INTRODUCTION

Brucellosis, is a highly contagious disease caused by the Gram-negative intracellular bacteria of the genus Brucella. It is one of the zoonotic diseases which could seriously endanger the safety of public sanitation around the world. Brucella is mainly divided into six species: B. melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae [1]. Oral infection is the main route of transmission of brucellosis. Infected animals can carry bacteria for a long time or even for life. Pathogenic bacteria can be discharged from milk, feces, and urine. Especially when animals abort, a large number of pathogenic bacteria will be discharged, which will pollute grasslands, livestock houses, drinking water, and feed. The susceptible animals exposed to the above pollutants can be infected through the digestive tract, respiratory tract, and damaged skin and mucosa [2,3,4].

Brucella outer membrane protein (Omp) can be divided into three groups according to its molecular weight: the first group is 36–38 kDa, such as Omp10 and Omp19, the second group is 31–34 kDa, such as Omp2, the third group is 25–34 kDa, such as Omp25 and Omp31 [5, 6]. The main virulence factors of Brucella include lipopolysaccharide (LPS) and Omp. Studies have shown that Omp25 is the virulence factor and immunogen of Brucella, and has a critical role in the survival and persistent infection of Brucella. Omp25-mediated miRNAs differentially expressed in porcine and murine macrophages, which affected Brucella intracellular survival by downregulating tumor necrosis factor α (TNF-α) secretion [7]. Brucella Omp25 promotes the BV2 microglial cells secretion of inflammatory cytokines such as interleukin-6 (IL-6) and TNF-α, and inhibits its apoptosis [8]. Type IV secretory system (T4SS) mediates intracellular viability and chronic infection of Brucella by regulating the expression of effector proteins, T4SS affects Omp25/Omp31 family may provide an important sight for Brucella to adapt rapidly to the intracellular environment [9].

Brucella Omp25 upregulated programmed death-1 (PD-1) and miR-155, miR-23b, and miR-21-5p expression and downregulated their target gene interleukin-12 (IL-12), which provided a new mechanism for monocyte/macrophage dysfunction caused by Brucella infection [10]. We previously reported that four miRNAs (the upregulation of mmu-miR-146a-5p and mmu-miR-155-5p and downregulation of mmu-miR-149-3p and mmu-miR-5126) were confirmed, and the differentially expressed genes (DEGs) profile was revealed in RAW264.7 macrophage cells infected with Brucella melitensis Omp25 deletion mutant (∆Omp25 B. melitensis) [11].

Herein, we predicted the target genes of the upregulation of mmu-miR-146a-5p and mmu-miR-155-5p and downregulation of mmu-miR-149-3p and mmu-miR-5126. GO and KEGG were used for functional enrichment analysis of DEGs profile to reveal the autophagic pathway-associated genes. Venn analysis was performed to identify the autophagic pathway-associated miRNA-mRNA networks and qRT-PCR was used to validate. The autophagic pathway-associated networks of mmu-miR-149-3p-Ptpn5, mmu-miR-149-3p-Ppp2r3c, and mmu-miR-146a-5p-Dusp16 were identified in RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis.

MATERIALS AND METHODS

Cells

RAW264.7 macrophage cell line was purchased from Shanghai cell bank of Chinese Academy of Science, and cultured with Dulbecco’s Modified Eagle Medium (DMEM) (Life Technology, USA) containing 10% FBS (Gibco Company, USA), penicillin (100 U/ml) (Thermo Fisher Scientific, USA), and streptomycin (100 mg/ml) (Thermo Fisher Scientific, USA), at 5% CO2, 37 °C.

Reconstruction of ∆Omp25 B. melitensis and Infection

As previous study, to reconstruct B. melitensis M5-90 Omp25 gene deletion mutant (∆Omp25 B. melitensis) [11], briefly, we reconstructed suicide plasmids using suicide vector pGEM-7ZF(+), the Kanar gene fragment which replaced the coding region of Omp25 gene of B. melitensis M5-90, ensured that the reading frame remains unchanged, and reconstructed B. melitensis M5-90 Omp25 gene deletion (∆Omp25 B. melitensis) by homologous recombination.

RAW264.7 macrophage cells respectively infected with B. melitensis M5-90 and ∆Omp25 B. melitensis for 4 h according to our previous study [11]. And the total RNAs were extracted according to the purelink™ RNA mini kit (Life Technology, USA) instructions to validate the genes of the autophagic pathway-associated miRNA-mRNA networks.

Prediction Target Genes of the Confirmed Four miRNAs

Three online databases of TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/microrna/home.do), and PicTar (http://www.pictar.org/) were used to predict target genes of the confirmed mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-149-3p, and mmu-miR-5126. Potential target genes for each miRNAs were the intersection of three online database predictions as previously described [11].

