Porcine parvovirus (PPV) infection primarily causes reproductive failure of pregnant swine and results in host cell death. Boars, as an important disseminator, shed PPV to sows via semen. PPV infects and numerously replicates in boar testicle, which results in damage of swine testicle in vivo. Reactive oxygen species (ROS), a mediator of cell apoptosis, play a crucial role in the mitochondria apoptotic pathway. However, whether PPV infection induces ST cells apoptosis and ROS accumulation is still unclear. To determine the effects of PPV infection on the apoptosis, we detected morphological changes, DNA ladder, activities of caspases, and expression of PARP in PPV-infected ST cells. Moreover, aiming to investigate the effect of PPV infection on the mitochondrial apoptotic pathway and ROS accumulation, we detected the Δψm, apoptosis-related genes, and ROS. To investigate the role of ROS in the process of PPV-induced apoptosis, the ST cells were infected with PPV and treated with the ROS antioxidants. The ROS level was measured using Reactive Oxygen Species Assay Kit and the Δψm, expression level of Bcl-2, translocation of Bax, and redistribution of mitochondria cytochrome c were tested. In this study, we demonstrated that PPV infection could induce apoptosis that was characterized by morphological changes, DNA fragmentation and activation of caspases. Moreover, PPV infection suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, decreased the mitochondrial transmembrane potential, and triggered the release of cytochrome c, which caused the subsequent activation of caspase-9 and caspase-3 and initiation of apoptosis. However, during the process of PPV-induced apoptosis, the protein levels of Fas and FasL were not affected. Further studies showed that PPV infection caused ROS accumulation. Inhibition of ROS could reduce mitochondrial transmembrane potential and could significantly block ST cells apoptosis via suppressing Bax translocation, cytochrome c and caspase-3 activation. All these results suggest that PPV-induced ROS accumulation mediates apoptosis in ST cells, which provided theoretical basis for the molecular pathogenesis of PPV infection.

Transmissible gastroenteritis virus (TGEV), a member of the coronaviridae family, could cause fatal diarrhea of piglets and result in numerous economic losses. Previous studies demonstrated that TGEV infection could lead to mitochondrial damage and upregulate miR-4331 level. So miR-4331 may play an important regulatory role in the control of mitochondrial function. To explore the potential role of miR-4331 in mitochondrial damage, we adopted a strategy consisting of quantitative proteomic analysis of porcine kidney (PK-15) cells in response to miR-4331 and TGEV infection. Eventually, 69 differentially expressed proteins were gained. The target of miR-4331 was identified. The effects of miR-4331 and its target RB1 on mitochondrial Ca²⁺ level, mitochondrial membrane potential (MMP), interleukin-1 receptor accessory protein (IL1RAP), p38 MAPK signaling pathway were investigated. The results showed that miR-4331 elevated mitochondrial Ca²⁺ level, reduced MMP, targets Retinoblastoma 1 (RB1), upregulated IL1RAP, and induced activation of p38 MAPK pathway during TGEV infection. RB1 was identified as the direct targets of miR-4331 and downregulated IL1RAP, suppressed the activation of p38 MPAK, and attenuated TGEV-induced mitochondrial damage. In addition, IL1RAP played a positive role in activating p38 MAPK signaling and negative role in TGEV-induced mitochondrial damage. The data indicate that miR-4331 aggravates TGEV-induced mitochondrial damage by repressing expression of RB1, promoting IL1RAP, and activating p38 MAPK pathway.

Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family. Our previous research showed that TGEV infection could induce mitochondrial dysfunction and up-regulate miR-222 level. Therefore, we presumed that miR-222 might be implicated in regulating mitochondrial dysfunction induced by TGEV infection. To verify the hypothesis, the effect of miR-222 on mitochondrial dysfunction was tested and we showed that miR-222 attenuated TGEV-induced mitochondrial dysfunction. To investigate the underlying molecular mechanism of miR-222 in TGEV-induced mitochondrial dysfunction, a quantitative proteomic analysis of PK-15 cells that were transfected with miR-222 mimics and infected with TGEV was performed. In total, 4151 proteins were quantified and 100 differentially expressed proteins were obtained (57 up-regulated, 43 down-regulated), among which thrombospondin-1 (THBS1) and cluster of differentiation 47 (CD47) were down-regulated. THBS1 was identified as the target of miR-222. Knockdown of THBS1 and CD47 decreased mitochondrial Ca²⁺ level and increased mitochondrial membrane potential (MMP) level. Reversely, overexpression of THBS1 and CD47 elevated mitochondrial Ca²⁺ level and reduced mitochondrial membrane potential (MMP) level. Together, our data establish a significant role of miR-222 in regulating mitochondrial dysfunction in response to TGEV infection.

With the widespread promotion of the green feeding concept of “substitution and resistance”, there is a pressing need for alternative products in feed and breeding industries. Employing lactic acid bacteria represents one of the most promising antimicrobial strategies to combat infections caused by pathogenic bacteria. As such, we analyzed the intestinal tract of Anhui local pig breeds, including LiuBai Pig, YueHei Pig, and HuoShou Pig, to determine the composition and diversity of intestinal microbiota using 16S rRNA. Further, the functionality of the pigs’ intestinal microbiota was studied through metagenomic sequencing. This study revealed that lactic acid bacteria were the primary contributors to the functional composition, as determined through a species functional contribution analysis. More specifically, the functional contribution of lactic acid bacteria in the HuoShou Pig group was higher than that of the LiuBai Pig and YueHei Pig. Subsequently, the intestinal contents of the HuoShou Pig group were selected for the screening of the dominant lactic acid bacteria strains. Out of eight strains of lactic acid bacteria, the acid-production capacity, growth curve, and tolerance to a simulated intestinal environment were assessed. Additional assessments included surface hydrophobicity, the self-aggregation capability, co-agglutination of lactic acid bacteria with pathogenic bacteria, and an in vitro bacteriostatic activity assay. Lactobacillus johnsonii L5 and Lactobacillus reuteri L8 were identified as having a strong overall performance. These findings serve as a theoretical basis for the further development of pig-derived probiotics, thereby promoting the application of lactic acid bacteria to livestock production.

The type VI secretion system (T6SS) of avian pathogenic Escherichia coli (APEC) can affect the functions of eukaryotic cells by secreting or injecting effectors. Hemolysin co-regulatory protein (Hcp), one of the markers of the T6SS, is both a structural protein and an effector protein of the T6SS. According to previous studies, mitochondria in eukaryotic cells are targeted by pathogenic bacteria. However, little is known about the regulation of mitochondria in eukaryotic host cells by the T6SS effector protein Hcp of APEC. In our study, DF-1 cells co-incubated with Hcp2a protein for 6 h showed decreased mitochondrial membrane potential, increased Ca²⁺ concentration, and increased cellular reactive oxygen species (ROS) levels. We therefore conclude that Hcp2a protein causes dysfunction to mitochondria in DF-1 cells. To explain the mechanism that causes mitochondrial dysfunction, we reanalyzed the Hcp2a interaction protein dataset in DF-1 cells, and the Leucine zipper EF-hand-containing transmembrane protein 1 (LETM1), which is associated with mitochondria, was screened. The protein and molecular docking results showed that Hcp2a protein and LETM1 protein have better binding. Finally, subcellular localization results showed that Hcp2a was localized to mitochondria. In summary, Hcp2a effector proteins caused dysfunction to DF-1 cellular mitochondria, and we hypothesize that the interaction of Hcp2a protein with LETM1 protein induces mitochondrial dysfunction and promotes mitochondrial localization of Hcp2a in DF-1 cells.

The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin–biotin affinity pull-down assay combined with LC–MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein–protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells.