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.

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.

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 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.

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.

An abattoir-based study on the prevalence of bovine tuberculosis from culled adult dairy cows in Wuhan, China.

Abattoir surveillance is an integral component of a bovine tuberculosis (bTB) eradication programme. The objective of this study was to determine the prevalence of bTB among culled adult dairy cows in Wuhan, China and to further assess two diagnostic procedures as an adjunct to the confirmation of M. bovis in animals with TB-like lesions. The study was conducted in an abattoir located in Wuhan, China over a period of 41 days from June to July 2019. A total of 171 culled adult dairy cows were sampled and inspected, and blood samples collected from 134 of these. The viscera and lymph nodes of the carcasses were visually inspected and palpated for TB-like lesions. A total of 28.1 % (48/171) of the carcasses had gross TB-like lesions. 89.6 % (43/48) of the animals with TB-like lesions were positive to a PCR procedure for bTB. The sensitivity and specificity for post-mortem examination for TB-like lesions using a Bayesian latent class analysis model was estimated to be 60.8 % and 86.6 %, respectively. A seroprevalence of 20.9 % (28/134) was recorded for antibody response to M. bovis antigens MPB70 and MPB83 based on an ELISA procedure. There was a low-moderate agreement between the ELISA and PCR results in the detection of bTB (Kappa = 0.46, 95 % CI: 0.24-0.67). The study confirms a high prevalence of bTB among culled adult dairy cows in the abattoir and highlights the need to implement surveillance for bTB based on post-mortem examination and ELISA and PCR methods in association with backward tracing of infected dairy herds.

MicroRNA-18b-5p Downregulation Favors Mycobacterium tuberculosis Clearance in Macrophages via HIF-1α by Promoting an Inflammatory Response.

The modulation of the interaction between macrophages and Mycobacterium tuberculosis (M.tb) through microRNA during M.tb infection is increasingly capturing the attention of researchers. However, the potential role of microRNA-18b-5p (miR-18b) is not elucidated yet. In this study, miR-18b was found to be downregulated in M.tb-infected macrophage cell lines (THP-1 and RAW264.7) in time- and dose-dependent manners. Furthermore, when the miR-18b mimic and inhibitor and small interfering RNA hypoxia-inducible factor 1α (si-HIF-1α) were transfected into the macrophages separately or in combination, it was found that miR-18b targeted hypoxia-inducible factor 1α (HIF-1α). During M.tb infection, the decrease in the expression of miR-18b facilitated HIF-1α expression, which led to the increased production of pro-inflammatory cytokines, such as IL-6, resulting in decreased bacterial survival in the host cells. Moreover, the phosphorylation of p38 MAPK and NF-κB p65 was activated by the miR-18b inhibitor. Our findings expand the current understanding of the M.tb-cell interaction mechanism and provide a potential target to control M.tb infection.

An Abattoir-Based Study on the Prevalence of Salmonella Fecal Carriage and ESBL Related Antimicrobial Resistance from Culled Adult Dairy Cows in Wuhan, China.

The objective of this study was to estimate the fecal carriage of Salmonella spp. among culled adult dairy cows presented to an abattoir in Wuhan, China and to evaluate their antimicrobial resistance profiles. Rectal swabs from 138 culled cows were cultured. Laboratory analysis involved the identification of Salmonella, the susceptibility assessment and the presence of Extended Spectrum ß-lactamases and mcr genes in the isolates. An overall prevalence of Salmonella of 29.0% was recorded with 63.4% (26/41) and 2.4% (1/41) of the isolates identified as S. Typhimurium and S. Dublin, respectively. The occurrence of Salmonella was higher (odd ratios: 3.3) in culled cows originating from the northeast zone of China than cows originating from the central and north zones. Twenty multi-drug resistant strains (resistant to three or more antimicrobial agents) were detected (48.8%) and overall, a high resistance to ampicillin (36/41) and tetracycline (15/41) was observed. Extended Spectrum ß-lactamases phenotypes were found in 7/41 isolates, of which all contained the blaCTX-M resistance gene, and no mcr genes were found by polymerase chain reaction. The high prevalence of Salmonella fecal carriage and antimicrobial resistance may contribute to an increased risk of Salmonella transmission to food.

