Regulation Of Mycobacterium Bovis PtpA Protein On NF-κB Signaling Pathway In RAW264.7 Cells

Bovine Tuberculosis(b TB)is a highly contagious and chronic zoonotic disease caused by Mycobacterium bovis,a member of the Mycobacterium tuberculosis complex.It is characterized by progressive weight loss,low-volatility fever,weakness,and loss of appetite.Nuclear transcription factor-κB(NF-κB)plays an important role in regulating the body’s immune response,inflammatory response and cell survival.Previous studies have shown that the NF-κB signaling pathway is involved in Mycobacterium tuberculosis pathogenic gene transcription factors and its regulatory network nodes.Ptp A is a secreted protein phosphatase that is essential for the pathogenicity of Mycobacterium tuberculosis(Mtb)and is involved in inhibiting phagosome maturation in host macrophages.Ptp A also has DNA binding ability,which can regulate the expression of certain genes related to innate immunity in the host and promote cell proliferation,thereby suppressing the host’s innate immunity.Mycobacterium Ptp A protein can utilize the host ubiquitin system and rely on Jnk,p38 and NF-κB signaling pathways to suppress innate immunity.Although M.bovis and M.tuberculosis have a gene similarity of more than99.95%,it is unclear whether the Ptp A protein of M.bovis has a similar effect to the Ptp A protein of M.tuberculosis.Exploring this effect can provide a new direction for the prevention and control of bovine tuberculosis.In this study,we constructed a recombinant lentivirus overexpression plasmid p CDH-Ptp A,co-transfected it with Ps Pa X2 plasmid and PMD2.G plasmid into HEK293 T cells to package the lentivirus.Lentivirus was collected to infect RAW264.7 cells.Drug-screening method was used to obtain the stable expression cell line Ptp A-RAW,and it was identified by RT-PCR,indirect immunofluorescence and Western Blot,and p CDH-RAW cell lines were obtained by the same method;Using overlapping extension PCR,ptp A was subjected to site-directed mutagenesis to construct the p CDH-ΔPtp A recombinant plasmid,and a ΔPtp A-RAW cell line was constructed;Cell Counting Kit-8was used to detect the growth curve of RAW264.7,Ptp A-RAW,p CDH-RAW andΔPtp A-RAW cells and the activity of Ptp A-RAW,p CDH-RAW and ΔPtp A-RAW cells;Laser confocal microscopy was used to locate the Ptp A protein in Ptp A-RAW;The constructed Ptp A-RAW and p CDH-RAW cell lines were used for tandem affinity purification(TAP)and mass spectrometry(MS)tests to screen the interaction proteins of Ptp A protein in RAW264.7 cells,and the results were bioinformatics analysis;RAW264.7cells were infected with the collected lentiviruses of p CDH-Ptp A and p CDH-empty vectors.The effect of Ptp A protein on the expression of cytokines related to NF-κB signaling pathway in RAW264.7 cells was detected by q RT-PCR;Total RNA of ΔPtp A-RAW and Ptp A-RAW cells was extracted separately,and the effect of Ptp A mutation on the regulation of NF-κB signaling pathway in RAW264.7 cells was detected by q RT-PCR.The test results found that the stable expression cell lines Ptp A-RAW,p CDH-RAW and ΔPtp A-RAW were successfully obtained;The growth of four kinds of cells accorded with the growth trend of normal cells;The viability of Ptp A-RAW cells increased,and the viability of ΔPtp A-RAW cells decreased.Ptp A protein is found in the cytoplasm and nucleus of RAW264.7;There are 71 proteins interacting with Ptp A protein in RAW264.7 cells.KEGG analysis found that these proteins are involved in 70 signal pathways,of which 6 signal pathways are related to the NF-κB signal pathway.The Ptp A interacting proteins involved in these 6 signal pathways are: C1 qb,C3,Hsp90ab1,Nppa,C1 qa,Gapdh.String analysis found that Gapdh,Hsp90ab1,and Nppa are directly related,and C1 qb,C3,and C1 qa are directly related.There is an indirect connection between them through Apoe;Apoptosis inhibitors(TRAF-1,TRAF-2,c-IAP 1)expression in the Ptp A group were significantly higher than those in the control group.In addition,the Ptp A group compared with the control group: Beclin expression decreased significantly when TNF-αwas added for 12 hours,and PIK3 CA and Beclin expression were significantly decreased when TNF-α was not added for 24 hours.When TNF-α was added for 24 hours,The expression of PIK3 CA and Beclin decreased significantly;Compared with the ΔPtp A-RAW group,the expression of apoptosis inhibitory factors in the Ptp A-RAW group increased significantly,the expression of autophagy factors decreased significantly,and the expression of TNF-α and IκBα significantly decreased.All test results show that: Mycobacterium bovis Ptp A protein exists in the cytoplasm and nucleus of RAW264.7.It may indirectly inhibit the activation of NF-κB signaling pathway through its interacting protein to promote the expression of apoptosis inhibitors and autophagy factors.The expression of RAW264.7 inhibits the apoptosis and autophagy of RAW264.7,thereby avoiding the body’s immune mechanism to achieve immune evasion and promote the survival of Mycobacterium bovis in macrophages.When Ptp A was mutated,the inhibitory effect on the activation of the NF-κB signaling pathway weakened or disappeared.