The VirB System Plays a Crucial Role in Brucella Intracellular Infection.

Brucellosis is a highly prevalent zoonotic disease caused by Brucella. Brucella spp. are gram-negative facultative intracellular parasitic bacteria. Its intracellular survival and replication depend on a functional virB system, an operon encoded by VirB1-VirB12. Type IV secretion system (T4SS) encoded by the virB operon is an important virulence factor of Brucella. It can subvert cellular pathway and induce host immune response by secreting effectors, which promotes Brucella replication in host cells and induce persistent infection. Therefore, this paper summarizes the function and significance of the VirB system, focusing on the structure of the VirB system where VirB T4SS mediates biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV), the effectors of T4SS and the cellular pathways it subverts, which will help better understand the pathogenic mechanism of Brucella and provide new ideas for clinical vaccine research and development.

The Mechanism of Facultative Intracellular Parasitism of Brucella.

Brucellosis is a highly prevalent zoonotic disease characterized by abortion and reproductive dysfunction in pregnant animals. Although the mortality rate of Brucellosis is low, it is harmful to human health, and also seriously affects the development of animal husbandry, tourism and international trade. Brucellosis is caused by Brucella, which is a facultative intracellular parasitic bacteria. It mainly forms Brucella-containing vacuoles (BCV) in the host cell to avoid the combination with lysosome (Lys), so as to avoid the elimination of it by the host immune system. Brucella not only has the ability to resist the phagocytic bactericidal effect, but also can make the host cells form a microenvironment which is conducive to its survival, reproduction and replication, and survive in the host cells for a long time, which eventually leads to the formation of chronic persistent infection. Brucella can proliferate and replicate in cells, evade host immune response and induce persistent infection, which are difficult problems in the treatment and prevention of Brucellosis. Therefore, the paper provides a preliminary overview of the facultative intracellular parasitic and immune escape mechanisms of Brucella, which provides a theoretical basis for the later study on the pathogenesis of Brucella.

The mechanism of chronic intracellular infection with Brucella spp.

Globally, brucellosis is a widespread zoonotic disease. It is prevalent in more than 170 countries and regions. It mostly damages an animal's reproductive system and causes extreme economic losses to the animal husbandry industry. Once inside cells, Brucella resides in a vacuole, designated the BCV, which interacts with components of the endocytic and secretory pathways to ensure bacterial survival. Numerous studies conducted recently have revealed that Brucella's ability to cause a chronic infection depends on how it interacts with the host. This paper describes the immune system, apoptosis, and metabolic control of host cells as part of the mechanism of Brucella survival in host cells. Brucella contributes to both the body's non-specific and specific immunity during chronic infection, and it can aid in its survival by causing the body's immune system to become suppressed. In addition, Brucella regulates apoptosis to avoid being detected by the host immune system. The BvrR/BvrS, VjbR, BlxR, and BPE123 proteins enable Brucella to fine-tune its metabolism while also ensuring its survival and replication and improving its ability to adapt to the intracellular environment.

Identifying Circular RNAs in HepG2 Expressing Genotype IV Swine Hepatitis E Virus ORF3 Via Whole Genome Sequencing.

Swine hepatitis E (SHE) is a new type of zoonotic infectious disease caused by swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key regulatory and virulent protein of SHEV. Circular RNAs (circRNAs) are a special kind of non-coding RNA molecule, which has a closed ring structure. In this study, to identify the circRNA profile in host cells affected by SHEV ORF3, adenovirus ADV4-ORF3 mediated the overexpression of ORF3 in HepG2 cells, whole genome sequencing was used to investigate the differentially expressed circRNAs, GO and KEGG were performed to enrichment analyze of differentially expressed circRNA-hosting gene, and Targetscan and miRanda softwares were used to analyze the interaction between circRNA and miRNA. The results showed adenovirus successfully mediated the overexpression of ORF3 in HepG2 cells, 1,105 up-regulation circRNAs and 1,556 down-regulation circRNAs were identified in ADV4-ORF3 infection group compared with the control. GO function enrichment analysis of differentially expressed circRNAs-hosting genes classified three main categories (cellular component, biological process and molecular function). KEGG pathway enrichment analysis scatter plot showed the pathway term of top20. The circRNAs with top10 number of BS sites for qRT-PCR validation were selected to confirmed, the results indicated that the up-regulated hsa_circ_0001423 and hsa_circ_0006404, and down-regulated of hsa_circ_0004833 and hsa_circ_0007444 were consistent with the sequencing data. Our findings first preliminarily found that ORF3 protein may affect triglyceride activation (GO:0006642) and riboflavin metabolism (ko00740) in HepG2 cells, which provides a scientific basis for further elucidating the effect of ORF3 on host lipid metabolism and the mechanism of SHEV infection.

Deep Insight Into Long Non-coding RNA and mRNA Transcriptome Profiling in HepG2 Cells Expressing Genotype IV Swine Hepatitis E Virus ORF3.

