Neospora caninum infection specifically suppresses the expression of a host lncRNA XR_001919077.1 to facilitate parasite propagation by modulating host cell mitochondrial function and autophagy

Abstract

Neospora caninum is one of the most common pathogens causing reproductive failure in ruminants (e.g., cattle and goats) worldwide. However, due to a poor understanding of the pathogenic mechanisms of N. caninum infection, no effective drugs and vaccines are currently available. Long non-coding RNAs (lncRNAs) have been reported to be important regulators involved in a great number of physiological and pathological processes. Our previous study found that N. caninum infection induced significantly aberrant expression of lncRNA profiles in caprine endometrial epithelial cells (EECs). In the present study, we found that N. caninum infection specifically suppressed the expression of a novel lncRNA, XR_001919077.1, and knockdown of XR_001919077.1 with small interfering RNA significantly promoted the propagation of N. caninum in caprine EECs. Rapid amplification of cDNA ends analysis generated six splice variants of XR_001919077.1, with lengths ranging from 592 to 694 nt. Transfection of the full length of each variant markedly inhibited the propagation of N. caninum in caprine EECs. Further study suggested that XR_001919077.1 acted as a sponge of Chi-miR-93-5p to promote the expression of sirt1, and the XR_001919077.1/Chi-miR-93-5p/sirt1 axis significantly delayed the in vitro growth of N. caninum in caprine EECs by regulating host cell mitochondrial function and autophagy. Our findings provide a novel insight to understand the interactions between N. caninum and host cells.IMPORTANCEThe uterus is an indispensable reproductive organ for embryo implantation and fetal growth. The endometrium is more vulnerable to infection by pathogenic microorganisms resulting in an increased risk of miscarriage. Neospora caninum is one of the most common pathogens causing miscarriage in ruminants and is able to naturally inhabit the uterus, with N. caninum tissue cysts found in the endometrium. Recent advances in N. caninum research have revealed aberrant expression of long non-coding RNA (lncRNA) profiles in infected caprine endometrial epithelial cells. In the present study, N. caninum, but not Toxoplasma gondii, which has similar morphological and biological features to N. caninum, specifically suppresses the expression of a host lncRNA, XR_ 001919077.1, to impair host's defense through the competitive endogenous RNA mechanism to modulate the host cell mitochondrial function and autophagy to facilitate parasite propagation. The findings suggest a novel immune evasion strategy of N. caninum to facilitate intracellular propagation and provide an alternative path to develop control strategies against neosporosis.

Keywords: Neospora caninum; XR_001919077.1; autophagy; mitochondrial function; propagation.

Fig 1

XR_001919077.1 is negatively correlated with the propagation of Neospora caninum in caprine endometrial epithelial cells. (A) The expression of XR_001919077.1 in caprine EECs during N. caninum infection for RT-qPCR. (B) The expression of XR_001919077.1 in caprine EECs infected with different MOIs (parasite:cell = 0:1, 1:1, 2:1, 3:1) of N. caninum tachyzoites at 48 h post infection (hpi) for RT-qPCR. (C) Interference efficiencies of two small interfering RNAs (si-XR_001919077.1–1 and si-XR_001919077.1–2) against XR_001919077.1 during N. caninum infection at 48 hpi for RT-qPCR. (D and E) Effect of knockdown of XR_001919077.1 on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (D) and 42 hpi (E). (F) Effect of knockdown of XR_001919077.1 on the number of N. caninum in 200 ng DNA in infected caprine EECs at 48 hpi using qPCR. (G and H) Effect of overexpression of each XR_001919077.1 variant on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (G) and 42 hpi (H). (I) Effect of overexpression of each XR_001919077.1 variant on the number of N. caninum in 200 ng DNA in infected caprine EECs at 48 hpi using qPCR. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. TZ represents the tachyzoite.

Fig 2

XR_001919077.1 localizes in both cytoplasm and nucleus in caprine endometrial epithelial cells. The nucleus was stained with DAPI. Scale bar: 10 µm.

Fig 3

XR_001919077.1 functions as a sponge for Chi-miR-93-5p in caprine endometrial epithelial cells infected with N. caninum. (A) The expression of Chi-miR-93-5p in caprine EECs infected with N. caninum by overexpression of each XR_001919077.1 variant at 48 h post-infection (hpi). (B) The expression of Chi-miR-93-5p in caprine EECs infected with N. caninum by interference with si-XR_001919077.1-2 at 48 hpi. (C) Potential binding sites between XR_001919077.1 and Chi-miR-93-5p predicted by miRanda database. (D) Sponging relationship between XR_001919077.1 and Chi-miR-93-5p identified using the dual-luciferase reporter assay. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05 and **P < 0.01.

Fig 4

Chi-miR-93-5p targets sirt1 in caprine endometrial epithelial cells infected with N. caninum. (A) mRNA level of sirt1 analyzed in caprine EECs infected with N. caninum tachyzoites from 6 to 48 h post infection (hpi) by RT-qPCR. (B) Protein level of sirt1 analyzed in caprine EECs infected with N. caninum tachyzoites from 6 to 48 hpi by western blotting. Relative protein levels of SIRT1 compared to β-actin were determined by densitometry. (C) Potential binding sites between Chi-miR-93-5p and 3′-UTR of sirt1. (D) Target relationship between sirt1 and Chi-miR-93-5p identified using the dual-luciferase reporter assay. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05 and **P < 0.01. TZ represents the tachyzoite.