Mycobacterium tuberculosis YrbE3A Promotes Host Innate Immune Response by Targeting NF-κB/JNK Signaling.

: Mycobacterium tuberculosis is considered a successful pathogen with multiple strategies to undermine host immunity. The YrbE3A is encoded by Rv1964 within the RD15 region present in the genome of Mtb, but missing in M. bovis, M. bovis BCG (Pasteur) strain, and M. smegmatis (Ms). However, little is known about its function. In this study, the YrbE3A gene was cloned into pMV261 and expressed in Ms and BCG, while the strains with the vector served as the controls. The YrbE3A was expressed on the mycobacterial membrane, and the purified protein could stimulate RAW264.7 cells to produce IL-6. Furthermore, the effect of the recombinant strains on cytokine secretion by RAW264.7 was confirmed, which varied with the host strains. Ms_YrbE3A increased significantly higher levels of TNF-α and IL-6 than did Ms_vec, while BCG_YrbE3A enhanced higher TNF-α than BCG_vec. The pathways associated with NF-κB p65 and MAPK p38/JNK, other than Erk1/2, regulated this process. In addition, mice were infected with Ms_YrbE3A and Ms-vec and were kinetically examined. Compared to Ms-vec, Ms_YrbE3A induced more serious inflammatory damage, higher levels of TNF-α and IL-6, higher numbers of lymphocytes, neutrophils, and monocytes in a time-dependent way, but lower lung bacterial load in lung. These findings may contribute to a better understanding of Mtb pathogenesis.

Upregulation of Cytokines and Differentiation of Th17 and Treg by Dendritic Cells: Central Role of Prostaglandin E2 Induced by Mycobacterium bovis.

Mycobacterium bovis (M. bovis) is a zoonotic pathogen that causes bovine and human tuberculosis. Dendritic cells play a critical role in initiating and regulating immune responses by promoting antigen-specific T-cell activation. Prostaglandin E2 (PGE2)-COX signaling is an important mediator of inflammation and immunity and might be involved in the pathogenesis of M. bovis infection. Therefore, this study aimed to reveal the character of PGE2 in the differentiation of naïve CD4+ T cells induced by infected dendritic cells (DCs). Murine bone marrow-derived DCs were pre-infected with M. bovis and its attenuated strain M. bovis bacillus Calmette-Guérin (BCG). Then, the infected DCs were co-cultured with naïve CD4+ T cells with or without the cyclooxygenase (COX) inhibitor indomethacin. Quantitative RT-PCR analysis and protein detection showed that PGE2/COX-2 signaling was activated, shown by the upregulation of PGE2 production as well as COX-2 and microsomal PGE2 synthase (mPGES1) transcription in DCs specifically induced by M. bovis and BCG infection. The further co-culture of infected DCs with naïve CD4+ T cells enhanced the generation of inflammatory cytokines IL-17 and IL-23, while indomethacin suppressed their production. Following this, the differentiation of regulatory T cells (Treg) and Th17 cell subsets was significantly induced by the infected DCs rather than uninfected DCs. Meanwhile, M. bovis infection stimulated significantly higher levels of IL-17 and IL-23 and the differentiation of Treg and Th17 cell subsets, while BCG infection led to higher levels of TNF-α and IL-12, but lower proportions of Treg and Th17 cells. In mice, M. bovis infection generated more bacterial load and severe abnormalities in spleens and lungs, as well as higher levels of COX-2, mPGE2 expression, Treg and Th17 cell subsets than BCG infection. In conclusion, PGE2/COX-2 signaling was activated in DCs by M. bovis infection and regulated differentiation of Treg and Th17 cell subsets through the crosstalk between DCs and naive T cells under the cytokine atmosphere of IL-17 and IL-23, which might contribute to M. bovis pathogenesis in mice.

Down-Regulation of miR-378d Increased Rab10 Expression to Help Clearance of Mycobacterium tuberculosis in Macrophages.

Mycobacterium tuberculosis (M. tb) can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of M. tb-infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during M. tb infection. This study aimed to investigate the roles of miR-378d in M. tb infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, M. tb infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, M. tb survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1ß, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during M. tb infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with M. tb infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during M. tb infection of macrophages, miR-378d was down-regulated and functioned on decreasing M. tb intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against M. tb infection.