Swine hepatitis E (swine HE) is a new type of zoonotic infectious disease caused by the swine hepatitis E virus (swine HEV). Open reading frame 3 (ORF3) is an important virulent protein of swine HEV, but its function still is mainly unclear. In this study, we generated adenoviruses ADV4-ORF3 and ADV4 negative control (ADV4-NC), which successfully mediated overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP, respectively, in HepG2 cells. High-throughput sequencing was used to screen for differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs). The cis-target genes of lncRNAs were predicted, functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]) was performed, and 12 lncRNAs with statistically significant different expressions (p ≤ 0.05 and q ≤ 1) were selected for further quantitative real-time reverse transcription (qRT-PCR) validation. In HepG2 cells, we identified 62 significantly differentially expressed genes (DEGs) (6,564 transcripts) and 319 lncRNAs (124 known lncRNAs and 195 novel lncRNAs) that were affected by ORF3, which were involved in systemic lupus erythematosus, Staphylococcus aureus infection, signaling pathways pluripotency regulation of stem cells, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and platinum drug resistance pathways. Cis-target gene prediction identified 45 lncRNAs corresponding to candidate mRNAs, among which eight were validated by qRT-PCR: LINC02476 (two transcripts), RAP2C-AS1, AC016526, AL139099, and ZNF337-AS1 (3 transcripts). Our results revealed that the lncRNA profile in host cells affected by ORF3, swine HEV ORF3, might affect the pentose and glucuronate interconversions and mediate the formation of obstructive jaundice by influencing bile secretion, which will help to determine the function of ORF3 and the infection mechanism and treatment of swine HE.

Immunological pathways of macrophage response to Brucella ovis infection.

As the molecular mechanisms of Brucella ovis pathogenicity are not completely clear, we have applied a transcriptome approach to identify the differentially expressed genes (DEGs) in RAW264.7 macrophage infected with B. ovis. The DEGs related to immune pathway were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) functional enrichment analysis. Quantitative real-time PCR (qRT-PCR) was performed to validate the transcriptome sequencing data. In total, we identified 337 up-regulated and 264 down-regulated DEGs in B. ovis-infected group versus mock group. Top 20 pathways were enriched by KEGG analysis and 20 GO by functional enrichment analysis in DEGs involved in the molecular function, cellular component, and biological process and so on, which revealed multiple immunological pathways in RAW264.7 macrophage cells in response to B. ovis infection, including inflammatory response, immune system process, immune response, cytokine activity, chemotaxis, chemokine-mediated signaling pathway, chemokine activity, and CCR chemokine receptor binding. qRT-PCR results showed Ccl2 (ENSMUST00000000193), Ccl2 (ENSMUST00000124479), Ccl3 (ENSMUST00000001008), Hmox1 (ENSMUST00000005548), Hmox1 (ENSMUST00000159631), Cxcl2 (ENSMUST00000075433), Cxcl2 (ENSMUST00000200681), Cxcl2 (ENSMUST00000200919), and Cxcl2 (ENSMUST00000202317). Our findings firstly elucidate the pathways involved in B. ovis-induced host immune response, which may lay the foundation for revealing the bacteria-host interaction and demonstrating the pathogenic mechanism of B. ovis.

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

Brucellosis is a zoonotic infectious disease caused by Brucella infection. Outer membrane protein 25 (Omp25) is closely related to the virulence and immunogenicity of Brucella. However, the molecular mechanism of Omp25 affecting Brucella-mediated macrophage autophagy remains unclear. Our previous study 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 revealed the differentially expressed genes (DEGs) profile in RAW264.7 macrophage cells infected with Brucella melitensis Omp25 deletion mutant (∆Omp25 B. melitensis). Here, we predicted the target genes of the four miRNAs by TargetScan, miRanda, and PicTar. GO and KEGG were used for functional enrichment analysis of DEGs profile to reveal the autophagic pathway-associated genes. The overlapped genes, which drawn the autophagic pathway-associated miRNA-mRNA networks by cytoscape software, were identified by intersecting with the predicted target genes and autophagic pathway-associated DEGs. qRT-PCR was performed to validate the mRNAs of networks. The results showed that 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. Our findings are of great significance in elucidating the function of Omp25, revealing the infection mechanism of Brucella and prophylaxising and treating brucellosis.

Transcriptome Landscape of Intracellular Brucella ovis Surviving in RAW264.7 Macrophage Immune System.

Brucella ovis infection results in genital damage and epididymitis in rams, placental inflammation and rare abortion in ewes, and neonatal mortality in lambs. However, the mechanism underlying B. ovis infection remains unclear. In the present study, we used prokaryotic transcriptome sequencing to identify the differentially expressed genes (DEGs) between wild-type B. ovis and intracellular B. ovis in RAW264.7 macrophages. Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed, and quantitative reverse transcriptase PCR (qRT-PCR) was used to validate the top 10 upregulated and downregulated DEGs. The results showed that 212 genes were differentially expressed, including 68 upregulated and 144 downregulated genes, which were mainly enriched in 30 GO terms linked to biological process, cellular component, and molecular function. KEGG analysis showed that the DEGs were enriched in the hypoxia-inducible factor 1 (HIF-1) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, beta-alanine metabolism, and quorum sensing pathway. BME_RS01160, BME_RS04270, BME_RS08185, BME_RS12880, BME_RS25875, predicted_RNA865, and predicted_RNA953 were confirmed with the transcriptome sequencing data. Hence, our findings not only reveal the intracellular parasitism of B. ovis in the macrophage immune system, but also help to understand the mechanism of chronic B. ovis infection.