protein levels of LC-3II determined by Western blotting. e–h The protein levels of p62 determined by western blotting. Caprine EECs were pre-treated with 50 μM resveratrol (RSV) (a, b, e, f) or 20 μM Ex 527 (c, d, g, h) for 1 h and then infected with N. caninum tachyzoites at a multiplicity of infection (MOI) of 3:1 (parasite:cell) for 48 h. Data are shown as mean ± standard deviation (SD) of three independent experiments. P-values were calculated using Student’s t test. *P < 0.05. NS no significant difference was observed

Fig. 5

Intracellular propagation of Neospora caninum tachyzoites in caprine endometrial epithelial cells (EECs). Caprine EECs were pre-treated with 10, 20 and 50 μM resveratrol (RSV, a, b) or 5, 10 or 20 µM Ex 527 (c, d) for 1 h and then infected with N. caninum tachyzoites at a multiplicity of infection (MOI) of 3:1 (parasite:cell) for 48 h. The numbers of N. caninum tachyzoites of vacuoles were counted by randomly selecting 100 parasitophorous vacuoles. Three independent experiments were conducted in triplicate. Data are shown as mean ± standard deviation (SD) of three independent experiments. P-values were calculated using Student’s t test. *P < 0.05. NS no significant difference was observed