| Mycoplasma gallisepticum（MG）is a pathogenic microorganism that can cause respiratory disease in chickens and is one of the common pathogens that threaten the healthy development of poultry industry.MG is difficult to be cleared completely after infection and is easily to form mixed infections with other pathogens,leading to increased mortality.The pathogenic mechanism of MG has not been fully revealed yet,and some studies have shown that MG can act on the host by secreting virulence proteins.As a virulence protein widely found in pathogenic microorganisms,nucleases can induce apoptosis in host cells while also mediate immune escape.Nucleases are also essential to the survival of the MG itself.Mycoplasma has no ability to synthesize nucleic acid precursors,and mainly provides nucleic acid precursors for its growth and metabolism by secreting nucleases to degrade exogenous nucleic acids.Therefore,it is important to investigate the biological functions of MG nucleases to reveal the pathogenic mechanism of MG.In this study,689 proteins identified by MG outer membrane vesicle proteomics were analyzed and toxicity prediction was performed using the Virulent Pred toxicity protein database,we combined the literature and identified TatD nuclease as a potential virulence factor for MG with a Virulent Pred prediction score of 0.8063.The MG TatD nuclease gene sequence was analyzed to determine the absence of signal peptide and the absence of TGA codon,and the prokaryotic expression system was used for expression.The optimal induction concentration of TatD nuclease recombinant protein IPTG was determined to be 0.5 mmol/L,the optimal induction time was 4 h,and the expression form was soluble expression.The nuclease activity was determined by nuclease degradation of nucleic acids,and the results showed that MG TatD nuclease is a nuclease with nucleic acid degradation activity,which can degrade host DNA,RNA and plasmid DNA with Mg2+-dependent activity and has good heat resistance activity in the temperature range of 29-59°C.Meanwhile,A rabbit polyclonal antibody to TatD nuclease was also prepared with an antibody potency above 16,000.To detect the toxic effects of TatD nuclease on chicken macrophages,HD11 cells were treated with different concentration gradients of TatD nuclease recombinant protein for 4,8,12 and 24 h.Cell viability was measured by CCK8,and the results showed that the viability of HD11 cells was significantly affected by the TatD nuclease dose of 40μg/m L.Laser confocal microscopy was used to observe the adhesion of TatD nuclease to HD11 cells,and it was observed that TatD nuclease could adhere to the surface of HD11 cells.Nuclei of HD11 cells stained with DAPI dye after TatD nuclease action were observed to undergo fragmentation.Further use of AO/EB staining and flow cytometry determined that TatD nuclease induced HD11 cells to undergo apoptosis.AO/EB staining showed that TatD nuclease action caused apoptosis in HD11 cells at 8 h and a large increase in apoptotic cells at 12 h.The effect of 20,40 and 80μg/m L of TatD nuclease interacted with HD11 cells for 12 h by flow cytometry,and TatD nuclease at 40μg/m L caused 41.8%of apoptosis in HD11 cells,and TatD nuclease at 80μg/m L caused 79%of apoptosis in HD11 cells.The effect of TatD nuclease on the level of lactate dehydrogenase release in HD11 cells was measured,and the results showed that the level of lactate dehydrogenase release increased with the prolonged action time,reaching a high level at 12 h.The effect of TatD nuclease on the level of reactive oxygen species in HD11 cells was detected,and the level of reactive oxygen species also increased with the duration of action,reaching a higher level at 12 h.Neutrophils capture microorganisms by releasing an extracellular trap net with DNA as the backbone.To test whether TatD nuclease could degrade the neutrophil extracellular trap net and mediate immune escape,neutrophils were isolated from chicken peripheral blood and stimulated with 2μm PMA for 3 h to produce the extracellular trap net,and the results showed that 10μg/m L of TatD nuclease could degrade the extracellular trap net after 1 h of action.In this study,a virtual screening of a compound library containing 4000 natural product small molecules was performed using TatD nuclease as a target.The three-dimensional structure of TatD nuclease was built using Alpha Fold 2 with a confidence level of more than 90%,and the Ramachandran plot showed that the structure accounted for 92.3%in the favorable region and 0%of residues in the disallowed region.The key amino acids of TatD nuclease activity were searched,compared and analyzed,His 9,His 11,His 133,His 157,Asp 211 were found to be the key amino acid residues,and the molecular docking pockets were set with the above key amino acids.The4,000 compounds were first evaluated for their drug-forming properties using Lipinski’s rule,and the 2425 compounds that passed the evaluation were screened virtually using Autodock vina and Sybyl Surflex-DOCK high-precision docking models.The compounds with high scores in both software were selected for ADME prediction,and 10 compounds with good properties were obtained.Then these 10 compounds were accurately docked,and finally 3 compounds were identified as candidates for molecular dynamics simulation.The results of molecular dynamics simulations showed that chrysosplenol D was able to bind stably to the TatD nuclease active pocket with good binding energy.The binding mode of TatD nuclease to Host DNA was simulated by molecular docking,and the docking results showed that TatD nuclease could bind to Host DNA with reasonable binding position.In conclusion,this study demonstrates that TatD nuclease is a virulence factor of Mycoplasma gallisepticum that causes apoptosis of chicken immune cells and mediates immune escape of Mycoplasma gallisepticum through its nuclease activity.The virtual screening based on TatD nucleases provides a basis for the development of new drugs targeting TatD nucleases,and provides a preliminary exploration of drug applications against Mycoplasma gallisepticum. |
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