GO and KEGG Functional Enrichment of the Differentially Expressed Genes (DEGs) Profile

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to functionally enrich the differentially expressed genes (DEGs) profile to identify the autophagic pathway-associated DEGs. GO is an internationally standardized classification system of gene functions. GO has three ontologies, which describe the molecular function of genes, cellular components, and biological processes. GO enrichment plot can visually show the percentage of target genes annotated to the same GO. GO enrichment scatter plot indicated for autophagy-related genes in DEGs. KEGG is the main public database for pathway, KEGG enrichment analysis for DEGs to further reveal the autophagic pathway-associated genes.

Venn Analysis and the Autophagic Pathway Associated miRNA-mRNA Networks Identification

The results of predicted target genes of the confirmed mmu-miR-146a-5p, mmu-miR-155-5p, mmu-miR-149-3p, and mmu-miR-5126 by three online databases, intersected the autophagic pathway-associated DEGs by GO and KEGG functional enrichment analysis to obtain the overlapped genes, which identified the autophagic pathway-associated miRNA and its potential target mRNA. miRNA-mRNA networks were drawn by cytoscape online software (http://www.cytoscape.org/).

qRT-PCR Validation for the Genes of the Autophagic Pathway-Associated miRNA-mRNA Networks

The high quality total RNAs of RAW264.7 macrophage cells-infected B. melitensis M5-90 and ∆Omp25 B. melitensis were used as the template to obtain cDNA according to the M-MLV first-strand synthesis kit (Life Technology, USA) instructions; the specific primers were designed and synthesized as shown in Table 2. qRT-PCR was used to validate the mRNAs of the autophagic pathway-associated miRNA-mRNA networks; GAPDH was used for an internal control. Each sample was repeated three times.

Statistical Analysis

The t test or one-way ANOVA was performed for statistical analysis of data. Independent sample t test was used for comparison within the group. One-way ANOVA was used for comparison between different groups. P < 0.05 was considered statistically significant. P < 0.01 was considered very statistically significant.

RESULTS

The Significantly Differentially Expressed Genes (DEGs) Profile of RAW264.7 Macrophage Cells Infected with ∆Omp25 B. melitensis

RAW264.7 macrophage cells were infected with B. melitensis M5-90 and ∆Omp25 B. melitensis, respectively. Total RNAs were extracted to perform the agilent transcriptome sequencing analysis experiment according to our pervious study. As shown in Fig. 1a, red dots represented the upregulated DEGs and green dots represented downregulated DEGs in RAW264.7 macrophage cells affected by ∆Omp25 B. melitensis infection. As shown in Fig. 1b, red dots represented the DEGs with log2 (fold change) (Omp25 vs M5-90) > 2.0, and green dots represented the DEGs with log2 (fold change) (Omp25 vs M5-90) < − 2.0 in RAW264.7 macrophage cells affected by ∆Omp25 B. melitensis infection.

Fig. 1
figure 1

The significantly differentially expressed genes (DEGs) profile of RAW264.7 macrophage cells infected with B. melitensis M5-90 and ∆Omp25 B. melitensis, respectively.a Scatter plot of DEGs. Red dots represented the upregulated DEGs, and green dots represented downregulated DEGs. b Volcano plot of DEGs. Red dots represented the DEGs with log2 (fold change) (Omp25 vs M5-90) > 2.0, and green dots represented the DEGs with log2 (fold change) (Omp25 vs M5–90) < − 2.0

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Venn Analysis Identified the Autophagic Pathway-Associated mRNAs in RAW264.7 Macrophage Cells Affected by ∆Omp25 B. melitensis Infection

The results of predicted target genes of the confirmed 4 miRNAs intersected the autophagic pathway-associated DEGs by GO and KEGG functional enrichment analysis. The overlapping part was the autophagic pathway-associated mRNAs in RAW264.7 macrophage cells affected by ∆Omp25 B. melitensis infection, As shown in Table 1 and Fig. 2, there were 6 genes, they were Dnase2b (deoxyribonuclease II beta), Mras (muscle and microspikes RAS), Ptpn5 (protein tyrosine phosphatase, non-receptor type 5), Dusp16 (dual specificity phosphatase 16), Nfkb1 (nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105), and Ppp2r3c (protein phosphatase 2, regulatory subunit B″, gamma).

Table 1 The autophagic pathway-associated mRNAs in RAW264.7 macrophage cells affected by ∆Omp25 B. melitensis infection by agilent transcriptome sequencing analysis
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Fig. 2
figure 2

Heat-map of the autophagic pathway-associated target mRNAs in RAW264.7 macrophage cells affected by ∆Omp25 B. melitensis infection. Group1 represented the three times repeated samples of RAW264.7 macrophage cells infected with B. melitensis M5-90, and Group2 represented the three times repeated samples of RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis, respectively.

