Soluble expression and purification of recombinant bovine ferritin H-chain.

Ferritin is a potential medicine delivery vehicle and vaccine platform, and its efficient expression is a prerequisite for widespread application. This study introduces a soluble expression strategy for recombinant bovine ferritin heavy chain (rFTH) in a prokaryotic system and an improved protein purification method. The amplified rFTH gene was ligated into the prokaryotic expression vector pET30a. The recombinant vectors with the N-terminal His-tag(N-His) or C-terminal His-tag(C-His) were translated and expressed separately. The results showed that the solubility of rFTH with C-His was significantly higher than that with N-His. The expression of rFTH with C-His was attempted at 37 °C and 16 °C, respectively. The results showed that the proportion of soluble protein expressed at 37 °C was more than 90%, higher than that expressed at 16 °C. Then rFTH with C-His was purified successfully using anion exchange chromatography, modified PEG precipitation, and dialysis. The rFTH protein was characterized using SDS-PAGE, Native-PAGE, Western blot, transmission electron microscopy, and dynamic light scattering. The results demonstrated that the purified rFTH protein self-assembled into ferritin nanoparticles with a regular shape and uniform size. This study sheds new light on the soluble expression of ferritin and provides a foundation for the construction of bovine ferritin nanoparticle production platforms.

Identification of Differential Circular RNA Expression Profiles and Functional Networks in Human Macrophages Induced by Virulent and Avirulent Mycobacterium tuberculosis Strains.

Circular RNAs (circRNAs) are noncoding RNAs with diverse functions. However, most Mycobacterium tuberculosis (M.tb)-related circRNAs remain undiscovered. In this study, we infected THP-1 cells with virulent and avirulent M.tb strains and then sequenced the cellular circRNAs. Bioinformatic analysis predicted 58,009 circRNAs in all the cells. In total, 2035 differentially expressed circRNAs were identified between the M.tb-infected and uninfected THP-1 cells and 1258 circRNAs were identified in the virulent and avirulent M.tb strains. Further, the top 10 circRNAs were confirmed by Sanger sequencing, among which four circRNAs, namely circSOD2, circCHSY1, circTNFRSF21, and circDHTKD1, which were highly differentially expressed in infected cells compared with those in uninfected cells, were further confirmed by ring formation, specific primers, and RNase R digestion. Next, circRNA-miRNA-mRNA subnetworks were constructed, such as circDHTKD1/miR-660-3p/IL-12B axis. Some of the individual downstream genes, such as miR-660-3p and IL-12B, were previously reported to be associated with cellular defense against pathological processes induced by M.tb infection. Because macrophages are important immune cells and the major host cells of M.tb, these findings provide novel ideas for exploring the M.tb pathogenesis and host defense by focusing on the regulation of circRNAs during M.tb infection.

Prevalence of bovine astroviruses and their genotypes in sampled Chinese calves with and without diarrhoea.

Bovine astrovirus (BoAstV) belongs to genus Mamastravirus (MAstV). It can be detected in the faeces of both diarrhoeal and healthy calves. However, its prevalence, genetic diversity, and association with cattle diarrhoea are poorly understood. In this study, faecal samples of 87 diarrhoeal and 77 asymptomatic calves from 20 farms in 12 provinces were collected, and BoAstV was detected with reverse transcription-polymerase chain reaction (RT-PCR). The overall prevalence rate of this virus in diarrhoeal and asymptomatic calves was 55.17 % (95 % CI: 44.13, 65.85 %) and 36.36 % (95 % CI: 25.70, 48.12 %), respectively, indicating a correlation between BoAstV infection and calf diarrhoea (OR=2.15, P=0.024). BoAstV existed mainly in the form of co-infection (85.53 %) with one to five of nine viruses, and there was a strong positive correlation between BoAstV co-infection and calf diarrhoea (OR=2.83, P=0.004). Binary logistic regression analysis confirmed this correlation between BoAstV co-infection and calf diarrhoea (OR=2.41, P=0.038). The co-infection of BoAstV and bovine rotavirus (BRV) with or without other viruses accounted for 70.77 % of all the co-infection cases. The diarrhoea risk for the calves co-infected with BoAstV and BRV was 8.14-fold higher than that for the calves co-infected with BoAstV and other viruses (OR=8.14, P=0.001). Further, the co-infection of BoAstV/BRV/bovine kobuvirus (BKoV) might increase the risk of calf diarrhoea by 14.82-fold, compared with that of BoAstV and other viruses (OR=14.82, P <0.001). Then, nearly complete genomic sequences of nine BoAstV strains were assembled by using next-generation sequencing (NGS) method. Sequence alignment against known astrovirus (AstV) strains at the levels of both amino acids and nucleotides showed a high genetic diversity. Four genotypes were identified, including two known genotypes MAstV-28 (n=3) and MAstV-33 (n=2) and two novel genotypes designated tentatively as MAstV-34 (n=1) and MAstV-35 (n=3). In addition, seven out of nine BoAstV strains showed possible inter-genotype recombination and cross-species recombination. Therefore, our results increase the knowledge about the prevalence and the genetic evolution of BoAstV and provide evidence for the association between BoAstV infection and calf diarrhoea.