Fig 5

XR_001919077.1 promotes the expression of sirt1 by sponging Chi-miR-93-5p in caprine endometrial epithelial cells following N. caninum infection. (A) The expression of the SIRT1 protein analyzed in caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor during N. caninum infection at 48 h post-infection (hpi) using western blotting. (B) The expression of the SIRT1 protein analyzed in caprine EECs transfected with recombinant plasmids of six XR_001919077.1 variants during N. caninum infection at 48 hpi using western blotting. (C) The expression of the SIRT1 protein analyzed in caprine EECs transfected with si-XR_001919077.1–2 during N. caninum infection at 48 hpi using western blotting. (D) The expression of the SIRT1 protein analyzed in caprine EECs co-transfected with recombinant plasmid of each of six XR_001919077.1 variants and Chi-miR-93-5p mimics during N. caninum infection at 48 hpi using western blotting. Relative protein level of SIRT1 compared to β-actin was determined by densitometry. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 6

The XR_001919077.1/Chi-miR-93-5p/sirt1 axis delays the propagation of N. caninum tachyzoites in caprine endometrial epithelial cells. (A and B) Effect of Chi-miR-93-5p mimics or inhibitor on the propagation of N. caninum tachyzoites in caprine EECs at 30 h post infection (hpi) (A) and 42 hpi (B). (C) The number of N. caninum in 200 ng DNA in infected caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor at 48 hpi using qPCR. (D and E) Effect of pcDNA3.1 (+)-sirt1 or si-sirt1 on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (D) and 42 hpi (E). (F) The number of N. caninum in 200 ng DNA in infected caprine EECs transfected with pcDNA3.1 (+)-sirt1 or si-sirt1 at 48 hpi using qPCR. (G and H) Effect of co-transfection of Chi-miR-93-5p mimics and each of the six XR_001919077.1 variants, or pcDNA3.1(+)-sirt1 on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (G) and 42 hpi (H). Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 7

The XR_001919077.1/Chi-miR-93-5p/sirt1 axis affects the ROS levels and MMP levels in caprine EECs following N. caninum infection. (A and B) ROS levels (A) and MMP levels (B) determined in caprine EECs transfected with recombinant plasmids of six XR_001919077.1 variants during N. caninum infection at 48 h post-infection (hpi). (C and D) ROS levels (C) and MMP levels (D) determined in caprine EECs transfected with si-XR_001919077.1-2 during N. caninum infection at 48 hpi. (E and F) ROS levels (E) and MMP levels (F) determined in caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor during N. caninum infection at 48 hpi. (G and H) ROS levels (G) and MMP levels (H) determined in caprine EECs transfected with pcDNA3.1 (+)-sirt1 or si-sirt1 during N. caninum infection at 48 hpi. Data were obtained in triplicate and were analyzed using the Student’s t test. Scale bar: 200 µm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 8

The XR_001919077.1/Chi-miR-93-5p/sirt1 axis affects the adenosine triphosphate (ATP) contents and mtDNA copy numbers in caprine endometrial epithelial cells following N. caninum infection. (A and B) ATP contents (A) and mtDNA copy numbers (B) determined in caprine EECs transfected with recombinant plasmids of six XR_001919077.1 variants during N. caninum infection at 48 h post-infection (hpi). (C and D) ATP contents (C) and mtDNA copy numbers (D) determined in caprine EECs transfected with si-XR_001919077.1-2 during N. caninum infection at 48 hpi. (E and F) ATP contents (E) and mtDNA copy numbers (F) determined in caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor during N. caninum infection at 48 hpi. (G and H) ATP contents (G) and mtDNA copy numbers (H) determined in caprine EECs transfected with pcDNA3.1 (+)-sirt1 or si-sirt1 during N. caninum infection at 48 hpi. Data were obtained in triplicate and were analyzed using the Student’s t test. Scale bar: 200 µm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 9

The XR_001919077.1/Chi-miR-93–5p/sirt1 axis affects the expression of the LC3B protein and p62 protein in caprine endometrial epithelial cells following N. caninum infection. (A–D) The protein levels of LC3B determined in caprine EECs transfected with recombinant plasmids of the six XR_001919077.1 variants (A), si-XR_001919077.1-2 (B), Chi-miR-93-5p mimics or inhibitor (C), pcDNA3.1 (+)-sirt1, or si-sirt1 (D) during N. caninum infection at 48 h post-infection (hpi) by western blotting. (E–H) The protein levels of p62 determined in caprine EECs transfected with recombinant plasmids of the six XR_001919077.1 variants (E), si-XR_001919077.1–2 (F), Chi-miR-93-5p mimics or inhibitor (G), pcDNA3.1 (+)-sirt1, or si-sirt1 (H) during N. caninum infection at 48 hpi by western blotting. The relative protein levels of LC3-II (A–D) or p62 (E–H) compared to β-actin were determined by densitometry. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 10

A hypothetical mechanism graph of XR_001919077.1/Chi-miR-93-5p/sirt1 axis regulates the propagation of N. caninum in vitro. N. caninum infection significantly downregulates the expression of XR_001919077.1 and sirt1 but upregulates the expression of Chi-miR-93-5p. XR_001919077.1 promotes the expression of sirt1 by sponging Chi-miR-93-5p in caprine EECs infected with N. caninum. The XR_001919077.1/Chi-miR-93-5p/sirt1 axis negatively affects the propagation of N. caninum tachyzoites in vitro by regulating mitochondrial function and autophagy.