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Bioinformatics Analysis of the Autophagic Pathway-Associated miRNA-mRNA Networks in RAW264.7 Macrophage Cells Infected with ∆Omp25 B. melitensis

Venn analysis results showed that there were six mRNAs-associated autophagic pathway, and the six mRNAs were the target genes of two miRNAs. miRNA-mRNA networks were drawn by cytoscape online software. The results of Fig. 3 indicated that there were 5 miRNA-mRNA network-associated autophagic pathways in RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis; they were mmu-miR-149-3p-Mars, mmu-miR-149-3p-Ptpn5, mmu-miR-149-3p-Ppp2r3c, mmu-miR-146a-5p-Dnase2b, and mmu-miR-146a-5p-Dusp16.

Fig. 3
figure 3

The autophagic pathway-associated miRNA-mRNA networks by bioinformatics analysis in RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis. Green arrow represented downregulated miRNAs, and the red dot represented their target genes associated with the autophagy pathway in RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis.

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qRT-PCR Validation and Identification the Autophagic Pathway-Associated miRNA-mRNA Networks in RAW264.7 Macrophage Cells Infected with ∆Omp25 B. melitensis

Specific primers were designed, as shown in Table 2, qRT-PCR was used to validate the relative expression level of the mRNA of the autophagic pathway-associated miRNA-mRNA networks. The confirmed mRNAs were Ptpn5, Dusp16, and Ppp2r3c, which demonstrated that the autophagic pathway-associated mmu-miR-149-3p-Ptpn5, mmu-miR-149-3p-Ppp2r3c, and mmu-miR-146a-5p-Dusp16 networks were identified in RAW264.7 macrophage cells infected with ∆Omp25 B. melitensis, as shown in Fig. 4.

Table 2 The primers designed for qRT-PCR validation for mRNAs of the autophagic pathway-associated miRNA-mRNA networks
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Fig. 4
figure 4

qRT-PCR validated the autophagy pathway-associated target mRNAs of the miRNA-mRNA networks

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DISCUSSION

Brucella is a Gram-negative intracellular bacteria, which can enter, survive, and proliferate in a variety of host cell types including macrophages and dendritic cells [12]. Brucella controls the transport of intracellular membrane, thus promoting its survival and growth in cells. When Brucella is absorbed by phagocytosis, it attaches to cell membrane and forms the Brucella-containing vacuole (BCV), which spreads along the endocytosis pathway in the first 8 h after infection and has limited fusion with lysosomes [13]. Although, the intracellular cycle of Brucella is replicated from the endoplasmic reticulum (ER) within 12 to 24 h postinfection [14,15,16,17,18]. However, the mechanism of intracellular parasitic reproduction and the process of autophagy mediated by Brucella are still unclear.

Omp25 is an important virulent protein and immunogen of Brucella. It has been reported in mice as an antigen to protect Brucella infection by inducing Th1 and Th2 immune responses. Studies have shown that the complex of Omp25 and other proteins, as a prospective and effective immunogen, can induce cell-mediated and humoral immune responses to brucellosis [19]. Omp25 affects the structural stability of Brucella and plays an important role in the activation of mitogen-activated protein kinase (MAPK) signal pathway by Brucella [20]. B. melitensis M5-90 can cause abortion of pregnant female and livestock, and its virulence is still strong. Maybe the ∆Omp25 B. melitensi-deleted strain will have more prospects and value for vaccine application.

There are few reports that Brucella Omp25 affects autophagy of macrophages or other target cells. We used bioinformatics software to analyze and predict the effect of Brucella Omp25 on the miRNA-mRNA regulatory networks associated with RAW264.7 macrophage cells autophagy. mmu-miR-149-3p-Ptpn5, mmu-miR-149-3p-Ppp2r3c, and mmu-miR-146a-5p-Dusp16 networks were identified. Ptpn5 plays an important role in neurodegenerative diseases and neuropsychiatric diseases, and is an attractive new target for the treatment of these diseases [21]. Ppp2r3c is a regulatory subunit of protein phosphatase; its dephosphorylation can bind to P-glycoprotein and affect the drug resistance of tumors [22]. Mycobacterium tuberculosis initiates the inhibition of c-Jun N-terminal kinases (JNK)-dependent autophagy by acetylating Dusp16/mitogen-activated protein kinase phosphatase-7 (MKP-7), which mediates the host immune response and improves the survival rate of mycobacterium in macrophages [23]. During the cold storm, the subcutaneous fat bank initiated miR-149-3p targeting regulation of PR domain containing 16 (PRDM16) expression to preserve energy and maintain balance [24]. mmu-miR-146a-5p plays an important regulatory role in aspergillus-induced central nervous system inflammation [25].

For the first time, we have identified three miRNA-mRNA networks associated with the autophagic pathway of macrophage cells infected with ∆Omp25 B. melitensis. Although bioinformatics analysis and prediction are false positive, follow-up double-luciferase reporter gene experiments should further validate these three networks. Our findings not only lay a foundation for further elucidating the molecular mechanism of Brucella Omp25 affecting macrophage autophagy, but also provide a new way for the prophylaxis and treatment of brucellosis.