Differential nitric oxide induced by Mycobacterium bovis and BCG leading to dendritic cells apoptosis in a caspase dependent manner.

Dendritic cells (DCs) are critical for both innate and adaptive immunity. Meanwhile, nitric oxide (NO) is a member of reactive nitrogen species (RNS) generally considered to play a key role in the bactericidal process in innate immunity against Mycobacterium tuberculosis complex infection. The present study therefore investigated the mechanism of NO production in murine DCs induced by Mycobacterium bovis (M.bovis) and its attenuated strain Bacillus Calmette-Guérin (BCG) infection. The expression of genes Slc7A1, Slc7A2, iNOS, and ArgI essential to NO synthesis was up-regulated in M.bovis/BCG infected DCs. IFN-γ addition further increased, while the iNOS inhibitor L-NMMA significantly inhibited their expression. Accordingly, the end products of arginine metabolism, NO and urea, were found to be significantly increased. In addition, BCG induced significantly higher levels of apoptosis in DCs compared to M.bovis shown by higher levels of DNA fragmentation using flow cytometry and release of mitochondrial Cytochrome C, and up-regulation of the genes caspase-3, caspase-8, caspase-9 and dffa critical to apoptosis by qRT-PCR detection and western blot analysis. Furthermore, IFN-γ increased, but L-NMMA decreased apoptosis of M.bovis/BCG infected DCs. In addition, mycobacterial intracellular survival was significantly reduced by IFN-γ treatment in BCG infected DCs, while slightly increased by L-NMMA treatment. Taken altogether, our data show that NO synthesis was differentially increased and associated with apoptosis in M.bovis/BCG infected DCs. These findings may significantly contribute to elucidate the pathogenesis of M.bovis.

Indirubin Treatment of Lipopolysaccharide-Induced Mastitis in a Mouse Model and Activity in Mouse Mammary Epithelial Cells.

Indirubin is a Chinese medicine extracted from indigo and known to be effective for treating chronic myelogenous leukemia, neoplasia, and inflammatory disease. This study evaluated the in vivo anti-inflammatory activity of indirubin in a lipopolysaccharide- (LPS-) induced mouse mastitis model. The indirubin mechanism and targets were evaluated in vitro in mouse mammary epithelial cells. In the mouse model, indirubin significantly attenuated the severity of inflammatory lesions, edema, inflammatory hyperemia, milk stasis and local tissue necrosis, and neutrophil infiltration. Indirubin significantly decreased myeloperoxidase activity and downregulated the production of tumor necrosis factor-α, interleukin-1ß (IL-1ß), and IL-6 caused by LPS. In vitro, indirubin inhibited LPS-stimulated expression of proinflammatory cytokines in a dose-dependent manner. It also downregulated LPS-induced toll-like receptor 4 (TLR4) expression and inhibited phosphorylation of LPS-induced nuclear transcription factor-kappa B (NF-κB) P65 protein and inhibitor of kappa B. In addition to its effect on the NF-κB signaling pathway, indirubin suppressed the mitogen-activated protein kinase (MAPK) signaling by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), P38, and c-jun NH2-terminal kinase (JNK). Indirubin improved LPS-induced mouse mastitis by suppressing TLR4 and downstream NF-κB and MAPK pathway inflammatory signals and might be a potential treatment of mastitis and other inflammatory diseases.

Host factor RBMX2 promotes epithelial cell apoptosis by downregulating APAF-1's Retention Intron after Mycobacterium bovis infection.

The Mycobacterium tuberculosis variant bovis (M. bovis) is a highly pathogenic environmental microorganism that causes bovine tuberculosis (bTB), a significant zoonotic disease. Currently, "test and culling" is the primary measure for controlling bTB, but it has been proven to be inadequate in animals due to their high susceptibility to the pathogen. Selective breeding for increased host resistance to bTB to reduce its prevalence is feasible. In this study, we found a vital host-dependent factor, RBMX2, that can potentially promote M. bovis infection. By knocking RBMX2 out, we investigated its function during M. bovis infection. Through transcriptome sequencing and alternative splicing transcriptome sequencing, we concluded that after M. bovis infection, embryo bovine lung (EBL) cells were significantly enriched in RNA splicing associated with apoptosis compared with wild-type EBL cells. Through protein/molecular docking, molecular dynamics simulations, and real-time quantitative PCR, we demonstrated that RBMX2 promotes the apoptosis of epithelial cells by upregulating and binding to apoptotic peptidase activating factor 1 (APAF-1), resulting in the alternative splicing of APAF-1 as a retention intron. To our knowledge, this is the first report of M. bovis affecting host epithelial cell apoptosis by hijacking RBMX2 to promote the intron splicing of downstream APAF-1. These findings may represent a significant contribution to the development of novel TB prevention and control strategies.

Mycobacterium tuberculosis FadD18 Promotes Proinflammatory Cytokine Secretion to Inhibit the Intracellular Survival of Bacillus Calmette-Guérin.

Mycobacterium tuberculosis causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a Bacillus Calmette-Guérin (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive fadD18 gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that fadD18 expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1ß, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.

Mycobacterium tuberculosis Fatty Acyl-CoA Synthetase fadD33 Promotes Bacillus Calmette-Guérin Survival in Hostile Extracellular and Intracellular Microenvironments in the Host.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tb), remains a significant global health challenge. The survival of M. tb in hostile extracellular and intracellular microenvironments is crucial for its pathogenicity. In this study, we discovered a Bacillus Calmette-Guérin (BCG) mutant B1033 that potentially affected mycobacterium pathogenicity. This mutant contained an insertion mutation gene, fadD33, which is involved in lipid metabolism; however, its direct role in regulating M. tb infection is not well understood. Here, we found that the absence of fadD33 reduced BCG adhesion and invasion into human pulmonary alveolar epithelial cells and increased the permeability of the mycobacterial cell wall, allowing M. tb to survive in the low pH and membrane pressure extracellular microenvironment of the host cells. The absence of fadD33 also inhibited the survival of BCG in macrophages by promoting the release of proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumors necrosis factor-α, through the mitogen-activated protein kinase p38 signaling pathway. Overall, these findings provide new insights into M. tb mechanisms to evade host defenses and might contribute to identifying potential therapeutic and vaccine targets for tuberculosis prevention.

The miR-100-5p Targets SMARCA5 to Regulate the Apoptosis and Intracellular Survival of BCG in Infected THP-1 Cells.

Mycobacterium tuberculosis (M. tb) is the causative agent of tuberculosis (TB) that leads to millions of deaths each year. Extensive evidence has explored the involvement of microRNAs (miRNAs) in M. tb infection. Limitedly, the concrete function of microRNA-100-5p (miR-100-5p) in M. tb remains unexplored and largely elusive. In this study, using Bacillus Calmette-Guérin (BCG) as the model strain, we validated that miR-100-5p was significantly decreased in BCG-infected THP-1 cells. miR-100-5p inhibition effectively facilitated the apoptosis of infected THP-1 cells and reduced BCG survival by regulating the phosphatidylinositol 3-kinase/AKT pathway. Further, SMARCA5 was the target of miR-100-5p and reduced after miR-100-5p overexpression. Since BCG infection down-regulated miR-100-5p in THP-1 cells, the SMARCA5 expression was up-regulated, which in turn increased apoptosis through caspase-3 and Bcl-2 and, thereby, reducing BCG intracellular survival. Collectively, the study uncovered a new molecular mechanism of macrophage to suppress mycobacterial infection through miR-100-5p and SMARCA5 pathway.

Mycobacterium tuberculosis Rv0309 Dampens the Inflammatory Response and Enhances Mycobacterial Survival.

To reveal functions of novel Mycobacterium tuberculosis (M. tb) proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of M. tb genome in inhibiting the host inflammatory response and the underlying mechanism, using in-vitro and in-vivo experiments. A recombinant M. smegmatis strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1ß, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1ß, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.