Objective: Evidence suggests that microRNAs (miRNAs) regulate the expression of genes involved in bone metabolism. This study aimed to investigate the role of miR-505 in the osteogenic differentiation of MC3T3-E1 cells. Methods: We performed miRNA sequencing to identify differentially expressed miRNAs between MC3T3-E1 cells treated with osteogenic induction medium (OIM) and control cells. Bioinformatics analysis was performed by using the TargetScan and miRDB databases. The expression of miR-505 in MC3T3-E1 cells was detected during osteogenic differentiation. After transfection with miR-505 mimic or miR-505 inhibitor, MC3T3-E1 cells were induced to differentiate into osteoblasts, and the expression of osteogenic differentiation markers (Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), and osterix (OSX)) was detected. Results: miR-505 was the most downregulated miRNA among the differentially expressed miRNAs. The RUNX2 gene was identified as a potential target of miR-505 using the target prediction program. miR-505 expression was downregulated during osteogenic differentiation of MC3T3-E1 cells. The expression of osteogenic marker genes was inhibited in MC3T3-E1 cells after transfection with miR-505. However, the expression of osteogenic marker genes was upregulated after transfection with miR-505 inhibitor.Conclusion: This study is the first to report miR-505 could bind to the RUNX2 gene and thus regulate partly the dysfunction of osteoblasts differentiation, which is expected to be targets for the treatment of osteoporosis.
Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the “type IX secretion system” (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2Δ gldM ) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2Δ gldM and that these genes were mainly involved in the secretion of proteins. Yb2Δ gldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography–tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2Δ gldM compared to Yb2. The complementation strain cYb2Δ gldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2Δ gldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence. Electronic supplementary material The online version of this article (10.1186/s13567-019-0660-0) contains supplementary material, which is available to authorized users.
In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear.
Riemerella anatipestifer is one of the major bacterial pathogens of ducks and causes significant economic losses in poultry agriculture. Usually, methods for detecting R. anatipestifer infection need specialized equipment and highly skilled personnel. In this study, a novel colloidal gold immunochromatographic strip was developed for rapid detection of R. anatipestifer in ducks. The monoclonal antibodies 2D5 and 2A6 against R. anatipestifer were used as colloidal gold-labeled protein and capture protein, respectively, to recognize the bacteria in tryptic soy broth medium culture and in hearts of infected ducks. The goat anti-mouse IgG antibody was labeled on nitrocellulose membrane as a control for C line. The labeling pH was optimized as 10.0, and the concentration of 2D5 labeled to colloidal gold particles was optimized as 18 μg/mL. The strip specifically detected serotypes 1, 2, and 10 R. anatipestifer strains and showed no cross-reaction with Escherichia coli , Salmonella enterica, and Pasteurella multocida strains. The sensitivity of the strip for detecting R. anatipestifer was 1.0 × 10 6 colony forming unit. The strips remained stable for up to 8 mo at 4°C, and the detection can be completed within 15 min. The strip can detect R. anatipestifer in hearts of the ducks experimentally infected with R. anatipestifer but not infected with E. coli , which were also confirmed with bacterial isolation followed by multiplex polymerase chain reaction. These results suggested that the strips are reliable methods for identification of R. anatipestifer in laboratories and in duck farms.
Biotin is essential for the growth and pathogenicity of microorganisms. Damage to biotin biosynthesis results in impaired bacterial growth and decreased virulence in vivo. However, the mechanisms of biotin biosynthesis in Riemerella anatipestifer remain unclear. In this study, two R. anatipestifer genes associated with biotin biosynthesis were identified. AS87_RS05840 encoded a BirA protein lacking the N-terminal winged helix-turn-helix DNA binding domain, identifying it as a Group I biotin protein ligase, and AS87_RS09325 encoded a BioX protein, which was in the helix-turn-helix xenobiotic response element family of transcription factors. Electrophoretic mobility shift assays demonstrated that BioX bound to the promoter region of bioF. In addition, the R. anatipestifer genes bioF (7-keto-8-aminopelargonic acid synthase), bioD (dethiobiotin synthase), and bioA (7,8-diaminopelargonic acid synthase) were in an operon and were regulated by BioX. Quantitative reverse-transcription PCR showed that transcription of the bioFDA operon increased in the mutant Yb2ΔbioX in the presence of excessive biotin, compared with that in the wild-type strain Yb2, suggesting that BioX acted as a repressor of biotin biosynthesis. Streptavidin blot analysis showed that BirA caused biotinylation of BioX, indicating that biotinylated BioX was involved in metabolic pathways. Moreover, as determined by the median lethal dose, the virulence of Yb2ΔbioX was attenuated a 500-fold compared with that of Yb2. To summarize, the genes bioA and bioX were identified in R. anatipestifer, and BioX was found to act as a repressor of the bioFDA operon involved in the biotin biosynthesis pathway and identified as a bacterial virulence factor. IMPORTANCE Riemerella anatipestifer is a causative agent of diseases in ducks, geese, turkeys, and various other domestic and wild birds. Our study reveals that biotin synthesis of R. anatipestifer is regulated by the BioX through binding to the promoter region of bioF gene to inhibit transcription of bioFDA operon. Moreover, bioX is required for R. anatipestifer pathogenicity, suggesting BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX is thus identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.
Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrate that bacterial virulence and secretion proteins of the T9SS mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, compare to the wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which absent in the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of the gene mutation and complementation strains. The virulence assessment showed a more than 1,000-folds attenuated virulence, and significantly reduced bacterial loads in blood of infected ducks for the mutant strain Yb2Δ00980. The bacterial virulence was recovered in the complementation strain cYb2Δ00980. Further study indicated that the AS87_RS00980 gene encodes a secretion protein metallophosphatase (MPPE), which displayed phosphatase activity and cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined as 7.0 and 60°C, respectively; and the Km and Vmax were determined as 3.53 mM and 198.1U/mg. The MPPE activity was activated by Zn2+, Cu2+, but inhibited by Fe3+, Fe2+, and EDTA. There are five conserved sites of N267, H268 H351, H389, and H391 in the MEEP domain. The mutant proteins of Y267-rMPPE and Y268-rMPPE retained 29.30% and 19.81% relative activity respectively, and mutant proteins Y351-rMPPE, Y389-rMPPE, and Y391-rMPPE lost almost all the MPPE activity. Taken together, these results indicated that R. anatipestifer AS87_RS00980 gene encodes a MPPE, which is a secretion protein of T9SS and plays an important role in bacterial virulence. Importance Riemerella anatipestifer T9SS is recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes are identified, but no effector is reported in the R. anatipestifer up to date. In this study, we identified the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase that displays phosphatases activity and associated with bacterial virulence. Enzymatic activity of the metallophosphoesterase was determined, and the Km and Vmax were 3.53 mM and 198.1U/mg respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated over 1000-fold, indicating it is an important virulence factor. In summary, we identified that R. anatipestifer AS87_RS00980 gene encodes an important T9SS effector MPPE, which plays an important role in bacterial virulence.
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